Erosion and Abrasion of Enamel and Dentin Associated With At-Home Bleaching : An In Vitro Study Kenneth Engle, Anderson

T. Hara, Bruce Matis, George J. Eckert and Domenick T. Zero JADA 2010;141;546-551 The following resources related to this article are available online at jada.ada.org ( this information is current as of October 12, 2011):
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Erosion and abrasion of enamel and dentin associated with at-home bleaching
An in vitro study
Kenneth Engle; Anderson T. Hara, DDS, MS, PhD; Bruce Matis, DDS, MS; George J. Eckert, MAS; Domenick T. Zero, DDS, MS

he decline of dental caries, the increase of tooth-retention rates and the high consumption of acidic beverages and foodstuffs has brought more attention to the risk of the occurrence of dental erosion. In addition, patients esthetic concerns have led to increasing interest in dental bleaching procedures.1 Some studies have shown a reduction in the surface microhardness of enamel and dentin after bleaching procedures2-8 and after erosive challenges.9-12 Once softened, the tooth surface becomes more susceptible to abrasive forces,13-14 such as those caused by toothbrushing, which can lead to surface wear by means of two-body and three-body abrasive wear processes.15,16 The resulting dental surface wear is irreversible and, if excessive, may reach pathological levels, affecting the dental pulp and modifying the form, function and esthetics of the teeth.17,18 Although the detrimental effects of erosion/abrasion and bleaching have been investigated, there are limited published data on the association among the processes. Additionally, to our knowledge, there is little published information regarding the relevance of the abrasive level of toothpaste to the development of erosive-abrasive lesions in either enamel or dentin. Research has shown that some dentifrices,

ABSTRACT

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CON

IO N

Background. Bleaching and erosive processes have been reported to soften dental surfaces, posN sibly increasing their susceptibility to toothbrush C U U A ING ED 4 abrasion. The authors conducted an investigation of RT ICLE the effect on enamel and dentin of the interaction among bleaching, erosion and dentifrice abrasivity. Methods. The authors prepared specimens of human enamel and root dentin and randomly divided them into eight groups that underwent 10 percent carbamide peroxide bleaching, erosion and dentifrice abrasion. The authors submitted the specimens to cycles of erosion, toothbrushing and bleaching for five days. They determined the change in surface loss by means of profilometry. Results. Dental erosion and the more abrasive dentifrice increased toothbrushing wear on enamel surfaces, while bleaching showed no deleterious effect. Dentin wear also increased after subjection to erosion and use of the more abrasive dentifrice. Bleaching increased surface loss on noneroded dentin and decreased loss on eroded dentin when it was brushed with the less abrasive dentifrice. Conclusion. Bleaching did not increase the susceptibility of enamel to erosive and abrasive wear, regardless of the dentifrice used. Dentin wear was modulated by the interaction of bleaching, erosion and dentifrice. Clinical Implications. Bleaching with 10 percent carbamide peroxide does not increase erosive and abrasive wear of enamel. However, it may change dentins abrasive wear, depending on erosive and abrasive challenges. Key Words. Dental erosion; toothbrushing; tooth bleaching; hydrogen peroxide; dentifrice; enamel; dentin. JADA 2010;141(5):546-551.
Mr. Engle is an undergraduate student, Biology Department, Wabash College, Crawfordsville, Ind. Dr. Hara is an assistant professor, Oral Health Research Institute, School of Dentistry, Indiana University, 415 N. Lansing St., Indianapolis, Ind. 46202-2876, e-mail ahara@iupui.edu. Address reprint requests to Dr. Hara. Dr. Matis is a professor, Department of Restorative Dentistry, School of Dentistry, Indiana University, Indianapolis. Mr. Eckert is a biostatistician supervisor, Division of Biostatistics, School of Medicine, Indiana University, Indianapolis. Dr. Zero is a professor; the director, Oral Health Research Institute; and the chair, Department of Preventive and Community Dentistry, School of Dentistry, Indiana University, Indianapolis.
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TABLE such as the whitening formulations, present Experimental groups, according to the agents used. different abrasive GROUP BLEACHING AGENT EROSIVE AGENT DENTIFRICE ABRASIVITY levels, most likely due 1 10 percent carbamide peroxide 1 percent citric acid (pH 3.8) High to the abrasive system 2 10 percent carbamide peroxide 1 percent citric acid (pH 3.8) Low used in their formula3 10 percent carbamide peroxide None High tions19 (A. Hara and colleagues, unpub4 10 percent carbamide peroxide None Low lished data, Sep5 None 1 percent citric acid (pH 3.8) High tember 2008). 6 None 1 percent citric acid (pH 3.8) Low Philpotts and col7 None None High leagues20 hypothesized 8 None None Low a positive correlation between tooth surface wear and dentifrices relaand root dentin), which we tested and analyzed tive abrasive indexes, radioactive dentin abraindependently. sivity (RDA) and radioactive enamel abrasivity Specimen preparation. We obtained 64 slabs (REA). Therefore, it is possible that more abrasive (each 4 4 2 cubic millimeters) of enamel and dentifrices can increase the risk of experiencing root dentin from extracted human molars, previdental surface loss owing to their higher abraously stored in 0.1 percent thymol (Sigmasivity, especially if the tooth substrate has been Aldrich, St. Louis) at 4oC. We flattened the pulpal compromised. and external sides of the slabs and embedded the We undertook a study to evaluate the potential specimens in acrylic resin, forming blocks that effects of bleaching on tooth surfaces under clinimeasured 10 10 8 mm3, with the external surcally relevant conditions. We investigated two face of the substrates exposed. Each acrylic block hypotheses: had two slabs (enamel and root dentin). We poldat-home overnight bleaching can increase the ished the exposed dental surfaces sequentially. susceptibility of the tooth surfaces to erosive wear, We placed adhesive unplasticized polyvinyl chlotoothbrushing-related abrasive wear or both; ride tape (TapeCase, Elk Grove Village, Ill.) on dthe abrasive level of dentifrices can modulate the each specimens surface, leaving an exposed censurface wear of bleached and eroded tooth surfaces. tral area of 1 4 mm2. The preparation is fully described elsewhere.10 MATERIALS AND METHODS Erosion-abrasion cycling model. We ranExperimental design. Our study received domly assigned specimens to the experimental approval from the institutional review board of groups, following a simple randomization table Indiana University-Purdue University Indigenerated in spreadsheet software (Excel 2007, anapolis. It consisted of a factorial 23 design, in Microsoft, Redmond, Wash.). We immersed each which the experimental factors were group for two minutes, static, in 120 milliliters of dbleaching, at two levels: yes and no; 1 percent citric acid (pH 3.8, adjusted with 1 derosion, at two levels: yes and no; normal sodium hydroxide) (Sigma-Aldrich) or ddentifrice, at two levels: less abrasive 120 mL of deionized water. We then immersed (Aquafresh Advanced, RDA 58.1 [6.5], 1,100 parts them in 120 mL of artificial saliva (1.45 millimoper million [ppm] fluoride in the form of sodium lars calcium, 5.4 mmol phosphate, 0.1 molar fluoride, GlaxoSmithKline, Moon Township, Pa.) tris(hydroxymethyl)aminomethane buffer and more abrasive (Aquafresh Advanced with (2.2 grams per liter porcine gastric mucin) (SigmaWhitening, RDA 123.6 [8.4], 1,100 ppm fluoride Aldrich) for 60 minutes, under constant stirring at in the form of sodium fluoride, GlaxoSmithKline). 100 revolutions per minute. We positioned speciThe association among all factors resulted in mens in the brushing machine and brushed them eight experimental groups (Table). for 40 strokes (a 200-g load) (Oral-B 40, Procter & We randomly assigned eight specimens, Gamble, Cincinnati). For brushing each specimen, described below, to each group. The response variwe used 100 milliliters of a suspension prepared able was surface loss, in micrometers, analyzed by means of optical profilometry. An additional ABBREVIATION KEY. RDA: Radioactive dentin abrasivity. REA: Radioactive enamel abrasivity. experimental factor was tooth substrate (enamel

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with the test dentifrices and artificial saliva (1:3, weight-volume ratio). After each procedure (demineralization, remineralization and toothbrushing), we rinsed specimens in deionized water and gently dried them with absorbent paper. We repeated the described cycling three times per day for five days at room temperature (23C variation of 1). Except for the specimens that received the overnight bleaching treatment, all specimens were immersed in artificial saliva overnight. Bleaching treatment. For specimens assigned to the bleaching groups, we applied a layer (approximately 15 microliters) of the bleaching gel (Opalescence 10%, Ultradent Products, South Jordan, Utah) on the surface of the specimens with a custom-made bleaching tray (Sof-Tray Classic Sheets, Ultradent Products). We then immersed the specimen-bleaching trays in saliva for a total of 10 hours. Before starting the erosiveabrasive cycling, we removed the bleaching gel gently with paper wipes (Kimwipes, KimberlyClark Professional, Roswell, Ga.), rinsed the specimens with deionized water and dried them. Surface wear analysis. We removed the pieces of adhesive tape and positioned the specimens in the optical profilometer (Proscan 2000, Scantron, Taunton, England), keeping the experimental surface parallel to the horizontal plane. We scanned an area of 2 1 mm2 (covering the two reference surfaces and the treated surface), using a horizontal resolution of 0.01 micrometers and 0.05 m, respectively, in the x and y directions. We analyzed images by using dedicated software (Proscan 2000, Scantron), which calculated the average height of the two reference areas and subtracted it from the experimental area. The difference, expressed in micrometers, was the response variable of this study. Statistical analysis. We performed separate analyses for the enamel and root dentin substrates. We analyzed the effects of the three factors (bleaching, erosion, dentifrice abrasivity) on surface loss by means of three-way analysis of variance (ANOVA). We examined pairwise comparisons of the treatment combinations for significance using the Fisher protected least significant differences method. The significance level used for this study was 5 percent.
RESULTS

for the enamel substrate by using an ANOVA on the ranks of the surface-loss measurements. Enamel. None of the interactions between the three factors was significant (P = .35 for the three-way interaction, P = .39 for bleach-byerosion, P = .08 for bleach-by-abrasive and P = .12 for erosion-by-abrasive) (Figure 1). Bleaching did not have a significant effect on surface loss (P = .47). Samples with erosion had significantly more surface loss (P < .001). The more abrasive dentifrice led to significantly more surface loss than did the less abrasive dentifrice (P < .001). Dentin. The interaction among all three factors was significant (P = .034) (Figure 2). For specimens without erosion, bleaching led to significantly more surface loss (P = .001). For specimens with erosion and exposed to a less abrasive dentifrice, bleaching led to significantly less surface loss (P = .004). For specimens with erosion and exposed to a more abrasive dentifrice, bleaching did not have a significant effect on surface loss (P = .23). Specimens with erosion had significantly more surface loss (P < .001), and the difference between specimens with and without erosion was larger for specimens that were not bleached and smaller for specimens that were bleached. Specimens treated with the more abrasive dentifrice had significantly more surface loss (P .001), and the difference between specimens treated with more and less abrasive dentifrices was largest for specimens that had erosion and had undergone bleaching and smallest for specimens that had erosion but had not undergone bleaching.
DISCUSSION

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Enamel data did not present normal distribution and homoscedasticity, with the identification of one outlier. Therefore, we performed the analyses
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The association among bleaching, abrasion and erosion seldom has been studied and reported in the literature. Considering the high demand for bleaching procedures, the increasing consumption of acidic foods and beverages and the availability of dentifrices with a large range of abrasive levels, it is possible that these three factors would interact with one another in the mouth, potentially leading to undesirable wear of dental surfaces. To investigate these interactions, we tested two hypotheses. We rejected the first main hypothesis formulated in this study for enamel; enamel experienced no deleterious effect as a result of the association of the bleaching treatment with erosion and abrasion. Our results showed that overnight bleaching with 10 percent carbamide peroxide did not increase the potential for surface

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loss caused by the erosive challenge. Pretty 7.5 No Bleach and colleagues21 observed a similar Bleach 6.0 effect. In addition, we observed that bleaching did not 4.5 increase the susceptibility of enamel to 3.0 toothbrushing-related abrasive wear, regard1.5 less of the abrasivity of the dentifrice used. The previously 0.0 reported softening of the enamel surface 1.5 due to bleaching5,8 did High Abrasion Low Abrasion High Abrasion Low Abrasion not seem to be relevant in increasing enamel surface loss in No Erosion Erosion the experimental conDENTIFRICE TYPE AND BRUSHING ABRASION ditions used. The magnitude of the surface Figure 1. Graphic representation of enamel surface loss (means and standard-deviation bars). microhardness changes caused by bleaching, if any, was 25.0 not controlled in this No Bleach study. Our main focus Bleach was on surface wear, 20.0 one of the potential * problems associated * with reduction of 15.0 enamel microhard13 ness. Interestingly, we observed no sur10.0 face loss in bleached * specimens not submitted to erosion, sug5.0 gesting that the bleaching protocol adopted can be viewed 0.0 High Abrasion Low Abrasion High Abrasion Low Abrasion as safe for enamel when surface wear is being considered. No Erosion Erosion Erosion and the use DENTIFRICE TYPE AND BRUSHING ABRASION of more abrasive dentifrice, on the other 2. Graphic representation of root dentin (means and standard-deviation bars). The hand, caused a signifi- Figure denotes a significant difference between surface loss that underwent bleaching and those that asterisk the groups cant increase in did not. enamel wear. In the absence of erosion, no meaningful enamel surface frice abrasion of enamel, in which investigators loss was caused by any of the dentifrices. This is reported that dentifrices did not cause any signifiin accordance with other studies regarding denticant damage to sound enamel.16,22 However, we
SURFACE LOSS (MICROMETERS) SURFACE LOSS (MICROMETERS)
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found that this drastically changed when erosion was associated with toothbrushing-related abrasion. In that sense, our results corroborated those of previous studies, in which the association of both erosion and toothbrushing-related abrasion significantly increased the detrimental effects on enamel.23-25 The study hypothesis based on the influence of dentifrice abrasivity on erosion and bleaching was rejected, because different dentifrice abrasivity levels were not able to modify the potential of erosion and bleaching to cause surface loss. It may be relevant to mention that when we brushed specimens with the more abrasive dentifrice, we observed a surface loss among the bleached specimens numerically greater than that among the nonbleached ones. However, this surface loss was not statistically significant. With the use of more abrasive dentifrices, this difference could be more evident, and this possibility deserves further investigation. For dentin, there was a significant interaction among the experimental factors studied, leading us to accept the two study hypotheses. Bleaching led to increasing surface loss of noneroded specimens when brushed with either more or less abrasive dentifrices. This detrimental effect possibly is related to the reduction of dentin microhardness previously reported to occur after bleaching treatment.2,3 Dentin seems to be more susceptible to demineralization than is enamel,22 and its ability to remineralize has been questioned.26 This may explain why the bleaching effects on dentin surface wear were more evident in dentin than in enamel in this study. We observed different results when bleached dentin specimens were submitted to the erosive challenges. Bleaching associated with toothbrushing with the more abrasive dentifrice did not increase dentin surface loss. It seems that the erosive effect was stronger than the bleaching effect, not allowing the latter to be as evident as it was when specimens were not eroded. Surprisingly, when bleaching was associated with the less abrasive dentifrice, we observed a reduction of dentin wear. Whereas we did not expect this, we can hypothesize that the bleaching gel could have remained on the specimens surface after the treatment, possibly entrapped in the dentinal tubules, acting as a mechanical barrier and protecting dentin from subsequent erosive challenges. Urea, produced as a byproduct by the breakdown reaction of carbamide peroxide, also could have played a role, because it degrades into
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ammonia and carbon dioxide, thus causing pH elevation and potentially buffering the erosive acid.27,28 The observed protection was partial, because eroded groups demonstrated more surface loss than did noneroded ones, but enough to provide bleached specimens with higher protection than those not bleached. The protective effect also would be more evident when associated with the less abrasive dentifrice, possibly because of the dentifrices lesser ability to abrade it off the dentin surfaces. We should mention that we carefully removed the bleaching gel manually with the aid of absorbent paper, followed by rinsing in deionized water and drying. The remaining gel layer, if present, was not visible to the naked eye. It is unknown whether this suggested protection mechanism would be relevant clinically, because the remainder of the bleaching gel would be subject to mechanical and chemical processes that could remove it. Testing with more clinically relevant models may clarify this hypothesis. Therefore, we accepted the study hypotheses for dentin, because bleaching interacted with erosion to modify the surface loss, either increasing it or decreasing it depending on the presence of erosive challenge or the abrasivity of the dentifrice. Another point of potential concern would be the numerically higher surface-loss values we observed for root dentin compared with enamel when submitted to the same erosive and abrasive challenges. If confirmed by more clinically relevant experimental models, these results could raise concerns for clinicians recommending dental bleaching for patients who have exposed root surfaces, especially those who have dental erosion. Although our cycling model is limited by its in vitro nature, we designed it to simulate high erosive and abrasive challenges, as expected from patients experiencing erosion or abrasion. We selected the erosive solution used (1 percent citric acid, pH 3.75) and the time (two minutes per challenge) and frequency (three times per day) of exposure to simulate the regular daily ingestion of citric beverages and juices. We immersed the specimens in saliva for 60 minutes to allow remineralization to occur before brushing for 40 strokes (14 seconds). Researchers have suggested that an interval of at least one hour elapse between erosive and abrasive procedures performed on enamel to reduce the abrasive effects of brushing.29 For dentin, it seems as if the protection of such a waiting period is limited30 or nonexistent.26 The use of commercial products with similar fluoride

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concentration (1,100 ppm fluoride in the form of sodium fluoride) with known RDA and REA values allowed for better standardization of the study. The less abrasive dentifrice we used had an RDA that would classify it as having low abrasivity, whereas the more abrasive dentifrice had an RDA similar to those of moderately abrasive formulations, according to a general consensus among manufacturers and researchers on the laboratorial interpretation of the RDA index. Although the use of more abrasive dentifrices could have changed the results of the study, most of the dentifrices on the U.S. market are classified as being between low and moderate abrasivity. Our bleaching protocol followed the standard recommendations for overnight home bleaching, with use of custom-made bleaching trays, small amounts of bleaching gel and immersion in artificial saliva at 37C for 10 hours. We selected the total number of five days in light of the fact that the more evident changes resulting from bleaching are observed in the first days of the process.4
CONCLUSION

Within the limits of this in vitro study, we can conclude that overnight bleaching with 10 percent carbamide peroxide did not increase the susceptibility of enamel to erosive and abrasive wear, regardless of the abrasive level of the dentifrice used. Dentinal wear was modulated by the interaction of bleaching, erosion and dentifrice. Bleaching increased dentin surface loss of noneroded surfaces and provided protection to eroded surfaces brushed with less abrasive dentifrice.
Disclosure. Dr. Matis has received reimbursement from Ultradent Products, South Jordan, Utah, for presentations he has given. He also has received honoraria from Ultradent, although he does not serve as a consultant for the company. Mr. Engle received a grant from the G. Michael Dill Fund, Wabash College, Crawfordsville, Ind., to conduct the study described in this article. 1. Hasson H, Ismail AI, Neiva G. Home-based chemically-induced whitening of teeth in adults. Cochrane Database Syst Rev 2006(4): CD006202. doi: 10.1002/14651858.CD006202. Accessed Feb. 26, 2010. 2. de Freitas PM, Turssi CP, Hara AT, Serra MC. Monitoring of demineralized dentin microhardness throughout and after bleaching. Am J Dent 2004;17(5):342-346. 3. de Freitas PM, Turssi CP, Hara AT, Serra MC. Dentin microhardness during and after whitening treatments. Quintessence Int 2004;35(5):411-417. 4. Chen HP, Chang CH, Liu JK, Chuang SF, Yang JY. Effect of fluoride containing bleaching agents on enamel surface properties. J Dent 2008;36(9):718-725. 5. Majeed A, Grobler SR, Moola MH, Rossouw RJ, van Kotze TJ. Effect of four different opalescence tooth-whitening products on enamel microhardness. SADJ 2008;63(5):282-284, 286.

6. Faraoni-Romano JJ, Da Silveira AG, Turssi CP, Serra MC. Bleaching agents with varying concentrations of carbamide and/or hydrogen peroxides: effect on dental microhardness and roughness. J Esthet Restor Dent 2008;20(6):395-404. 7. Faraoni-Romano JJ, Turssi CP, Serra MC. Effect of a 10 percent carbamide peroxide on wear resistance of enamel and dentine: in situ study. J Dent 2009;37(4):273-278. 8. Azer SS, Machado C, Sanchez E, Rashid R. Effect of home bleaching systems on enamel nanohardness and elastic modulus. J Dent 2009;37(3):185-190. 9. Barbour ME, Finke M, Parker DM, Hughes JA, Allen GC, Addy M. The relationship between enamel softening and erosion caused by soft drinks at a range of temperatures. J Dent 2006;34(3):207-213. 10. Hara AT, Gonzlez-Cabezas C, Creeth J, Zero DT. The effect of human saliva substitutes in an erosion-abrasion cycling model. Eur J Oral Sci 2008;116(6):552-556. 11. Rios D, Honrio HM, Francisconi LF, Magalhes AC, de Andrade Moreira Machado MA, Buzalaf MA. In situ effect of an erosive challenge on different restorative materials and on enamel adjacent to these materials. J Dent 2008;36(2):152-157. 12. Ren YF, Amin A, Malmstrom H. Effects of tooth whitening and orange juice on surface properties of dental enamel. J Dent 2009;37(6):424-431. 13. Attin T, Koidl U, Buchalla W, Schaller HG, Kielbassa AM, Hellwig E. Correlation of microhardness and wear in differently eroded bovine dental enamel. Arch Oral Biol 1997;42(3):243-250. 14. Hemingway CA, Parker DM, Addy M, Barbour ME. Erosion of enamel by non-carbonated soft drinks with and without toothbrushing abrasion. Br Dent J 2006;201(7):447-450. 15. Mair LH. Wear in dentistry: current terminology. J Dent 1992;20(3):140-144. 16. Wiegand A, Schwerzmann M, Sener B, et al. Impact of toothpaste slurry abrasivity and toothbrush filament stiffness on abrasion of eroded enamel: an in vitro study. Acta Odontol Scand 2008;66(4): 231-235. 17. Kuroiwa M, Kodaka T, Kuroiwa M. Microstructural changes of human enamel surfaces by brushing with and without dentifrice containing abrasive. Caries Res 1993;27(1):1-8. 18. Vanuspong W, Eisenburger M, Addy M. Cervical tooth wear and sensitivity: erosion, softening and rehardening of dentineeffects of pH, time and ultrasonication. J Clin Periodontol 2002;29(4):351-357. 19. Vicentini BC, Braga SR, Sobral MA. The measurement in vitro of dentine abrasion by toothpastes. Int Dent J 2007;57(5):314-318. 20. Philpotts CJ, Weader E, Joiner A. The measurement in vitro of enamel and dentine wear by toothpastes of different abrasivity. Int Dent J 2005;55(3 suppl 1):183-187. 21. Pretty IA, Edgar WM, Higham SM. The effect of bleaching on enamel susceptibility to acid erosion and demineralisation. Br Dent J 2005;198(5):285-290. 22. Hooper S, West NX, Pickles MJ, Joiner A, Newcombe RG, Addy M. Investigation of erosion and abrasion on enamel and dentine: a model in situ using toothpastes of different abrasivity. J Clin Periodontol 2003;30(9):802-808. 23. Davis WB, Winter PJ. The effect of abrasion on enamel and dentine and exposure to dietary acid. Br Dent J 1980;148(11-12):253-256. 24. Turssi CP, Faraoni JJ, Rodrigues Jr AL, Sera MC. An in situ investigation into the abrasion of eroded dental hard tissues by a whitening dentifrice. Caries Res 2004;38(5):473-477. 25. Hara AT, Gonzlez-Cabezas C, Creeth J, Parmar M, Eckert GJ, Zero DT. Interplay between fluoride and abrasivity of dentifrices on dental erosion-abrasion. J Dent 2009;37(10):781-785. 26. Hara AT, Turssi CP, Teixeira EC, Serra MC, Cury JA. Abrasive wear on eroded root dentine after different periods of exposure to saliva in situ. Eur J Oral Sci 2003;111(5):423-427. 27. Stephan RM. The effect of urea in counteracting the influence of carbohydrates on the pH of dentin plaques. J Dent Res 1943;22(1): 63-71. 28. Leonard Jr. RH, Austin SM, Haywood VB, Bentley CD. Change in pH of the plaque and 10% carbamide peroxide solution during nightguard vital bleaching treatment. Quintessence Int 1994;25(12):819-823. 29. Jaeggi T, Lussi A. Toothbrush abrasion of erosively altered enamel after intraoral exposure to saliva: an in situ study. Caries Res 1999;33(6):455-461. 30. Attin T, Siegel S, Buchalla W, Lennon AM, Hannig C, Becker K. Brushing abrasion of softened and remineralised dentin: an in situ study. Caries Res 2004;38(1):62-66.

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