Journal of Baghdad College of Dentistry, Vol. 34, No. 4 (2022), ISSN (P): 1817-1869, ISSN (E): 2311-5270 Journal of Baghdad College of Dentistry, Vol. 34, No. 4 (2022), ISSN (P): 1817-1869, ISSN (E): 2311-5270 34 Research Article Impact of different types of storage media on enamel surface roughness and granularity distribution of avulsed teeth (In vitro study) Rawaa S. Obeid 1* , Muna S. Khalaf 2 1Master Student, Department of Pediatric and Preventive Dentistry, College of Dentistry, University of Baghdad. 2Assistant professor, Department of Pediatric and Preventive Dentistry, College of Dentistry, University of Baghdad. Bab-Almoadham, P.O. Box 1417, Baghdad, Iraq.  Correspondence: srwa3713@gmail.com Abstract: Background: To investigate the effect of different types of storage media on enamel surface microstructure of avulsed teeth by using atomic force microscope.Materials and methods : Twelve teeth blocks from freshly extracted premolars for orthodontic treatment were selected . The study samples were divided into three groups according to type of storage media :A-egg white , B- probiotic yogurt , and C-bovine milk . All the samples were examined for changes in surface roughness and surface granularity distribution using atomic force microscope, at two periods: baseline, and after 8 hours of immersing in the three types of storage media. Results: Milk group had showed a significant increase in the mean of the roughness values at the test period, while the egg white and the probiotic yogurt groups showed decrease in the surface roughness at the test period. No significant changes was found in the grain size of enamel surface of the avulsed tooth in any types of three storage media at eight hours interval. The use of egg white and probiotic yogurt to store the samples may be beneficial in that they contain various ions and proteins that fill up enamel valleys, while the longer periods of milk exposure encourage the bacteria to continue fermenting lactose, resulting in continual acid generation and increased demineralization. Conclusion : Milk group demonstrated the highest roughness values, while the egg white group demonstrated the lowest roughness values of the teeth. No significant changes in the grain size of enamel surface of the tested teeth in any types of three storage media at eight hours interval . Keywords: Atomic force microscope, Avulsion, Bovine milk, Egg white, Granularity distribution , Probiotic yogurt, Storage media , Surface roughness. Introduction The most extreme of all sorts of traumatic dental injuries is avulsion of permanent teeth, which occurs when the tooth is completely dislodged from its position, causing extensive harm to the supportive tissue, vascular, and nerve components (1). The survival of the periodontal ligament (PDL) that are cells residual on the root surface, the integrity of the root cementum, and limited microbial contamination, all of which are circumstances specifically linked to the extra-alveolar period. Sort of storage after avulsion, and root surface changes, can determine the outcome of a replanted tooth as well as its preservation on the dental arch for the lengthiest probable period , the ideal treatment for an avulsed permanent tooth is its immediate replantation into the socket. Despite its acknowledged therapeutic efficacy, clinical practice has demonstrated that immediate replanting is uncommon owing to injury related circumstances, such as the existence of life-threatening incidents, the recipient location if subjected to significant damage, the patient's emotive state at the time of incident, or easily an insufficient information or trust in replantation Received: date: 01-03-2022 Accepted: date: 10-04-2022 Published: date: 15-12-2022 Copyright: © 2022 by the authoruthors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY) liscence (https://creativecommons.org/li censes/by/4.0/). https://doi.org/10.26477 /jbcd.v34i4.3275 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://doi.org/10.26477/jbcd.v34i1.3089 https://doi.org/10.26477/jbcd.v34i1.3089 J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 35 techniques among the common people or even practitioners(2). Enamel is the tooth's exterior protective layer. Tooth demineralization is accelerated in acidic environment, which leads to cavity formation(3). Surface texture includes roughness. The variations in the orientation of a practical surface's vector from its perfect form are used to assess it. The surface is rough if the deviations are considerable; smooth if the variances are minimal(4). When periodontal ligament cells are out of their alveolar socket, there are solutions that can prolong their lives. These options must be employed if immediate reimplantation is not possible (5). Dry storage (up to 30 minutes) can induce more root resorption and is more detrimental to maintaining a normal PDL as compared to wet storage (6). Natural products had been investigated as potential pharmacological substitutes, and as interest in complementary and alternative medicine has grown, numerous studies on the use of natural products for tissue healing had been carried out (7). Because natural ingredients are easily available and have the ability to keep PDL cells alive for longer periods of time, they can be employed as storage media (8). Egg white is a viable alternative as a storage medium for teeth undergoing delayed replantation because of its high protein, vitamin, and water content, lack of microbial contamination, and ease of access (9). Milk has several benefits as a storage medium for the avulsed teeth, including being an isotonic liquid with physiological osmolality and pH, containing growth factors and vital nutrients for cells, and being widely available and inexpensive(9). Probiotics Bifidobacterium can also contribute to good health by creating a microbiological balance in the oral and decreasing bacteria's acidogenicity via oral defense systems like the peroxidase system , because Bifidibacterium animalis DN 173010 appears to be an alternative for temporary preservation of avulsed teeth. This is due to the high number of PDL cells that remain viable on the root surface of the avulsed tooth, probiotics may be a potential transport medium for avulsed teeth (10). There is no available data related to the effect of different types of storage media on surface roughness and surface granularity distribution of the enamel of avulsed teeth. The aim of this study was to evaluate and compare the effect of different types of storage media on enamel microstructure of avulsed teeth by using atomic force microscope at two different time intervals. Research question Is there a difference in the effect of varies storage media on the enamel surface microstructure of the avulsed teeth? Materials and Methods Premolar teeth that had just been extracted as part of a treatment plan for orthodontics were chosen. The teeth were gathered as soon as possible after the extraction, were preserved wet by placement in a universal glass tubes and kept in distilled water. At all times without any additional sanitizing treatment. Following extraction, dental forceps were used to hold the coronal section of each tooth while a dental curette was used to scrape the PDL, rinsing with normal saline was done to remove blood cells (10). The procedure began with a naked-eye to do a visual examination of the teeth in favorable light. Any teeth that were observed to be damaged or fractured were eliminated(11). To prevent the bacterial growth, the distilled water was changed once a week until the experiment was finished(12). Non-fluoridated pumice was used to polish the teeth used for sample preparation (Pumice Powder, I Dental Company ,Lithuanian) using a rubber cup (Denmark), and a low-speed hand piece (STRONG 90- Saeshin Precision Co., Ltd., Korea) prior to experiment to remove any residual material from the tooth surface , then rinsed in distilled water and dried using cotton pads (China)(13). The crowns of the premolar teeth were separated from their roots at cementoenamel junction . The samples were prepared using a double-sided J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 36 diamond disc ( Dental lab diamond disc, Guangdong, China) at 4 x 4 mm width, and 2 mm thickness, and a low-speed handpiece with water cooling . To obtain correct dimensions, they were determined with an electronic digital caliper (SL01-1/-2, China). Except for the outer enamel surface that was to be examined, all of the sample surfaces were painted with nail varnish all over, so that variations in texture of enamel could only be determined from the exposed surface. Twelve teeth blocks from freshly extracted premolars for orthodontic treatment with closed apices and no caries were included in this study. The study samples were divided into three groups according to the type of the storage medium: • Egg white group ; Group A consists of four teeth blocks stored in green -colored universal tubes and immersed in egg white (Fresh Iraqi egg, Mazarie Albalad) for 8 hours. • Probiotic yogurt group ; Group B consists of four teeth blocks stored in pink-colored universal tubes and immersed in probiotic yogurt (Al Safi-Danone Iraq LLC Erbil-Iraq) for 8 hours. • Bovine milk group ; Group C consists of four teeth blocks stored in orange-colored universal tubes and immersed in bovine milk (Qirat Iraq) for 8 hours. Determination of the pH values of the storage media The physico-chemical parameters (salinity and pH ) were calculated using a wissenschaftlich technische werkstatten (WTW) multi-parameter inolab720, but only after standardizing the tool with standard pH and conductivity of electricity. WTW multi-parameter inolab 720 (WTW Gmbh, Weilheim, Germany) is powered by two sensors: one that measures pH and the other that measures salinity(14). The glass electrode was inserted in it and the pH level was displayed on the meter is shown in Table 1 . Between readings the electrode was washed with distilled water and wiped with cotton pads to avoid of mixture of the materials (15). Table 1: pH values of the storage media Storage media pH Egg white 9.5 Probiotic yogurt 5.5 Bovine milk 7.5 The surface topography properties of the enamel were examined using an atomic force microscope to evaluate the changes in the surface topography, which were determined before the experiment and after eight hours of exposure to three different natural storage media (egg white, probiotic yogurt, and bovine milk). The atomic force microscope examination was set in contact mode to obtain a topographic image from the surface. For each sample, pictures were obtained at 3080 × 3080 nm to create two-dimensional (2- D) and three-dimensional (3-D) images. These measurements involved roughness parameters expressed in nanometers as follows: 1. Average roughness (Sa) : from a mean line, the arithmetic mean of peak heights and valley depths (13). 2. Root mean square roughness (Sq): the distribution of heights in relative to the mean line (13). J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 37 3. Average maximum height (Sz): Within the sampling length, represents the profile's average maximum height, calculated over the five highest peaks Zp and five deepest valleys Zv (16). Measurement periods:- 1-Baseline measurements :- which were performed before starting the experiment, and considered as the control group. 2. eight hours’ interval measurements , which were made after exposure to the three types of storage media for eight hours. According to these periods, this study had a total of twenty four measurements for the twelve samples blocks used; divided into twelve measurements as baseline measurements for control group, and twelve measurements for experimental groups. Granularity cumulation distribution, in addition to determining the average size of the nanoparticles that made up the surface, the reports of granularity cumulation shows the distribution of particle diameters over volume ratios of the samples )17). Statistical analysis Statistical Package for Social Science (SPSS version 21) was used to describe, analyze, and present the data. Statistical analyses can be categorized into two classes:- Descriptive statistics: Minimum, maximum, mean, standard deviation (SD) for quantitative variable Inferential statistics: A. Shapiro Wilk test : Test the normality distribution of the quantitative variable. B. Levene test : Check if the variance is homogeneous between groups. C. One Way Analysis Of Variance (ANOVA) : Test the difference between k independent groups D. Paired T test : Test the difference between two measurements on the same subject or two related points. When the p value was < 0.05 it was considered as significant for all the statistical analysis. Results Normality test The normality of data was tested by Shapiro-Wilk test (Table 2 and 3) which revealed that the samples were normally distributed. J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 38 Table 2 : Normality test of roughness among storage media and periods Periods Variabl es Storage Shapiro-Wilk Statistic P value ^ Periods Periods 8hr 0.907 0.763 0.825 0.927 0.766 0.867 0.956 0.866 0.927 0.997 0.866 0.862 1.000 0.907 0.820 0.927 0.918 0.851 8hr 0.468 0.051 0.156 0.578 0.054 0.286 0.753 0.284 0.578 0.991 0.284 0.267 0.999 0.467 0.144 0.575 0.524 0.229 Baseline Sa Egg white Probiotic yogurt Bovine milk Sq Egg white Probiotic yogurt Bovine milk Sz Egg white Probiotic yogurt Bovine milk After 8 hours Sa Egg white Probiotic yogurt Bovine milk Sq Egg white Probiotic yogurt Bovine milk Sz Egg white Probiotic yogurt Bovine milk Table 3: Normality test of granularity diameter among periods and storage media Variables Storage media Shapiro-Wilk Statistic P value 0.888 0.918 0.775 0.902 0.997 0.896 0.347 0.446 0.056 0.393 0.892 0.372 Baseline Egg white Probiotic yogurt Bovine milk After 8 hours Egg white Probiotic yogurt Bovine milk J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 39 Table 4 shows the minimum, maximum ,mean and standard deviation (SD) of surface roughness values for egg white ,probiotic yogurt and bovine milk at eight hours period. Statistical analysis of the results using (ANOVA) test demonstrates that no significant difference was found among egg white and probiotic yogurt groups in all the roughness parameters (Sa, Sq and Sz) and at the test period. No significant difference was seen in milk group in (Sa and Sq) where as a significant difference was found for Sz parameter (P = 0.025).At the test periods, the lowest recorded minimum , maximum ,mean and standard deviation values at the test periods of surface roughness in egg white group while the highest values were at the test period in surface roughness in milk group. Table 4 : Descriptive and statistical test of roughness among periods by storage media Storage periods 8 hours Min. Max. Mean ±SD F P alue Egg white Baseline Sa 27.955 115.000 80.414 37.198 3.072 0.096 Sq 33.630 137.000 94.358 43.713 3.576 0.072 SZ 88.385 533.000 333.346 183.976 2.740 0.118 After 8 hours Sa 28.034 79.800 52.934 21.770 1.100 0.374 Sq 33.248 92.200 62.437 24.826 1.100 0.374 SZ 89.149 319.000 209.787 107.410 .971 0.415 Probiotic yogurt Baseline Sa 51.900 201.000 93.375 71.930 1.413 0.293 Sq 59.900 260.000 115.675 96.430 1.329 0.312 SZ 207.000 550.000 335.250 149.107 1.817 0.217 After 8 hours Sa 48.000 98.200 66.550 21.956 1.022 0.398 Sq 56.800 113.000 85.950 26.843 1.452 0.284 SZ 126.000 393.000 282.000 123.129 1.023 0.398 Bovine milk Baseline Sa 44.675 81.600 56.969 17.038 2.185 0.168 Sq 56.700 97.300 71.360 18.060 2.583 0.130 SZ 189.198 421.000 316.050 107.684 4.048 0.094 After 8 hours Sa 83.700 93.119 88.380 4.719 2.812 0.113 Sq 97.000 130.126 107.957 15.287 2.665 0.123 SZ 347.000 426.468 376.867 34.395 5.752 0.025* *Significant P value = 0.025, Sa- average roughness, Sq- root mean square roughness, Sz-average height Difference in surface roughness before and after immersion and within each medium can be seen in Table 5 Comparison of the roughness change for egg white , probiotic yogurt and bovine milk at the test period revealed that there was no statistical significant difference for the egg white and probiotic yogurt groups, where it was a significant difference for the milk group. Table 5 : Descriptive and statistical test of roughness before and after immersion and within each. J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 40 Periods Egg white Probiotic yogurt Bovine milk Mean ±SD Mean ±SD Mean ±SD F P value 8hr Presa 80.414 37.198 93.375 71.930 56.969 17.038 0.597 0.571 postsa 52.934 21.770 66.550 21.956 88.380 4.719 3.922 0.060 p 0.119 0.545 0.031* presq 94.358 43.713 115.675 96.430 71.360 18.060 0.511 0.616 postsq 62.437 24.826 85.950 26.843 107.957 15.287 3.959 0.058 p 0.119 0.554 0.061 presz 333.346 183.976 335.250 149.107 316.050 107.684 0.020 0.980 postsz 209.787 107.410 282.000 123.129 376.867 34.395 3.022 0.099 p 0.125 0.675 0.446 *significant at p<0.05 . Pre =Prior(before). Post=after Difference in granularity diameter between storage media before and after immersion and within each media is shown (Table 6) , (Table 7) revealed that there were no significant difference in all groups on the test period. Table 6 : Descriptive and statistical test of granularity diameter among periods by storage media Storage periods 8 hr F P value ^ Min. Max. Mean ±SD Egg white Before 93.340 118.790 103.450 13.507 0.485 0.638 After 101.820 151.840 122.077 26.330 2.225 0.189 Probiotic Before 104.100 150.800 123.423 24.369 0.439 0.664 After 82.830 192.080 135.673 54.712 1.716 0.257 Milk Before 101.260 109.630 104.060 4.824 2.122 0.201 After 96.290 129.610 109.667 17.604 0.736 0.518 Table 7 : Descriptive and statistical test of granularity diameter among storage media and periods . Periods Egg white Probiotic yogurt Bovine milk F P value ^ Mean ±SD Mean ±SD Mean ±SD 8hr Before 103.45 13.50 123.42 24.36 104.06 4.824 1.45 0.30 After 122.07 26.33 135.67 54.71 109.66 17.60 0.38 0.69 Paired T test 1.035 0.539 0.460 P value 0.410 0.644 0.691 Discussion The three roughness parameters Roughness (Sa, Sq and Sz) assessment were used to quantitatively evaluate the surface topography of each storage media of avulsed teeth. J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 41 A decrease in surface roughness (represented by mean Sa, Sq and Sz values) from baseline periods measured by atomic force microscope was observed following immersing of the avulsed tooth in egg white. But the result were not statistically significant. This result came in agreement with Hemingway et al.,( 2008) who reported that ovalbumin lowers the rate of hydroxyapatite dissolution under conditions that simulate tooth erosion caused by citrus-based soft beverages with a range of pH and calcium concentrations (18). This can be explained in that egg white contains various ions that fill up enamel valleys; creating a smoother surface and also alkaline pH and stability of pH in the all times of preserving of avulsed teeth . The primary protein contained in the white of hens' eggs is ovalbumin. Bovine enamel adsorbs ovalbumin (19). Probiotic yogurt decreased the surface roughness in terms of Sa, Sq and Sz from baseline period which was observed after immersing of avulsed tooth in its. But the result was not statistically significant. This result was in agreement with Ferrazzano et al., in 2008 who demonstrated that yogurt is a good source of calcium and phosphorus and has a greater protein level than milk. The advantageous ionic form of calcium is due to that the yogurt's lower pH as compared to milk. Furthermore, because of the proteolytic activity of the microbe found in yogurt, the concentration of casein phosphopeptide is greater than in milk (20). The ionic form of calcium keeps the calcium in the tooth structure and the fluids around it in equilibrium (21). Due to their natural presence of casein, calcium, and phosphorus, they are also thought to be safe for teeth, with possible favorable effects on both salivary microbial composition and caries development(22). In spite of the low pH of yogurt but it has high calcium and phosphate content which decrease the rate of erosion by remineralization of the tooth . Nevertheless; the results of the current study were in disagreement with Shen et al., in 2020 who reported that the high concentration of lactic acid, sugar, and live lactic acid generating bacteria in the processed yogurt causes considerable enamel subsurface demineralization(23). A significant increase in surface roughness (represented by mean Sa, Sq and Sz values) from baseline period was noticed following immersing of avulsed tooth in bovine milk. The roughness values showed increase which suggested that more erosion occurs with increased exposure time to bovine milk. The results of this study were in agreement with Lee et al., in 2011 who reported that micro-hardness was lower in the human and formula milk groups than in the control. In saliva and water , scan electron microscope (SEM) observation demonstrated higher surface roughness and loss of inorganic substance in the formula milk group than in human milk(24). Longer periods of milk exposure encourage bacteria to continue fermenting lactose, resulting in continual acid generation and increased demineralization. As a result, the amount of time the biofilm is immersed in milk is crucial (25). Average diameter of grain size of enamel surface shown no significant changes in grain size of enamel surface of the avulsed tooth in any types of three storage media at eight hours interval . Egg white and probiotic yogurt groups showed slight increase in the average diameter of grain size of enamel surface, but the results were not significant .This results might be explained in terms of the possibility of surface remineralization of enamel(19). J. Bagh. Coll. Dent. Vol. 34, No. 4. 2022 Obeid and Khalaf 42 Conclusion Depending on the findings of this study, one can conclude that bovine milk group demonstrated the highest roughness values of the teeth at the test period, milk was highly erosive in comparison with remaining types of storage media. Egg white group demonstrated the lowest roughness values of the teeth at the test periods and probiotic yogurt group showed the next lower roughness values. Because chicken eggs and probiotic yogurt are readily available in markets, inexpensive, and can be obtained in almost every home, individuals can be taught how to preserve avulsed teeth by immersing them in these storage medium as soon as possible after an accident that results in tooth avulsion. References 1. Gopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D. RETRACTED: Comparison of Coconut Water, Propolis, HBSS, and Milk on PDL Cell Survival. Elsevier; 2008;34: 587-589. 2. De Carvalho Cardoso L, Poi WR, Panzarini SR, Sonoda CK, da Silveira Rodrigues T, Manfrin TM. Knowledge of firefighters with special paramedic training of the emergency management of avulsed teeth. Dental Traumatology. 2009;25(1):58-63. 3. Tsai M-T, Wang Y-L, Yeh T-W, Lee H-C, Chen W-J, Ke J-L, et al. 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Caries Research. 2021: 55:260–267 ( مختبرية سطح المينا لألسنان المخلوعة )دراسةخشونة وتوزيع حبيبات أنواع مختلفة من وسائط التخزين على فعالية: العنوان منى سليم خلف , عبيد( : رواء صادق الباحثون : المستخلص سطح المينا لألسنان المخلوعة باستخدافوائد حبيبات سطح و توزيع خشونة وسائط التخزين على من مختلفة مجهر القوة الذرية م : تقييم تأثير أنواع سنًا من الضواحك المقلوعة حديثًا للمعالجة التقويمية لألسنان . تم تقسيم عينات الدراسة إلى ثالث مجموعات اثنا عشر اريالمواد والطرق : لقد تم أخت وسائط التخزين ب-أ -حسب نوع ج -بياض البيض خشو -زبادي بروبيوتيك جميع العينات لمعرفة التغيرات في تم فحص . و نةحليب بقري السطح نان المخلوعة باستخدام مجهر القوة الذرية: قبل بدء التجربة و ثمانية ساعات من االنغماس في األنواع الثالثة لوسائط توزيع حبيبات السطح المينا لألس فتر النتائج التخزين في الخشونة قيم متوسط في معنوية زيادة الحليب مجموعة أظهرت زبادي ة: و البيض بياض مجموعات أظهرت االختبار. االختبار. ال توجد تغيرات كبيرة في حجم حبيبات سطح مينا األسنان المخلوعة في أي نوع من ةالبروبيوتيك انخفاًضا في متوسط قيمة الخشونة في فتر قد يكون استخدام زبادي نا.قطر حجم الحبوب لسطح الميسط في مجموعات بياض البيض والزبادي بروبيوتيك تظهر زيادة طفيفة في متو .وسائط تخزين وديان المينا, بينما يشجع التعرض للحليب ل مختلفة تمأل وبروتينات أيونات مفيدًا ألنه يحتوي على والبروبيوتيك لتخزين العينات فترات أطول البيض توليد األحماض وزيادة فقدان المعادن راريؤدي إلى استم البكتيريا على االستمرار في تخمير الالكتوز, مما : إعتمادا على النتائج المذكورة سابقا تم التوصل الى أن مجموعة الحليب أظهرت أعلى قيم خشونة بينما أظهرت مجموعة بياض البيض أقل اتاالستنتاج فترة االختبار في قيم خشونة لمينا األسنان . لم يكن للمجموعات الثالث أي تأثير على قيم توزيع حبيبات السطح