Demineralization properties of newly erupted and mature premolars around orthodontic brackets: An in-vivo study

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1 ONLINE ONLY Demineralization properties of newly erupted and mature premolars around orthodontic brackets: An in-vivo study Tancan Uysal, a Mihri Amasyali, b Suat Ozcan, c and Deniz Sagdic d Kayseri and Ankara, Turkey Introduction: The aims of this study were to evaluate the in-vivo reaction of newly erupted enamel to demineralization around orthodontic brackets and to compare it with that of mature enamel. Methods: Thirteen orthodontic patients scheduled to have 4 first premolars extracted for orthodontic reasons were divided into 2 groups. Group 1 included 7 younger patients with newly erupted teeth (4 boys, 3 girls; mean age, years; range, years). Group 2 contained 6 adults with mature teeth (5 men, 1 woman; mean age, years; range, years). Brackets were placed, and, 30 days later, the teeth were extracted. These teeth were longitudinally sectioned, and demineralization was assessed by cross-sectional microhardness. Determinations were made at the bracket-edge composite limits and at occlusal and cervical points 100 mm away. Evaluations under the brackets and at the lingual surfaces were made as controls. In all these positions, 6 indentations were made at depths from 10 to 90 mm from the enamel surface. Analysis of variance (ANOVA) and Tukey tests were used for statistical evaluation at the P \0.05 level. Results: ANOVA showed statistically significant differences for tooth type, position, depth, and their interactions (P \0.05), except the tooth type and position interaction. The multiple comparison test showed less demineralization in the enamel around orthodontic brackets bonded to mature teeth campared with newly erupted teeth (P \0.05). Conclusions: During the 30-day study period, the tooth enamel in the adult orthodontic patients was more resistant to demineralization than that of the younger patients. (Am J Orthod Dentofacial Orthop 2010;137:582.e1-582.e6) a Associate professor and chair, Department of Orthodontics, Faculty of Dentistry, Erciyes University, Kayseri, Turkey; visiting professor, King Saud University, Riyadh, Saudi Arabia. b Research assistant, Department of Orthodontics, Center of Dental Sciences, Gülhane Military Medical Academy, Ankara, Turkey. c Research assistant, Department of Conservative Dentistry and Endodontics, Faculty of Dentistry, Gazi University, Ankara, Turkey. d Professor and chair, Department of Orthodontics, Center of Dental Sciences, Gülhane Military Medical Academy, Ankara, Turkey. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. Reprint requests to: Tancan Uysal, Erciyes Üniversitesi, Disx Hekimliği Fakültesi, Ortodonti Anabilim Dalı, 38039, Melikgazi, Kayseri, Turkey; , tancanuysal@yahoo.com. Submitted, June 2009; revised and accepted, August /$36.00 Copyright Ó 2010 by the American Association of Orthodontists. doi: /j.ajodo One of the most difficult problems in orthodontic treatment with fixed appliances is the control of enamel demineralization around brackets. 1 The bands and brackets and the various orthodontic elements (elastics, power chains, sleeves, springs) make the patient s oral hygiene more difficult and the accumulation of plaque easier. 2 Studies have documented significant increases in oral bacteria during orthodontic treatment. 3 Demineralization takes place when specific bacteria are retained for a long time on the enamel surface. 4 Patients with fixed orthodontic appliances have an elevated risk of caries, and enamel lesions can occur within a month, irrespective of mechanical plaque control and whether fluoridated dentifrices are used. 5-8 Previous studies have shown that the rate of demineralization in orthodontic patients is higher than in those without orthodontic treatment, and teenagers have a higher risk of demineralization than do adults. 9,10 Odontogenesis is a complex process: a series of events from bud formation until the completion of calcification and the maturation of the tooth. 11 Upon eruption, the outermost layer of enamel is immature and not completely calcified, and it then starts to calcify from the effects of salivary minerals. 11 Because of wear and replacement of organic material by minerals during the maturation process, the enamel surface of old teeth might have a different composition than that of newly erupted teeth. 12 In newly erupted teeth, high sodium and magnesium levels are thought to contribute to the relatively high solubility of enamel. 13 Mineral content in the enamel surface is transformed mainly to calcium phosphate, with little sodium and magnesium remaining during posteruptive maturation. 14 These changes are thought to occur mostly during the first few years after 582.e1

2 582.e2 Uysal et al American Journal of Orthodontics and Dentofacial Orthopedics May 2010 Fig. Diagram of positions and depths of indentations. eruption. 15 It was shown in the literature that decreases in enamel pore size and increases in the calcification of enamel matrix occur over time. 16 Also, during aging, hydroxyapatite crystals increase in size because of the incorporation of ions from the surrounding saliva. 17 This is attributed to the mineralization of calcium and phosphate ions from saliva and bacterial acid products. 17 Imanishi and Nishino 18 showed altered surface and subsurface enamel in newly erupted premolars when compared with premolars from 18-year-olds. Palamara et al 19 used scanning electron microscopy to study the superficial and deep layers of enamel from unerupted and erupted teeth. They also found marked differences in the unerupted surface enamel structure compared with erupted enamel. All these changes during the posteruptive maturation might influence the etching, bonding, and demineralization properties of newly erupted and mature enamel. Previous studies on posteruptive maturation and its effects on caries development showed decreases in the incidence of caries as the subjects age, confirming the continuous mineralization and maturation of the enamel. 17,18 The structural characteristics of enamel and their role in the bonding mechanism have been studied previously, but no in-vivo studies have investigated the demineralization properties of newly erupted and mature teeth around orthodontic brackets. Therefore, the aims of this study were to evaluate the in-vivo reaction of newly erupted enamel to demineralization around orthodontic brackets and to compare it with mature enamel quantitatively. In this study, the null hypothesis assumed that newly erupted enamel showed significantly higher demineralization around orthodontic brackets than do the mature teeth evaluated in the mouth. MATERIAL AND METHODS This study was approved by the Ethical Committee on Research of the Gulhane Military Medical Academy, Ankara, Turkey. Thirteen orthodontic patients, scheduled to have 4 first premolars extracted for orthodontic reasons, were invited to participate in the study, and consent forms were signed. A power analysis established by G*Power software (version , Franz Faul Universität, Kiel, Germany). Based on a 1:1 ratio between groups, a sample size of 13 patients would give more than 80% power to detect significant differences with 0.40 effect size and at the a significance level. For group standardization, before starting the study, all patients teeth were evaluated clinically and radiographically to determine the baseline caries risk. They were divided into 2 groups according to age. Group 1 (newly erupted teeth) included 4 boys and 3 girls (mean age, years; range: years); group 2 (mature teeth) included 5 men and 1 soman (mean age, years; range, years). The patients salivary flow rates and buffer capacities were recorded. The criteria for including the patients were no active caries lesions, developmental defects, or fluorosis; and normal salivary flow rate (.1.0 ml/min) and buffer capacity (final ph, ). All patients received a full-mouth cleaning to remove plaque in preparation for bonding. For evaluating the baseline demineralization values of all selected teeth, a portable battery-powered laser fluorescence device, DIAGNOdent Pen (KaVo Dental, Biberach/Rib, Germany) was used. 4 The 2 groups scores were less than 13, indicating no demineralization; they were equivalent for caries risk. Orthodontic brackets were bonded with Transbond XT (3M Unitek, Monrovia, Calif), a resin-based composite. A 37% phosphoric acid gel (3M Dental Products, St Paul, Minn) was used for 15 seconds. The teeth were rinsed with water for 30 seconds and dried with an oilfree source for 20 seconds. Transbond XT primer (3M Unitek) was applied to the etched surface in a thin film and not cured. Adhesive paste was applied to the bracket base (Dyna-Lok series, 100-gauge mesh, 3M Unitek), and the bracket was positioned on the tooth and pressed firmly into place. The excess adhesive was removed around the bracket with a scaler, and the

3 American Journal of Orthodontics and Dentofacial Orthopedics Uysal et al 582.e3 Volume 137, Number 5 Table I. ANOVA results Source Sum of squares df Mean square F Significance Tooth type (newly erupted/mature) * Position * Depth * Tooth type/position Tooth type/depth * Position/depth * Tooth type/position/depth * Adjusted R *Statistically significant (P \0.05). Table II. Descriptive statistics of microhardness values determined from different depths and positions for newly erupted and mature teeth Cervical 100 mm Cervical 0 mm Under Bracket Occlusal 0 mm Occlusal 100 mm Tooth type Depth n Mean (VHN) SD Mean (VHN) SD Mean (VHN) SD Mean (VHN) SD Mean (VHN) SD Newly erupted 10 mm mm mm mm mm mm Mature 10 mm mm mm mm mm mm adhesive was light cured from the mesial and distal aspects for 10 seconds each (total time, 20 seconds). A light-emitting diode unit (Elipar Freelight 2, 3M ESPE, St Paul, Minn) was used for curing the specimens. Twenty-eight brackets were bonded in group 1 (14 maxillary and 14 mandibular first premolars), and 24 brackets were bonded in group 2 (12 maxillary and 12 mandibular first premolars). After 30 days, the teeth were extracted and stored in a refrigerator in flasks containing gauze dampened with 2% formaldehyde (ph 7.0) until the analysis. Demineralization in enamel around the brackets was evaluated with the cross-sectional microhardness method according to the literature. 8,20,21 During the experimental period and 3 weeks previously, all subjects brushed their teeth with a nonfluoridated dentifrice. They received no instructions regarding oral hygiene, kept their usual habits, and were instructed not to use any antibacterial substance. One operator (S.O.), blinded to the group allocations, evaluated demineralization. The roots were removed 2 mm apical to the cementoenamel junction, and the crowns were hemisectioned vertically into mesial and distal halves with a 15 high cut (large) wafering blade on an Isomet low-speed saw (Buehler, Lake Bluff, Ill) directly through the slot of the bracket, leaving gingival and incisal portions. The teeth were embedded in self-curing EpoKwick epoxy resin (Buehler), leaving the cut face exposed. The half-crown sections were polished with 3 grades of abrasive paper discs (320, 600, and 1200 grit); final polishing was done with a 1-mm diamond spray and a polishing cloth disc (Buehler). A microhardness tester (HMV-700, Shimadzu, Kyoto, Japan) under a 2N load for 15 seconds was used for the microhardness analysis. Thirty-six indentations were made on each half crown at 6 positions (Fig). On the buccal surface, the first indentations were made under the bracket. In the occlusal and cervical regions, the indentations were made at the edge (0 mm) of the bracket and 100 mm away. Indentations were also made in the middle third of the lingual surface of each half crown as another control. In all these positions, 6 indentations were made at

4 582.e4 Uysal et al American Journal of Orthodontics and Dentofacial Orthopedics May 2010 Table III. Descriptive statistics and multiple comparisons of microhardness for newly erupted and mature teeth at different depths from enamel surface Interaction of tooth type/ depth Newly erupted Mature Mean (VHN) SD Mean (VHN) SD Multiple comparisons 10 mm * 20 mm * 30 mm NS 50 mm NS 70 mm NS 90 mm NS *P \0.05; Tukey test; NS, not significant. depths of 10, 20, 30, 50, 70, and 90 mm from the external surface of the enamel. These values in the 2 half crowns were averaged. Statistical analysis Data analyses were performed with the Statistical Package for Social Sciences, (version 13.0, SPSS, Chicago, Ill) and Excel 2007 (Microsoft, Redmond, Wash). The Shapiro-Wilks normality test and the Levene variance homogeneity test were applied to the demineralization data. The data showed normal distribution, and there was homogeneity of variances between the groups. Analysis of variance (ANOVA) was used to evaluate the effect of tooth type (newly erupted and mature), depths from the enamel surface (10, 20, 30, 50, 70, and 90 mm), positions (under the bracket, on the buccal surface in the occlusal and cervical regions at 0 and 100 mm from the brackets, and on the lingual surface), and their interactions. For multiple comparisons, the Tukey post-hoc test was used. Significance was predetermined at P \0.05. For evaluating intraobserver and interobserver agreement, the demineralization measurements were made by 2 investigators (S.O. and T.U.) using the same instrument at 2 separate times, and the Cohen kappa scores were determined. RESULTS The kappa scores for the assessment of intraexaminer and interexaminer agreement were higher than 0.80; this implies substantial agreement between the observers. ANOVA showed statistically significant difference for the factors of tooth type, position, and depth (P\0.05). The interactions (tooth type/depth, and position/depth) and (tooth type/position/depth) were also statistically significant (P \0.05) (Table I). ANOVA to evaluate the interactions of demineralization for tooth type at various positions under, occlusal, and cervical to the brackets on the labial and lingual (control) surfaces indicated no statistically significant interaction between tooth type and position (P.0.05) (Table I). All descriptive statistics of demineralization at the various depths and positions for newly erupted and mature teeth are shown in Table II. The lowest demineralization values were determined at the occlusal ( VHN ) and cervical ( VHN ) margins (at 0-mm position) at 10-mm depths for newly erupted premolars. Multiple comparisons of demineralization for newly erupted and mature teeth at different depths from enamel surface are shown in Table III. The interaction between tooth type and depth had significant differences between tooth type at the depths of 10 and 20 mm from the enamel surface. Less demineralization was found in enamel around the brackets bonded to mature teeth compared with newly erupted teeth. The Tukey post-hoc test applied to the triple interaction (tooth type/position/depth) and the results are given in Table IV. Statistically significant differences at all positions were found for both the cervical and occlusal margins on the buccal surfaces at 10 mm from the surface of the enamel. No significant difference between the tooth types in the demineralization was observed at the lingual surfaces of untreated teeth. Thus, according to our findings, the null hypothesis that the newly erupted enamel showed significantly higher demineralization around orthodontic brackets than the mature teeth could not be rejected. DISCUSSION In the literature, many investigations have compared the bonding properties of newly erupted and mature teeth. Previous in-vitro studies evaluated bond strengths, but no investigation has determined the demineralization differences of newly erupted and mature enamel around orthodontic brackets. 11,14,22 We evaluated quantitatively the demineralization properties of newly erupted and mature teeth around orthodontic brackets. Sheen et al 23 reported that bond strengths in older permanent teeth were greater than in younger teeth, regardless of etching time. Bhaskar 24 found that the enamel surfaces of unerupted and recently erupted teeth were completely covered with pronounced perikymata and rod ends. With age, the perikymata and rod ends can wear away. As a result of time, age-related changes in the organic portion of the enamel, presumably near

5 American Journal of Orthodontics and Dentofacial Orthopedics Uysal et al 582.e5 Volume 137, Number 5 Table IV. Microhardness for tooth types and positions at depth of 10 mm Newly erupted Mature Depth Position Mean (VHN) SD Mean (VHN) SD Multiple comparisons 10 mm Cervical 100 mm * Cervical 0 mm * Under bracket * Occlusal 0 mm * Occlusal 100 mm * Lingual NS *P \0.05; Tukey test; NS, not significant. the surface, might cause the teeth might to become harder and more resistant to demineralization. 24 In this study, the mineral loss was assessed by crosssectional microhardness, an accepted analytic method. This method was preferred to evaluate demineralization and caries, because a strong correlation coefficient (r ) was found between enamel microhardness and the percentage of mineral loss in the caries lesions. 25 In the past, to use fewer patients and for ethical considerations, preventive effects of various products such as fluoride-releasing materials against demineralization were investigated by using a split-mouth study design. 26 But a split-mouth design was unsuitable for this investigation. We divided the subjects into 2 groups according to age. The baseline clinical, radiologic, salivary, and laser fluorescence examinations were done for standardization. It was determined that all patients in both groups were equivalent with regard to caries risk or demineralization activity. Instead of in-vitro studies with extracted bovine and human teeth, our model had several advantages: the development of the caries lesions was studied in vital teeth; it required minimal patient cooperation and no special diet; and, because the protected enamel surface allowed the accumulation of thick plaque, no other site was at risk of caries with this procedure. 20 The only disadvantage of this procedure was the limited study period of 30 days, because of ethical considerations, as with most other caries models. A 30-day experimental period was used, because measurable demineralization can be observed around orthodontic appliances 1 month after bonding. 5,6 The demineralization values of enamel under 2 internal controls (under the bracket and at the lingual surface) bonded to the 2 types of teeth were used to evaluate the effect of acid etching and enamel demineralization. 8 Additionally, for extensive and controlled evaluation, the indentations were made at 10, 20, 30, 50, 70, and 90 mm from the external surface of the enamel to observe mineral changes at the outermost part of the enamel. Orthodontic attachments make a patient s dental hygiene more difficult and the accumulation of plaque easier around the brackets. Our results confirmed this by showing the lowest demineralization values at the occlusal ( VHN ) and cervical ( VHN ) margins (at 0 mm position) at 10-mm depths for newly erupted premolars. The development of demineralization around the brackets, with significant differences between 2 types of teeth up to the 20-mm depth of the enamel surface, is shown in Table III. Statistically significant differences were found between newly erupted and mature premolars at depths of 10 and 20 mm from the enamel surface. Newly erupted teeth had lower demineralization values, indicating more mineral loss than mature teeth. These findings were similar to previous ones at the 30-mm depth but differed from these of de Moura et al, 20 who found lesion depths up to 70 mm from the enamel surface. This could be explained by the experimental model used; they allowed more plaque accumulation and impaired its removal by tooth brushing. Multiple comparisons of demineralization for tooth type and position at the 10-mm depth from the enamel surface showed statistically significant differences at all positions in both the cervical and occlusal margins evaluated on the buccal surface. The differences in enamel demineralization under the brackets bonded to newly erupted or mature teeth might be attributed to the difference in acid etching pattern during bonding with the resin. By using a subjective measurement of etching patterns when viewed under a scanning electron microscope, Nordenvall et al 12 reported that more retentive surfaces were obtained with conventional etching of newly erupted teeth for 15 seconds and mature teeth for 60 seconds. Our demineralization results showed that mature teeth have significantly less enamel mineral loss when compared with newly erupted teeth in a group of orthodontic patients. This suggests that, in the short term, the teeth of adults undergoing orthodontic treatment are

6 582.e6 Uysal et al American Journal of Orthodontics and Dentofacial Orthopedics May 2010 more resistant to demineralization than those of younger patients. Younger patients might have a higher risk for demineralization because their oral hygiene tends to be less than ideal. Often, patients have brackets on all teeth, not just 4 first premolars, with wires and elastics compounding the plaque buildup, so that the difference in the demineralization effects on tooth types would probably be even more apparent. 21 CONCLUSIONS Enamel loss from demineralization around orthodontic brackets on mature teeth extracted from adults was significantly lower compared with that from newly erupted teeth during a 30-day period. Longer follow-up evaluations are needed. We thank Medifarm (Alin Kuyumciyan) and Guney Dental (Ertan Seçkin) for their support of this project. REFERENCES 1. Zabokova-Bilbilova E, Stafilov T, Sotirovska-Ivkovska A, Sokolovska F. Prevention of enamel demineralization during orthodontic treatment: an in vitro study using GC tooth mousse. Balk J Stom 2008;12: Basdra EK, Huber H, Komposch G. Fluoride released from orthodontic bonding agents alters the enamel surface and inhibits enamel demineralization in vitro. Am J Orthod Dentofacial Orthop 1996;109: Bloom RH, Brown LR. A study of the effects of orthodontic appliances on the oral microbial flora. Oral Surg 1964;17: Uysal T, Amasyali M, Koyuturk AE, Sagdic D. Efficiency of amorphous calcium phosphate-containing orthodontic composite and resin modified glass ionomer on demineralization evaluated by a new laser fluorescence device. Eur J Dent 2009;3: O Reilly MM, Featherstone JDB. Demineralization and remineralization around orthodontic appliances: an in vivo study. Am J Orthod Dentofacial Orthop 1987;92: Øgaard B, Rolla G, Arends J. Orthodontic appliances and enamel demineralization. Part 1. Lesion development. Am J Orthod Dentofacial Orthop 1988;94: Øgaard B, Rolla G, Arends J, ten Cate JM. Orthodontic appliances and enamel demineralization. Part 2. Prevention and treatment of lesions. Am J Orthod Dentofacial Orthop 1988;94: Pascotto RC, Navarro MFL, Filho LC, Cury JA. In vivo effect of a resin-modified glass ionomer cement on enamel demineralization around orthodontic brackets. Am J Orthod Dentofacial Orthop 2004;125: Mizrahi E. Enamel demineralization following orthodontic treatment. Am J Orthod 1982;82: Kukleva MP, Shetkova DG, Beev VH. Comparative age study of the risk of demineralization during orthodontic treatment with brackets. Folia Med 2002;44: Almy D. Bonding properties of newly erupted and mature human premolars [thesis]. Richmond, Va: Virginia Commonwealth University; Nordenvall KJ, Brannstrom M, Malmgren O. Etching of deciduous teeth and young and old permanent teeth: a comparison between 15 and 60 seconds of etching. Am J Orthod 1980;78: Driessens FC, Heyligers HJ, Woltgens JH, Verbeeck RM. X-ray diffraction of enamel from human premolars several years after eruption. J Biol Buccale 1982;10: Tüfekçi E, Almy DM, Carter JM, Moon PC, Lindauer SJ. Bonding properties of newly erupted and mature premolars. Am J Orthod Dentofacial Orthop 2007;131: Theuns HM, van Dijk JW, Driessens FC, Groeneveld A. The surface layer during artificial carious lesion formation. Caries Res 1984;18: ten Bosch JJ, Fennis-le Y, Verdonschot EH. Time-dependent decrease and seasonal variation of the porosity of recently erupted sound dental enamel in vivo. J Dent Res 2000;79: Dirks OB. Posteruptive changes in dental enamel. J Dent Res 1966;45: Imanishi H, Nishino M. Post eruptive maturation of immature young permanent enamel. J Int Assoc Dent Child 1983;14: Palamara J, Phakey PP, Rachinger WA, Orams HJ. Electron microscopy of surface enamel of human unerupted and erupted teeth. Arch Oral Biol 1980;25: de Moura MS, de Melo Simplıcio AH, Cury JA. In-vivo effects of fluoridated antiplaque dentifrice and bonding material on enamel demineralization adjacent to orthodontic appliances. Am J Orthod Dentofacial Orthop 2006;130: Gorton J, Featherstone JDB. In-vivo inhibition of demineralization around orthodontic brackets. Am J Orthod Dentofacial Orthop 2003;123: Oliver RG. Bond strength of orthodontic attachments to enamel from unerupted and erupted young permanent teeth. Eur J Orthod 1986;8: Sheen DH, Wang WN, Tarng TH. Bond strength of younger and older permanent teeth with various etching times. Angle Orthod 1993;63: Bhaskar SN. Orban s oral histology and embryology. 9th ed. St Louis: C.V. Mosby; p Featherstone JBD, ten Cate JM, Shariati M, Arends J. Comparison of artificial caries-like lesion by quantitative microradiography and microhardness profiles. Caries Res 1983;17: Twetman S, McWilliam JS, Hallgren A, Oliveby A. Cariostatic effect of glass ionomer retained orthodontic appliances. An in vitro study. Swed Dent J 1997;21: