Photomedicine and Laser Surgery Volume 26, Number 6, 2008 © Mary Ann Liebert, Inc. Pp. 559–563 DOI: 10.1089/pho.2007.2231 Effect of Nd:YAG Irradiation and Fluoride Application on Dentine Resistance to Erosion in Vitro Ana Carolina Magalhães, Ph.D.,1 Daniela Rios, Ph.D.,2 Maria Aparecida De Andrade Moreira Machado, Ph.D.,3 Salete Moura Bonifácio Da Silva, Ph.D.,3 Rosane De Fátima Zanirato Lizarelli, Ph.D.,4 Vanderlei Salvador Bagnato, Ph.D.,4 and Marília Afonso Rabelo Buzalaf, Ph.D.2 Abstract Objective: In this paper we evaluated the effect of two fluoridated agents and Nd:YAG irradiation separately and in combination on dentine resistance to erosion. Background Data: The morphological changes in dentin induced by laser treatment may reduce the progression of erosive lesions. Due to the possibility of a synergis- tic effect of laser with fluoride, this study was conducted. Materials and Methods: Eighty bovine dentine sam- ples (4 � 4 mm) were randomly divided into eight groups, according to the following treatments: G1: untreated (control); G2: acidic phosphate fluoride gel (APF 1.23%) for 4 min; G3: fluoride varnish (NaF 2.26%) for 6 h; G4: 0.5 W Nd:YAG laser (250 �sec pulse, 10 Hz, 35 J/cm2, 30 sec); G5: 0.75 W Nd:YAG laser (52.5 J/cm2); G6: 1.0 W Nd:YAG laser (70 J/cm2); G7: APF � 0.75 W Nd:YAG laser; and G8: NaF � 0.75 W Nd:YAG laser. Af- ter the treatments, half of each dentine surface was protected with nail varnish. The samples were stored in ar- tificial saliva (30 mL/sample) for 24 h and submitted to four erosive 1-min cycles. Between the erosive attacks, the blocks were maintained in artificial saliva for 59 min. The erosive wear was evaluated by profilometry. Re- sults: The mean wear (�SD, �m) was: G1: 1.20 � 0.20; G2: 0.47 � 0.06; G3: 0.81 � 0.11; G4: 1.47 � 0.32; G5: 1.52 � 0.24; G6: 1.49 � 0.30; G7: 0.49 � 0.11; and G8: 1.06 � 0.31 (Tukey’s test, p � 0.05). Conclusions: Laser ir- radiation was not able to reduce dentine erosion. However, fluoride application was able to increase the den- tine’s resistance to erosion, and APF showed better results than fluoride varnish. 559 Introduction CERVICAL HYPERSENSITIVITY IS A COMMON PROBLEM in den- tistry, and is related to the exposure of root dentine and consequent dentin wear, which can be induced by erosion or abrasion.1 Dental erosion is thought to be the most im- portant etiological factor, as increased consumption of acidic foods and beverages can be observed.2,3 Initially, the erosive wear of dentine is characterized by demineralization of the interface between the intertubular and peritubular dentin. With increasing exposure time, the erosive attack results in a hollowing and funneling of the dentinal tubules. Finally, the peritubular dentin is com- pletely dissolved. The erosive demineralization results in the exposure of an outer layer of fully demineralized organic matrix, followed by a partly demineralized zone, until the sound inner dentin is reached.4,5 The high number of open and widened dentinal tubules may allow fluid movement, which stimulates the nerve endings located in the preden- tine and the odontoblast layer. As a consequence, painful hy- persensitivity of the tooth may occur, especially during acid exposure.6 As it is difficult to control all of the etiological factors con- tributing to dental erosion, such as the intake of acidic bev- erages or other damaging agents,7 many strategies have been developed to prevent or arrest dentine erosion and conse- quently, dentine hypersensitivity. Studies have shown that the application of fluoride8,9 and Nd:YAG laser treatment10 can promote dentinal tubule occlusion, which in turn pre- vents dentinal fluid movement. The beneficial effect of flu- oride is associated with the formation of a calcium fluoride- like precipitate, which may occlude the dentinal tubules.11 On the other hand, the ability of lasers to melt the dentin, reduce the tubular diameter, or seal the opened dentinal 1Department of Child and Social Dentistry, School of Dentistry of Araçatuba, São Paulo State University, Araçatuba, 2Department of Bi- ological Sciences, Bauru School of Dentistry, University of São Paulo, 3Department of Pediatric Dentistry, Orthodontic and Public Health, Bauru School of Dentistry, Bauru, and 4Department of Physics and Materials Science, Institute of Physics of São Carlos, São Carlos, Brazil. tubules, may be a potential tool for treatment of hypersen- sitivity.10,12–14 The morphological changes induced by laser treatment of dental surfaces, such as the reduction of the carbonate con- tent15 and closure of dentinal tubules,10 might also reduce the progression of erosive lesions. Due to the possibility of a synergistic effect between different treatments, it is specu- lated that it may be possible to increase their beneficial ef- fects using the combination of fluoride application and laser irradiation. Therefore, the aim of the present in vitro study was to evaluate the effect of two fluoridated agents (gel and varnish) and Nd:YAG irradiation (0.5, 0.75, and 1.0 W) sep- arately and in combination on dentine’s resistance to ero- sion. Materials and Methods Dentine sample preparation Eighty root dentine samples (4 � 4 � 3 mm) were pre- pared from extracted bovine incisors, which were previously stored in 2% formaldehyde solution (pH 7.0) for 30 d at room temperature. One sample was cut from each root using an IsoMet Low Speed Saw (Buehler Ltd., Lake Bluff, IL, USA) with two diamond disks (Extec Corp., Enfield, CT, USA), which were separated by a 4-mm spacer. The labial dentine surfaces were ground flat with water-cooled abrasive discs (320-, 600-, and 1200-grit Al2O3 papers; Buehler, Lake Bluff, IL, USA) and polished with felt paper and diamond spray (1 �m; Buehler). This procedure resulted in removal of a layer of dentine about 200 �m deep. The surface micro- hardness was determined by performing five indentations (Knoop diamond, 10 g, 5 sec, HMV-2000; Shimadzu Corp., Tokyo, Japan) in the center of the dentine surface, for selec- tion and randomized distribution purposes. Dentine samples with microhardness ranging from 34–60 Knoop hardness (KHN) (45.90 � 6.33 KHN) were randomly distributed into eight groups (n � 10 each): G1: untreated (controls); G2: acidic phosphate fluoride gel (APF; 1.23% fluoride) for 4 min; G3: fluoride varnish (NaF; 2.26% fluoride) for 6 h; G4: 0.5 W Nd:YAG laser (250 �sec pulse, 10 Hz, 35 J/cm2, 30 sec); G5: 0.75 W Nd:YAG laser (52.5 J/cm2); G6: 1.0 W Nd:YAG laser (70 J/cm2); G7: APF � 0.75 W Nd:YAG laser; and G8: NaF � 0.75 W Nd:YAG laser. Experimental procedure In group 1, the dentine samples were not treated and served as controls. In group 2, the acidic phosphate fluoride gel (pH 3.5; Flugel, DFL, Rio de Janeiro, Brazil) was applied to each dentine surface with a microbrush and left on for 4 min. After the time elapsed, the excess gel was removed with cotton swabs. In group 3, the fluoride varnish (pH 4.5; Du- raphat, Colgate, São Paulo, Brazil) was applied in a thin layer with a microbrush to each dentine surface. After 6 h, the var- nish was carefully removed using acetone and a scalpel blade.16,17 In groups 4, 5, and 6, the samples were irradiated with an Nd:YAG laser (Twinlight; Fotona Medical Lasers, Ljublijana, Slovenia), in contact and non-contact mode, perpendicularly to the sample via a quartz fiber with a diameter of 300 �m. This laser system has a pulse length of 250 �sec and repeti- tion rate from 5–10 Hz. The laser device was fixed above a horizontally moving table, which allowed scanning move- ments in four directions (mesial-distal and apical-occlusal; 5 � 5 mm) with uniform velocity for 30 sec for each appli- cation. For laser application, the samples were moved by the table, allowing the fixed laser beam to scan the entire sur- face. The irradiation conditions for the laser groups were: G4: 0.5 W Nd:YAG laser (250 �m, 10 Hz, 35 J/cm2); G5: 0.75 W Nd:YAG laser (52.5 J/cm2); and G6: 1.0 W Nd:YAG laser (70 J/cm2). In group 7, the samples were first treated with APF as de- scribed above, and afterwards they were irradiated with the Nd:YAG laser (0.75 W). Group 8 samples were first treated with fluoride varnish as described above, and afterwards were irradiated with the Nd:YAG laser (0.75 W). After all of the treatments, two layers of nail varnish were applied to half of the surface of the dentine of each sample in order to maintain reference surfaces for lesion depth de- termination. For 24 h, the samples were kept in artificial saliva (30 mL/sample), and then were immersed in Sprite Light® (30 mL/sample; Coca-Cola Co., Spal, Porto Real, RJ, Brazil) for 1 min, four times at room temperature.18 Between the ero- sive attacks, the samples were kept in artificial saliva for 59 min. The Sprite Light (pH 2.87) had a buffering capacity of 0.375 � 0.01, which is equivalent to 0.375 mL of 0.2 M NaOH/3 mL beverage, for an increase of one pH unit.18 The composition of the artificial saliva was: 1.5 mM Ca(NO3)2 � 4 H2O, 0.9 mM NaH2PO4 � 2 H2O, 150 mM KCl, 0.1 M Tris buffer, 0.03 ppm fluoride, pH 7.0].19 Wear assessment The dentine wear was determined by profilometry in re- lation to the reference surface, which was previously pro- tected by nail varnish (Hommel Tester T1000; Hommel Tech, VS, Schwenningen, Germany). The nail varnish on the ref- erence surfaces was carefully removed with acetone-soaked cotton.20 Five readings were taken on each sample by scan- ning from the reference portion to the exposed surface. The lowest area of the eroded area compared to the reference sur- face was calculated by the software of the device and was considered as wear. The mean values of five readings for each group were averaged. Statistical analysis We used GraphPad Instat for Windows (GraphPad, San Diego, CA, USA) software to perform the statistical analy- sis. The assumptions of equality of variances and normal dis- tribution of errors were checked for all the variables tested. Since the assumptions were satisfied, ANOVA and Tukey’s tests were carried out for statistical comparisons and statis- tical significance was set at p � 0.05. Results Table 1 shows the mean wear (�m � SD) for each group. Significantly higher dentine wear (1.20–1.52 �m) was recorded for groups G5 (0.75 W laser), G1 (control), G4 (0.5 W laser), and G6 (1 W laser). Group G5 significantly differed from G1, G4, and G6, which did not differ significantly from one another. Group G8 (varnish � laser) had less wear com- pared to the other laser groups G4, G5, and G6 (p � 0.05), MAGALHÃES ET AL.560 but it was also similar to controls. Only G2 (APF), G3 (var- nish), and G7 (APF � laser) had significantly less dentine wear (0.47–0.81 �m) compared to controls (p � 0.05). How- ever, groups G2 and G7 (APF) were not significantly differ- ent from each other, but showed better results compared to group G3 (fluoride varnish) (p � 0.05). Discussion Fluoride treatment is an established tool in the prevention of demineralization of dental hard tissues.11,21 The most com- mon fluoridated agents in clinical use are gels and varnishes. As fluoride varnishes exhibit a better capability to adhere to the tooth surface than fluoride gels, the fluoride varnish was applied for 6 h, while the gel was applied for only 4 min in the present study. However, the efficacy of fluoride var- nishes and gels in decreasing demineralization and enhanc- ing remineralization of dental hard tissues is similar.22,23 On the other hand, the protective effects of laser irradia- tion of enamel and dentine from demineralization have gained increasing attention in recent years. Several types of lasers, such as ruby, CO2, Nd:YAG, and argon, with differ- ent operational modes and energy outputs have been inves- tigated.24 Most of the studies of the effects of laser irradia- tion on dental hard tissue demineralization have been conducted in vitro using the Nd:YAG laser, due to its ease of application in the oral cavity.24 However, microscopic analyses have been used in most studies that tested this laser,12,25,26 and this method is unable to quantify the loss of dental hard tissue. Naylor et al.12 showed that irradiation with the Nd:YAG laser produces obliteration of and reduction in the numbers of dentinal tubules, thus modifying its original structure. The authors suggested that dentine irradiated with 0.6 W of Nd:YAG laser energy had a higher resistance to acidic bev- erages such as colas and passion fruit juice, but they did not evaluate erosion quantitatively. Kimura et al.27 showed that Nd:YAG irradiation (1–5 J/cm2) induced morphological changes (melting and smoothing) of the dentine’s surface. However, Nd:YAG irradiation was unable to protect dentine against caries, as shown by scattering light microscopy. This result indicates that the effect of laser irradiation is limited to the surface. This observation underlines the need for stud- ies evaluating the effects of laser irradiation quantitatively. In the present study, bovine dentine was used as a sub- stitute for human teeth, as has been done in several previ- ous studies.25,28,29 Despite the morphological differences be- tween bovine and human substrates, bovine dentine offers similar acid resistance to that of human dentine in situ.30 With regard to the polished dentine samples we used in the present study, it must also be considered that polished sam- ples have been shown to be more susceptible to demineral- ization than natural surfaces, as the fluoride-rich enamel sur- face layer is removed by the polishing.31 This aspect has to be carefully considered when the results are extrapolated to an in-vivo situation. Despite these limitations, in vitro stud- ies have to be conducted before clinical testing of new meth- ods can begin. The type of laser and the intensity used were chosen in accordance with previous studies.12,26,27,32 The samples were briefly eroded (60 sec) by a common soft drink containing citric acid to simulate the sipping or drinking of an erosive beverage. The artificial saliva we used allows rehardening of the samples between erosive challenges. The profilometry technique we used in the present study is a quantitative method that is able to measure dentine loss induced by the cyclic pH changes,33 thus reflecting the cumulative effect of the erosive challenges. In this study, we found that laser irradiation was unable to reduce erosive dentine wear. The efficacy of the laser ir- radiation may have been affected by many factors, such as the laser wavelength and energy output, and dentine surface conditions (dry or wet). The proper laser wavelength and en- ergy output may change the dentine’s surface and reduce the progression of demineralization.12 Regarding the energy output, it can be assumed that the high laser power used in this study (0.5–1 W) may induce cracks and fissures instead of melting and resolidification of the dentinal surface. This cracked surface may be more susceptible to acid challenge.24 Lan et al.26 showed that energy levels inducing crater for- mation by the Nd:YAG laser ranged from 100–150 mJ, which is much lower than the energy levels we used in the present study. Similarly to the results of the present study, Tsai et al.24 found no protective effect of Nd:YAG laser treatment on enamel demineralization in vitro. The pulsed Nd:YAG laser (83.33 J/cm2) was unable to increase the enamel’s re- sistance to acid challenge (lactate buffer solution, pH 4.5, for 24 and 72 h), as shown by calcium analysis and polarized light microscopy. Therefore, further studies are required to test the effects of lower laser power levels (�0.5 W) on den- tal erosion. To understand the mechanism of action of Nd:YAG irradiation, it is also important to analyze the su- perficial and structural changes of laser-irradiated dentine at different laser power levels. Besides crack formation, thermal changes caused by ele- vations in temperature in the irradiated region may cause pulp damage in the clinical setting.26 White et al.32 suggested using laser power levels no higher than 1 W to avoid these thermal changes. According to Kimura et al.,27 the intrapul- pal temperature increases may exceed 5.5°C during irradia- tion at energy densities of 2.0 J/cm2. However, the power levels (0.5–1.0 W) and the type of laser system (nanosecond pulse) used in the present study may not cause pulpal heat- ing to damaging levels. In contrast to the laser treatment and in accordance with previous studies, fluoride application was effective in in- creasing the dentine’s resistance to erosive losses.34,35 As ero- sive lesions do not cause subsurface lesions that make rem- EFFECT OF LASER AND FLUORIDE ON DENTINE EROSION 561 TABLE 1. DENTIN WEAR (�M � SD) FOR EACH EXPERIMENTAL GROUP Group Wear (�m) G1: Control 1.20 � 0.20a,c G2: APF gel 0.47 � 0.06b G3: NaF varnish 0.81 � 0.11c G4: 0.5 W Nd�YAG laser 1.47 � 0.32a G5: 0.75 W Nd�YAG laser 1.52 � 0.24d G6: 1.0 W Nd�YAG laser 1.49 � 0.30a G7: APF gel � 0.75 W Nd�YAG laser 0.49 � 0.11b G8: NaF varnish � 0.75 W Nd�YAG laser 1.06 � 0.31c,e Values followed by the same superscript letters indicate statistical significance (p � 0.05). ineralization possible, the action of fluoride is mainly attrib- utable to precipitation of a calcium fluoride-like material on dental surfaces.34,35 Although the fluoride concentration as well as the time of contact is lower for the gel (4 min) com- pared to the varnish (6 h), the APF gel (1.23% fluoride, pH 3.5) had better results compared to those of the varnish (2.26% fluoride, pH 4.5). This may be explained by their dif- fering pH levels. It is well known that the formation of the calcium fluoride reservoir is increased under acidic com- pared to neutral pH levels.11,21 Conclusion With the protocol we used in this study, laser irradiation was unable to reduce beverage-induced dentinal erosion. However, fluoride application was able to increase the den- tine’s resistance to erosion, and the acidic phosphate fluo- ride gel achieved better results than the fluoride varnish. Acknowledgments The authors thank Prof. Rafael Lia Mondelli for the use of the profilometer, and Master Student in Electric Engineering Mardoqueu Martins Costa for the use of his device to stan- dardize our laser irradiation scanning protocol. Disclosure Statement No conflicting financial interests exist. References 1. Addy, M., and Shellis, R.P. (2006). Interaction between at- trition, abrasion and erosion in tooth wear. Monogr. Oral Sci. 20, 17–31. 2. Lussi, A., Jaeggi, T., and Zero, D. (2004). The role of diet in the etiology of dental erosion. Caries Res. 38, 34–44. 3. Lussi, A. (2006). Erosive tooth wear—a multifactorial con- dition of growing concern and increasing knowledge. Monogr. Oral Sci. 20, 1–8. 4. Kinney, J.H., Balooch, M., Haupt, D.L. Jr., Marshall, S.J., and Marshall, G.W. Jr. (1995). Mineral distribution and dimen- sional changes in human dentin during demineralization. J. Dent. Res. 74, 1179–1184. 5. Meurman, J.H., Drysdale, T., and Frank, R.M. (1991). Ex- perimental erosion of dentine. Scand. J. Dent. Res. 99, 457–462. 6. Addy, M., Absi, E.G., and Adams, D. (1987). Dentin hyper- sensitivity. The effects in vitro of acids and dietary sub- stances on root-planed and burred dentin. J. Clin. Peri- odontol. 14, 274–279. 7. Johansson, A.K., Lingstrîm, P., Imfeld, T., and Birkhed, D. (2004). Influence of drinking method on tooth-surface pH in relation to dental erosion. Eur. J. Oral Sci. 112, 484–489. 8. White, D.J., Lawless, M.A., Fatade, A., et al. (2007). Stannous fluoride/sodium hexametaphosphate dentifrice increases dentin resistance to tubule exposure in vitro. J. Clin. Dent. 18, 55–59. 9. Ritter, A.V., Dias, W.L., Miguez, P., Caplan, D.J., and Swift, E.J. Jr. (2006). Treating cervical dentin hypersensitivity with fluoride varnish: a randomized clinical study. J. Am. Dent. Assoc. 137, 1013–1020. 10. Lan, W.H., and Liu, H.C. (1996). Treatment of dentin hy- persensitivity by Nd:YAG laser. J. Clin. Laser Med. Surg. 14, 89–92. 11. ten Cate, J.M. (1997). Review on fluoride, with special em- phasis on calcium fluoride mechanisms in caries prevention. Eur. J. Oral Sci. 105, 461–465. 12. Naylor, F., Aranha, A.C., Eduardo, C.P., Arana-Chavez, V.E., and Sobral, M.A.P. (2006). Micromorphological analy- sis of dentinal structure after irradiation with Nd:YAG laser and immersion in acidic beverages. Photomed. Laser Surg. 24, 745–752. 13. Yonaga, K., Kimura, Y., and Matsumoto, K. (1999). Treat- ment of cervical dentin hypersensitivity by various methods using pulsed Nd:YAG laser. J. Clin. Laser Med. Surg. 17, 205–210. 14. Ciaramicoli, M.T., Carvalho, R.C., and Eduardo, C.P. (2003). Treatment of cervical dentin hypersensitivity using neodymium: yttrium-aluminum-garnet laser. Clinical eval- uation. Laser Surg. Med. 33, 358–362. 15. Nelson, D.G.A., Wefel, J.S., Jongebloed, W.L., and Feather- stone, J.D.B. (1987). Morphology, histology and crystallog- raphy of human dental enamel treated with pulsed low-en- ergy infrared laser radiation. Caries Res. 21, 411–426. 16. Delbem, A.C.B., Bergamaschi, M., Sassaki, K.T., and Cunha, R.F. (2006). Effect of fluoridated varnish and silver diamine fluoride solution on enamel demineralization: pH-cycling study. J. Appl. Oral Sci. 14, 88–92. 17. Vieira, A., Lugtenborg, M., Ruben, J.L., and Huysmans, M.C. (2006). Brushing abrasion of eroded bovine enamel pre- treated with topical fluorides. Caries Res. 40, 224–230. 18. Sales-Peres, S.H.C., Magalhães, A.C., Machado, M.A.A.M., and Buzalaf, M.A.R. (2007). In vitro evaluation of variables related to the erosive potential of five soft drinks. Eur. J. Dent. 1, 10–13. 19. Vieira, A.E.M., Delbem, A.C.B., Sassaki, K.T., Rodrigues, E., Cury, J.A., and Cunha, R.F. (2005). Fluoride dose response in pH-cycling models using bovine enamel. Caries Res. 39, 514–520. 20. Attin, T., Buchalla, W., Gollner, M., and Hellwig, E. (2000). Use of variable remineralization periods to improve the abrasion resistance of previously eroded enamel. Caries Res. 34, 48–52. 21. ten Cate, J.M. (1999). Current concepts on the theories of the mechanism of action of fluoride. Acta. Odontol. Scand. 57, 325–329. 22. Vieira, A., Jager, D.H., Ruben, J.L., and Huysmans, M.C. (2007). Inhibition of erosive wear by fluoride varnish. Caries Res. 41, 61–67. 23. Seppa, L. (1988). Effects of a sodium fluoride solution and a varnish with different fluoride concentrations on enamel remineralisation in vitro. Scand. J. Dent. Res. 96, 304–309. 24. Tsai, C.L., Lin, Y.T., Huang, S.T., and Chang, H.W. (2002). In vitro acid resistance of CO2 and Nd:YAG laser-treated hu- man tooth enamel. Caries Res. 36, 423–429. 25. Bachmann, L., Craievich, A.F., and Zezell, D.M. (2004). Crys- talline structure of dental enamel after Ho:YLF laser irradi- ation. Arch. Oral Biol. 49, 923–929. 26. Lan, W.H., Chen, K.W., Jeng, J.H., Lin, C.P., and Lin, S.K. (2000). A comparison of the morphological changes after Nd-YAG and CO2 laser irradiation of dentin surfaces. J. En- dod. 26, 450–453. 27. Kimura, Y., Wilder-Smith, P., Arrastia-Jitosho, A.M., Liaw, L.H., Matsumoto, K., and Berns, M.W. (1997). Effects of nanosecond pulsed Nd:YAG laser irradiation on dentin re- sistance to artificial caries-like lesions. Laser Surg. Med. 20, 15–21. 28. Hara, A.T., Ando, M., Gonzalez-Cabezas, C., Cury, J.A., Serra, M.C., and Zero, D.T. (2006). Protective effect of the MAGALHÃES ET AL.562 dental pellicle against erosive challenges in situ. J. Dent. Res. 85, 612–616. 29. Hannig, C., Becker, K., Hausler, N., Hoth-Hannig, W., At- tin, T., and Hannig, M. (2007). Protective effect of the in situ pellicle on dentin erosion—an ex vivo pilot study. Arch. Oral Biol. 52, 444–449. 30. Hara, A.T., Queiroz, C.S., Paes Leme, A.F., Serra, M.C., and Cury, J.A. (2003). Caries progression and inhibition in hu- man and bovine root dentine in situ. Caries Res. 37, 339–344. 31. Ganss, C., Klimek, J., and Schwarz, N. (2000). A compara- tive profilometric in vitro study of the susceptibility of pol- ished and natural human enamel and dentine surfaces to erosive demineralization. Arch. Oral Biol. 45, 897–902. 32. White, J.M., Goodis, H.E., Setcos, J.C., Eakle, S., Hulscher, B.E., and Rose, C.L. (1993). Effects of pulsed Nd:YAG laser energy on human teeth: a three-year follow-up study. J. Am. Dent. Assoc. 124, 45–51. 33. Attin T. (2006). Methods for assessment of dental erosion. Monogr. Oral Sci. 20, 152–172. 34. Ganss, C., Klimek, J., SchÑffer, U., and Spall, T. (2001). Ef- fectiveness of two fluoridation measures on erosion pro- gression in human enamel and dentine in vitro. Caries Res. 35, 325–330. 35. Ganss, C., Klimek, J., Brune, V., and Schumann, A. (2004). Effects of two fluoridation measures in erosion progression on enamel and dentine in situ. Caries Res. 38, 561–566. Address reprint requests to: Professor Marília Afonso Rabelo Buzalaf, Ph.D. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Al. Octavio Pinheiro Brissola 9-75 Bauru, SP, Brazil 17012-910 E-mail: mbuzalaf@fob.usp.br EFFECT OF LASER AND FLUORIDE ON DENTINE EROSION 563 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile () /CalRGBProfile (Adobe RGB \0501998\051) /CalCMYKProfile (Japan Web Coated \050Ad\051) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Off /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJDFFile false /CreateJobTicket true /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails true /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize false /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo true /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Remove /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile (None) /AlwaysEmbed [ true /ACaslon-AltBold /ACaslon-AltBoldItalic /ACaslon-AltItalic /ACaslon-AltRegular /ACaslon-AltSemibold /ACaslon-AltSemiboldItalic /ACaslon-Bold /ACaslon-BoldItalic /ACaslon-Italic /ACaslon-Ornaments /ACaslon-Regular /ACaslon-Semibold /ACaslon-SemiboldItalic /ACaslon-SwashBoldItalic /ACaslon-SwashItalic /ACaslon-SwashSemiboldItalic /ACaslonExp-Bold /ACaslonExp-BoldItalic /ACaslonExp-Italic /ACaslonExp-Regular /ACaslonExp-Semibold /ACaslonExp-SemiboldItalic /AGaramond-BoldItalic /AGaramond-Italic /AGaramond-Regular /AGaramond-Semibold /AGaramond-SemiboldItalic /ATTorino-Bold /ATTorino-Condensed /Americana /Americana-Bold /Americana-ExtraBold /Americana-Italic /AmericanaBT-Bold /AmericanaBT-ExtraBold /AmericanaBT-ExtraBoldCondensed /AmericanaBT-Italic /AmericanaBT-Roman /AvantGarde-Bold /AvantGarde-BoldObl /AvantGarde-Book /AvantGarde-BookOblique /AvantGarde-CondBold /AvantGarde-CondBook /AvantGarde-CondDemi /AvantGarde-CondMedium /AvantGarde-Demi /AvantGarde-DemiOblique /AvantGarde-ExtraLight /AvantGarde-ExtraLightObl /AvantGarde-Mdm /AvantGarde-Medium /AvantGarde-MediumObl /BauerBodoni-Black /BauerBodoni-BlackCond /BauerBodoni-BlackItalic /BauerBodoni-Bold /BauerBodoni-BoldCond /BauerBodoni-BoldItalic /BauerBodoni-Italic /BauerBodoni-Roman /Belwe-Bold /Belwe-Condensed /Belwe-Light /Belwe-Medium /BelweT-LighItal /Berkeley-Black /Berkeley-BlackItalic /Berkeley-Bold /Berkeley-BoldItalic /Berkeley-Book /Berkeley-BookItalic /Berkeley-Italic /Berkeley-Medium /BiffoMT /Bodoni /Bodoni-Bold /Bodoni-BoldCondensed /Bodoni-BoldItalic /Bodoni-Book /Bodoni-BookItalic /Bodoni-Italic /Bodoni-Poster /Bodoni-PosterCompressed /Bodoni-PosterItalic /Bookman-Bold /Bookman-BoldItalic /Bookman-Demi /Bookman-DemiItalic /Bookman-Light /Bookman-LightItalic /Bookman-Medium /Bookman-MediumItalic /BorderPi-OneFiveOneFiveNine /BrushScript /Carta /CaslonTwoTwentyFour-Black /CaslonTwoTwentyFour-BlackIt /CaslonTwoTwentyFour-Bold /CaslonTwoTwentyFour-BoldIt /CaslonTwoTwentyFour-Book /CaslonTwoTwentyFour-BookIt /CaslonTwoTwentyFour-Medium /CaslonTwoTwentyFour-MediumIt /Century-Bold /Century-BoldCond-DTC /Century-BoldCondItalic-DTC /Century-BoldCondensed /Century-BoldCondensedItalic /Century-BoldItalic /Century-Book /Century-BookCond-DTC /Century-BookCondItalic-DTC /Century-BookCondensed /Century-BookCondensedItalic /Century-BookItalic /Century-Light /Century-LightCond-DTC /Century-LightCondIta-DTC /Century-LightCondensed /Century-LightCondensedItalic /Century-LightItalic /Century-Ultra /Century-UltraCond-DTC /Century-UltraCondIta-DTC /Century-UltraCondensed /Century-UltraCondensedItalic /Century-UltraItalic /Century751BT-ItalicB /Century751BT-RomanB /CenturyExpanded /CenturyExpanded-Italic /CenturyExpandedBT-Bold /CenturyExpandedBT-BoldItalic /CenturyExpandedBT-Italic /CenturyExpandedBT-Roman /CenturyOldStyle-Bold /CenturyOldStyle-Italic /CenturyOldStyle-Regular /CgTorinoOutline /Cheltenham-Bold /Cheltenham-BoldCond /Cheltenham-BoldItalic /Cheltenham-Book /Cheltenham-BookCond /Cheltenham-BookCondItalic /Cheltenham-BookItalic /Cheltenham-Light /Cheltenham-LightCond /Cheltenham-LightCondItalic /Cheltenham-LightItalic /Cheltenham-Ultra /Cheltenham-UltraItalic /Clarendon /Clarendon-Bold /Clarendon-Light /ClarendonBT-BoldCondensed /ClarendonBT-RomanCondensed /CompactaBoldPlain /CompactaItalicPlain /CompactaOnlShaD-ReguItal /CooperBlack /CooperBlack-Italic /Courier /Courier-Bold /Courier-BoldOblique /Courier-Oblique /DINEngschrift-Alternate /EdwardianScriptITC /ElectraLH-Bold /ElectraLH-Cursive /ElectraLH-Regular /Fenice-Bold /Fenice-BoldOblique /Fenice-Light /Fenice-LightOblique /Fenice-Regular /Fenice-RegularOblique /Fenice-Ultra /Fenice-UltraOblique /FranklinGothic-Book /FranklinGothic-BookOblique /FranklinGothic-Condensed /FranklinGothic-CondensedOblique /FranklinGothic-Demi /FranklinGothic-DemiOblique /FranklinGothic-ExampleNumber /FranklinGothic-ExtraCond /FranklinGothic-Heavy /FranklinGothic-HeavyOblique /FranklinGothic-Roman /Frutiger-Black /Frutiger-BlackCn /Frutiger-BlackItalic /Frutiger-Bold /Frutiger-BoldCn /Frutiger-BoldItalic /Frutiger-Cn /Frutiger-ExtraBlackCn /Frutiger-Italic /Frutiger-Light /Frutiger-LightCn /Frutiger-LightItalic /Frutiger-Roman /Frutiger-UltraBlack /Futura /Futura-Bold /Futura-BoldOblique /Futura-Book /Futura-BookOblique /Futura-CondExtraBoldObl /Futura-Condensed /Futura-CondensedBold /Futura-CondensedBoldOblique /Futura-CondensedExtraBold /Futura-CondensedLight /Futura-CondensedLightOblique /Futura-CondensedOblique /Futura-Heavy /Futura-HeavyOblique /Futura-Light /Futura-LightOblique /Futura-Mdm /Futura-MdmCnd /Futura-MdmCndItl /Futura-MdmItl /Futura-Oblique /FuturaBT-Bold /FuturaBT-BoldCondensed /FuturaBT-BoldCondensedItalic /FuturaBT-BoldItalic /FuturaBT-Book /FuturaBT-BookItalic /FuturaBT-ExtraBlack /FuturaBT-ExtraBlackCondItalic /FuturaBT-ExtraBlackCondensed /FuturaBT-ExtraBlackItalic /FuturaBT-Heavy /FuturaBT-HeavyItalic /FuturaBT-Light /FuturaBT-LightCondensed /FuturaBT-LightItalic /FuturaBT-Medium /FuturaBT-MediumCondensed /FuturaBT-MediumItalic /FuturaBoldModified /FuturaBoldObliqueModified /FuturaEF-DemiBold /FuturaEF-DemiBoldObl /FuturaModified /FuturaObliqueModified /Garamond-Bold /Garamond-BoldCondensed /Garamond-BoldCondensedItalic /Garamond-BoldItalic /Garamond-Book /Garamond-BookCondensed /Garamond-BookCondensedItalic /Garamond-BookItalic /Garamond-Light /Garamond-LightCondensed /Garamond-LightCondensedItalic /Garamond-LightItalic /Garamond-Ultra /Garamond-UltraCondensed /Garamond-UltraCondensedItalic /Garamond-UltraItalic /GillSans /GillSans-Bold /GillSans-BoldCondensed /GillSans-BoldExtraCondensed /GillSans-BoldItalic /GillSans-Condensed /GillSans-ExtraBold /GillSans-ExtraBold-ScholasticLg /GillSans-ExtraBold-ScholasticSm /GillSans-ExtraBoldDisplay /GillSans-Italic /GillSans-Light /GillSans-LightItalic /GillSans-LightShadowed /GillSans-Shadowed /GillSans-UltraBold /GillSans-UltraBoldCondensed /GillSansExtraBoldItalic /GillSansUltraBoldItalic /Giovanni-Black /Giovanni-BlackItalic /Giovanni-Bold /Giovanni-BoldItalic /Giovanni-Book /Giovanni-BookItalic /Glypha /Glypha-Bold /Glypha-BoldOblique /Glypha-Oblique /Goldwater /Goudy /Goudy-Bold /Goudy-BoldItalic /Goudy-BoldItalicOsF /Goudy-BoldOsF /Goudy-ExtraBold /Goudy-Heavyface /Goudy-HeavyfaceItalic /Goudy-Italic /Goudy-ItalicOsF /Goudy-SC /GoudyCatalog-Itl /GoudyCatalog-Rgl /Helvetica /Helvetica-Black /Helvetica-BlackOblique /Helvetica-Bold /Helvetica-BoldOblique /Helvetica-Compressed /Helvetica-Condensed /Helvetica-Condensed-Black /Helvetica-Condensed-BlackObl /Helvetica-Condensed-Bold /Helvetica-Condensed-BoldObl /Helvetica-Condensed-Light /Helvetica-Condensed-LightObl /Helvetica-Condensed-Oblique /Helvetica-ExtraCompressed /Helvetica-Light /Helvetica-LightOblique /Helvetica-Narrow /Helvetica-Narrow-Bold /Helvetica-Narrow-BoldOblique /Helvetica-Narrow-Oblique /Helvetica-Oblique /Helvetica-UltraCompressed /HelveticaNeue-Black /HelveticaNeue-BlackCond /HelveticaNeue-BlackCondObl /HelveticaNeue-BlackExt /HelveticaNeue-BlackExtObl /HelveticaNeue-BlackItalic /HelveticaNeue-Bold /HelveticaNeue-BoldCond /HelveticaNeue-BoldCondObl /HelveticaNeue-BoldExt /HelveticaNeue-BoldExtObl /HelveticaNeue-BoldItalic /HelveticaNeue-Condensed /HelveticaNeue-CondensedObl /HelveticaNeue-ExtBlackCond /HelveticaNeue-ExtBlackCondObl /HelveticaNeue-Extended /HelveticaNeue-ExtendedObl /HelveticaNeue-Heavy /HelveticaNeue-HeavyCond /HelveticaNeue-HeavyCondObl /HelveticaNeue-HeavyExt /HelveticaNeue-HeavyExtObl /Humanist521BT-Roman /Industria-Inline /Industria-InlineA /Industria-Solid /Industria-SolidA /ItcEras-Bold /ItcEras-Book /ItcEras-Demi /ItcEras-Light /ItcEras-Medium /ItcEras-Ultra /ItcKabel-Bold /ItcKabel-Book /ItcKabel-Demi /ItcKabel-Medium /ItcKabel-Ultra /Kaufmann /Kaufmann-Bold /Linoscript /LubalinGraph-Book /LubalinGraph-BookOblique /LubalinGraph-Demi /LubalinGraph-DemiOblique /LucidaSans /LucidaSans-Bold /LucidaSans-Italic /Machine /MediciScript /Minion-Black /Minion-Bold /Minion-BoldItalic /Minion-DisplayItalic /Minion-DisplayRegular /Minion-Italic /Minion-Ornaments /Minion-Regular /Minion-Semibold /Minion-SemiboldItalic /Minion-SwashDisplayItalic /Minion-SwashItalic /Minion-SwashSemiboldItalic /MinionExp-Black /MinionExp-Bold /MinionExp-BoldItalic /MinionExp-DisplayItalic /MinionExp-DisplayRegular /MinionExp-Italic /MinionExp-Regular /MinionExp-Semibold /MinionExp-SemiboldItalic /Modern735BT-RomanA /Modern880BT-Bold /Modern880BT-Italic /Modern880BT-Roman /NewBaskerville-Black /NewBaskerville-BlackItalic /NewBaskerville-Bold /NewBaskerville-BoldItalic /NewBaskerville-BoldItalicOsF /NewBaskerville-BoldSC /NewBaskerville-Italic /NewBaskerville-ItalicOsF /NewBaskerville-Roman /NewBaskerville-SC /NewBaskerville-SemiBold /NewBaskerville-SemiBoldIta /NewCaledonia-Black /NewCaledonia-BlackItalic /NewCaledonia-Bold /NewCaledonia-BoldItalic /NewCaledonia-BoldItalicOsF /NewCaledonia-BoldSC /NewCaledonia-Italic /NewCaledonia-ItalicOsF /NewCaledonia-SC /NewCaledonia-SemiBold /NewCaledonia-SemiBoldItalic /Nofret-Bold /Nofret-BoldItalic /Nofret-Italic /Nofret-Light /Nofret-LightItalic /Nofret-Medium /Nofret-MediumItalic /Nofret-Regular /Novarese-Bold /Novarese-BoldItalic /Novarese-Book /Novarese-BookItalic /Novarese-Medium /Novarese-MediumItalic /Novarese-Ultra /OCRA /OCRB /OfficinaSans-Bold /OfficinaSans-BoldItalic /OfficinaSans-Book /OfficinaSans-BookItalic /OfficinaSerif-Bold /OfficinaSerif-BoldItalic /OfficinaSerif-Book /OfficinaSerif-BookItalic /OnyxMT /Optima /Optima-Bold /Optima-BoldOblique /Optima-Oblique /PalaceScriptMT /PalaceScriptMT-SemiBold /Palatino-Black /Palatino-BlackItalic /Palatino-Bold /Palatino-BoldItalic /Palatino-BoldItalicOsF /Palatino-BoldOsF /Palatino-Italic /Palatino-ItalicOsF /Palatino-Light /Palatino-LightItalic /Palatino-Medium /Palatino-MediumItalic /Palatino-Roman /Revival565BT-Bold /Revival565BT-BoldItalic /Revival565BT-Italic /Revival565BT-Roman /RomanaBT-Bold /RomanaBT-Roman /Sabon-Bold /Sabon-BoldItalic /Sabon-BoldItalicOsF /Sabon-BoldOsF /Sabon-Italic /Sabon-ItalicOsF /Sabon-Roman /Sabon-RomanSC /SmileyFace /Sonata /StoneInformal /StoneInformal-Bold /StoneInformal-BoldItalic /StoneInformal-Italic /StoneInformal-Semibold /StoneInformal-SemiboldItalic /StoneSans /StoneSans-Bold /StoneSans-BoldItalic /StoneSans-Italic /StoneSans-Semibold /StoneSans-SemiboldItalic /StoneSerif /StoneSerif-Bold /StoneSerif-BoldItalic /StoneSerif-Italic /StoneSerif-Semibold /StoneSerif-SemiboldItalic /StoneSerifTn4 /Syntax-Black /Syntax-Bold /Syntax-Italic /Syntax-Roman /Syntax-UltraBlack /Techno /TimelessCyrillic-Bold /TimelessCyrillic-Light /TimelessCyrillic-LightIta /Times-Bold /Times-BoldItalic /Times-BoldItalicOsF /Times-BoldSC /Times-ExtraBold /Times-Italic /Times-ItalicOsF /Times-PhoneticAlternate /Times-PhoneticIPA /Times-Roman /Times-RomanSC /Times-Semibold /Times-SemiboldItalic /TimesEuropa-Bold /TimesEuropa-BoldItalic /TimesEuropa-Italic /TimesEuropa-Roman /TimesNewRomanMT-BoldCond /TimesNewRomanMT-Cond /TimesNewRomanMT-CondItalic /TimesNewRomanPS /TimesNewRomanPS-Bold /TimesNewRomanPS-BoldItalic /TimesNewRomanPS-Italic /ToonRoman /TorinoModern-Bold /TorinoModern-BoldItalic /TorinoModern-Italic /TorinoModern-Roman /Trajan-Bold /Trajan-Regular /Univers /Univers-Black /Univers-BlackExt /Univers-BlackExtObl /Univers-BlackOblique /Univers-BoldExt /Univers-BoldExtObl /Univers-BoldOblique /Univers-Condensed /Univers-CondensedBold /Univers-CondensedBoldOblique /Univers-CondensedLight /Univers-CondensedLightOblique /Univers-CondensedOblique /Univers-Extended /Univers-ExtendedObl /Univers-ExtraBlack /Univers-ExtraBlackExt /Univers-ExtraBlackExtObl /Univers-ExtraBlackObl /Univers-LightOblique /Univers-LightUltraCondensed /Univers-Oblique /UniversityRoman /VAGRounded-Black /VAGRounded-Bold /VAGRounded-Light /VAGRounded-Thin /WoodtypeOrnaments-One /WoodtypeOrnaments-Two ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 150 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages false /ColorImageDownsampleType /Average /ColorImageResolution 300 /ColorImageDepth 8 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /FlateEncode /AutoFilterColorImages false /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 150 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages false /GrayImageDownsampleType /Average /GrayImageResolution 300 /GrayImageDepth 8 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /FlateEncode /AutoFilterGrayImages false /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages false /MonoImageDownsampleType /Average /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName (http://www.color.org) /PDFXTrapped /Unknown /SyntheticBoldness 1.000000 /Description << /JPN /FRA /DEU /PTB /DAN /NLD /ESP /SUO /ITA /NOR /SVE /ENU (Use these settings to create PDF documents for RR Donnelley Book plants. The PDF documents can be opened with Acrobat and Reader 5.0 and later.) >> >> setdistillerparams << /HWResolution [2400 2400] /PageSize [612.000 792.000] >> setpagedevice