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Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro
Journal of Dentistry (2004) 32, 635–641
www.intl.elsevierhealth.com/journals/jden
Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro S.A. Teppera, M. Zehndera, G.F. Pajarolab, P.R. Schmidlina,* a
Department of Preventive Dentistry, Periodontology and Cariology, Dental Institute, University of Zurich, Plattenstrasse 11, Zurich 2028, Switzerland b Department of Oral and Maxillofacial Surgery, Dental Institute, University of Zurich, Plattenstrasse 11, Zurich 2028, Switzerland Received 16 September 2003; received in revised form 23 June 2004; accepted 24 June 2004
KEYWORDS CO2 laser; Fluoride uptake; Acid resistance; Enamel; SEM
Summary Objectives. The aim of the current in vitro study was to evaluate the effect of CO2-laser treatment immediately after applying amine fluoride solution on enamel. It was hypothesized that such a treatment would increase enamel fluoride uptake, and reduce dissolution rate and thermal surface alterations. Methods. Fluoride uptake was determined in 40 human enamel sections randomly assigned to four groups (nZ10), which were either left untreated (1), exposed to a 1% amine fluoride solution for 15 s without irradiation (2), irradiated for 15 s with a continuous-wave carbon dioxide laser (3), or laser-treated for 15 s through the amine fluoride solution applied immediately beforehand (4). Fluoride uptake was determined with an ion selective electrode after acid dissolution of the specimens (surface and subsurface layers). For the determination of acid resistance, another 40 enamel sections were treated according to the above protocol. Acid resistance was determined in surface and subsurface layers by measuring eluted calcium upon 3% lactic acid exposure with atomic absorption spectrometry. Enamel surface alterations after laser irradiation were monitored using scanning electron microscopy. Results. Laser irradiation through the fluoride solution led to significantly higher fluoride contents in the surface enamel layer compared to fluoride treatment alone or laser treatment alone (pZ0.002). Laser treatment with or without fluoride resulted in an increased acid resistance of enamel specimens. Fewer surface alterations were observed upon SEM examination of specimens irradiated through the amine fluoride solution compared to counterparts treated with laser only. Conclusions. CO2 laser light application through an amine fluoride solution may be useful and effective in the prevention of caries. q 2004 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: C41-1-634-08-46; fax: C41-1-634-43-08. E-mail address: [email protected] (P.R. Schmidlin). 0300-5712/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2004.06.010
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S.A. Tepper et al.
Introduction
Materials and methods
Many studies have been carried out since Stern and Sognnaes (1972) first suggested the use of laser irradiation to inhibit dental caries.1 Several investigations have demonstrated that treatment with various lasers can reduce the rate of subsurface demineralization in enamel.2–6 For this purpose, CO2 lasers appear to be most efficient.1 Different explanations for the increased acid-resistance of laser-treated enamel have been suggested, such as decreased enamel permeability,7,8 alterations in chemical composition,9–11 or a combination of both.6 CO2 laser irradiation of human dental enamel at 10.6 mm and continuous-wave mode led to significantly reduced initial dissolution rates.12 Similar results have been found when investigating the effect of additional fluoride application before or after laser treatment, leading to an increased fluoride uptake and decreased rate of dissolution in acidic solution.13,14 Various studies support findings of an increased fluoride uptake of lasertreated dental enamel.15,16 Instantaneous transformation of hydroxyapatite to fluorapatite by irradiation with a high-energy CO2 laser in the presence of amine fluoride has been demonstrated in vitro.17 While laser treatment in combination with fluoride application appears to have several advantages over fluoride application alone, some untoward effects are observed on enamel surfaces after laser treatment, such as surface cracking and melted areas.18,19 The aim of the present study was to evaluate the effect of high-energy CO 2 laser when irradiating through topically applied amine fluoride, in hope to find a regimen that would combine the advantages of both treatments and reduce untoward laser effects. Fluoride content of human enamel specimens irradiated with laser light through an amine fluoride solution was compared to enamel fluoride content after laser treatment alone or exposure to the amine fluoride solution alone. A second set of enamel specimens was used to test acid resistance of enamel after these treatments. In addition, surface alterations were studied using scanning electron microscopy. In contrast to other studies where laser irradiation was performed prior to or after topical fluoridation, the current study investigated a simultaneous application by irradiating through a layer of topically applied amine fluoride solution.
Tooth sample preparation and laser treatment Twenty extracted, non-carious human mandibular molars, stored in 0.1% thymol solution, were used for this study. All subjects enrolled in this research responded to an informed-consent protocol, which had been approved by the Ethics committee of the Dental Center of the University of Zu ¨rich. After removal of the root, teeth were cut in half longitudinally (disto-mesial) and in the bucco-oral direction. Four specimens were obtained from each tooth. These sections were comparable with respect to chemical composition of the enamel structure. Tooth sections were embedded in chemically-curing acrylic resin, and the enamel surface was cleaned by using a non-fluoridated polishing paste (Pellex, Hawe-Neos Dental, Bioggio, Switzerland) for 30 s with a standardized load of 200 g. Stereomicroscopic examination of the surface of all specimens was performed to ensure that there was no residual resin remaining on the surface area. The enamel specimens were randomly allocated to four test groups as follows: untreated control (Group 1), 15 s amine fluoride solution application alone (1%, Elmex fluid, GABA International AG, Basel, Switzerland) (Group 2), 15 s laser treatment alone (Group 3), and laser treatment performed through amine fluoride solution for 15 s (Group 4). Samples in Groups 3 and 4 were treated with a commercially available continuous-wave CO2 laser (Sharplan 15F, Lumenis Inc., Santa Clara, CA, USA). The laser light wavelength was 10.6 mm at an output of 2 W. The entire enamel surface of the samples was irradiated for 15 s by one calibrated dentist by moving the laser probe tip continuously at a standardized distance of 4 mm from the sample surface. Before and after laser treatment, a polyvinyl siloxane impression (President Plus Jet light body, Colte `ne AG, Altsta ¨tten, Switzerland) was made from each sample for SEM analysis of the enamel surface structure using a replica model (see below). Amine fluoride solution was applied immediately before starting the laser treatment. After treatment, all specimens were rinsed with deionized water for 30 min.
Evaluation of fluoride uptake Forty enamel specimens treated as described above (10 per group) were used for this experiment. For each of the 40 specimens, a circular enamel window with a diameter of 3 mm was created using
Increased fluoride uptake and acid resistance by CO2 laser-irradiation an acid-resistant adhesive tape. The adhesive tape was fixed to the specimen and its carrier with polyvinyl siloxane of low viscosity (President Plus Jet light body). For the determination of fluoride in the outer enamel surface, specimens were etched with 10 ml 2 M HCl for 5 s, and, in a second step, for another 10 s. This protocol allowed fluoride determination in the surface layer and in the underlying subsurface stratum. Acid and dissolved enamel components were collected using three blotting paper disks of standardized size, which were immediately transferred into 2 ml of TISAB solution (Total Ionic Strength Adjustment Buffer). For the determination of the fluoride concentration in these solutions, measurements were performed with an ion-selective electrode against blank measurements with paper disks only.20
Acid resistance Forty specimens (10 per group) treated as described above, were covered with adhesive tape with a 5 mm circular window. Samples were fixed on a shaker, and 120 ml of 0.005 M lactic acid, pH 3.0, was applied to each specimen. The lactic acid remained on the surface for 5 min under constant shaking. Subsequently, the acid and dissolved enamel components were removed with a pipette and collected in a test tube. Immediately after acid exposure, the sample surface was rinsed with 50 ml of deionized water, which was also added to the test tube, and the sample surface was dried.21 This procedure was repeated once to investigate enamel dissolution of two consecutive layers (surface and subsurface). Calcium content in these solutions was immediately measured using atomic absorption
637
spectroscopy at 422.7 nm. Phosphorous was masked with strontium chloride (0.25%).
Scanning electron microscopy (SEM) examination Using the polyvinyl siloxane impressions, replicas of the surfaces were cast and bonded to SEM mounts. The mounted replicas were gold-coated and analyzed using a scanning electron microscope (Amray 1810/T, Amray Inc. Bedford, USA) at low magnification (500!).
Statistical analysis For both test parameters, fluoride determination and evaluation of acid resistance, the subgroups were statistically analyzed using ANOVA. Individual post hoc comparisons were performed using a Student’s t-test followed by Bonferroni correction for multiple comparisons. Significance was set at 95% (p%0.05).
Results Fluoride uptake Determination of fluoride content of enamel surface layers revealed a statistically significant difference between the laser irradiation through amine fluoride solution regimen (Group 4) and the three other groups (pZ0.002, Fig. 1). No differences were found between the three controls (Groups 1–3). The fluoride content of the untreated control group was 531G131 ppm (mean valueG standard deviation), whereas laser treatment
Figure 1 Box plots depicting fluoride content in surface and subsurface layers after different treatments. (Box-plot explanation: horizontal bars: medians; boxes: inter-quartile areas; error bars: 10th and 90th percentile; dots: extreme values). NZ10 for each subgroup, ANOVA analysis and Bonferroni post hoc correction for multiple comparisons. Significant differences are marked with an asterisk.
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Figure 2 Determination of enamel dissolution after lactic acid exposure (pH 3) at 5 min per layer, expressed as micrograms of calcium. NZ10 for each subgroup, ANOVA analysis and Bonferroni post hoc correction for multiple comparisons. Significant differences are marked with an asterisk.
performed through amine fluoride solution resulted in 1064G198 ppm fluoride. A tendency of the laser combined with fluoride solution treatment to result in an elevated fluoride uptake compared to controls was also observed in the subsurface layer; however, these differences were not statistically significant.
cracking was markedly reduced. Few fused and melted enamel areas were observed, which were markedly smaller in diameter (Fig. 3).
Acid resistance
The current study suggested beneficial effects when enamel surfaces were wetted with a fluoride solution before they were laser-irradiated. The use of fluorides is one of the most effective methods in caries prophylaxis. Fluoride helps control decay by enhancing remineralization and altering the structure of the tooth, making the surface less soluble.22 Recent papers confirm that topical fluoride application is more effective in caries prevention than systemic fluoride application.23,24 A 20–40% caries-preventive effect of topical fluoride agents was shown in clinical studies.25 Several studies demonstrated the superiority of amine fluoride agents compared to inorganic fluoride solutions like sodium fluoride and SMFP.26 Chan and co-workers investigated the effect of fluoride uptake after topical fluoridation of sound enamel with an 1% amine fluoride solution in vitro.27 After an application time of 3 min, fluoride concentration reached a mean value of 1062G 59 ppm in the superficial 2.5 mm. Mean fluoride uptake was calculated to be 622G288 ppm within the most superficial 5 mm. These findings are in line with the results of the present study. The current study suggests that, with respect to the surface enamel layer, laser treatment significantly increases fluoride uptake when irradiated through topically applied amine fluoride. Several combinations of different laser types and topically applied fluorides have been tested in comparable laboratory studies.28,29 Irrespective of laser type and fluoride combination used, results showed an
Enamel acid resistance was measured by assaying the amount of dissolved calcium from enamel specimens upon exposure to lactic acid. In the surface layer, a statistically significant difference between untreated controls (9.6G1.3 mg of dissolved calcium per specimen) and the laser-treated groups, with or without topical amine fluoride application (7.9G1.0 and 7.0G1.1 mg, respectively) was observed (pZ0.002, Fig. 2). Fluoride treatment alone also significantly (pZ0.004) increased acid resistance. No significant differences were found between test groups (Group 2–4). Results of the subsurface layer revealed statistically significant differences in mean values between both laser-treated test groups and untreated controls (pZ0.002). In this layer, Fluoride treatment alone did not significantly increase acid-resistance (pZ0.012).
Scanning electron microscopy (SEM) examination Evaluation of micromorphological surface changes after laser treatment at a magnification of 500! showed a pattern of microscopic mosaic cracks and isolated spots where the enamel was melted, in contrast to the smooth and sound enamel surfaces in the control groups without laser irradiation. When amine fluoride solution was applied immediately prior to laser treatment, enamel surface
Discussion
Increased fluoride uptake and acid resistance by CO2 laser-irradiation
639
Figure 3 Representative SEM micrographs at a magnification of 500!, showing enamel surface alterations after different treatment modalities. Panel A: untreated smooth surface (Group 1). Panels B and C: laser irradiation for 15 s (Group 3) resulting in a pattern of mosaic cracks and extensive melting areas. Panel D: laser-treatment through amine fluoride solution for 15 s, showing less surface cracks and smaller melted areas (arrow) compared to laser treatment of dry surface (Panels B, C).
increased resistance against caries formation when compared to laser irradiation alone. Crystalline phase transformation,17 other changes in chemical composition,10,12 and reduced acid permeability caused by surface alterations (i.e. fusion and melting of crystallites)4,6 are discussed as fundamental principles for reduced acid solubility of dental enamel after laser treatment. Laboratory studies have shown that the critical pH for enamel dissolution (pH 5.5) is lowered considerably by laser irradiation (pH 4.8). In the presence of as little as 0.1 ppm fluoride, lased enamel will not undergo dissolution until a critical pH of 4.3 is reached.30,31 A significant synergism between laser and fluoride in the reduction of enamel solubility was confirmed in the present study. Using synthetic hydroxyapatite, it has been shown that CO2 laser treatment could transform hydroxyapatite into fluorapatite in the presence of fluoride.17 However, it remains uncertain as to whether the firmly-bound or the loosely-bound fluoride plays the major role in the laser-induced increase of fluoride uptake. Superficially located fluoride may be lost again in vivo by back exchange, back diffusion, and migration from the mineral to the surrounding tissue fluid, saliva, or plaque fluid;32 such possible in vivo phenomena could not be evaluated in the present study. Surface cracking and melted areas after laser treatment, as observed by SEM in the current investigation, correlate well with findings by other
authors.18,19 The laser-treated specimens that received irradiation through amine fluoride solution showed less surface cracking on SEM images. This may be caused by a cooling effect of the amine fluoride solution due to the heat absorbing capacity of liquids. In another in vitro study, enamel was treated with a 2% NaF topical fluoride.33 After 4 min, fluoride was wiped off with tissue papers and teeth were irradiated with a CO2 laser. This combination of laser and fluoride treatment was found to induce more surface alterations than the laser alone. While not tested in the present study, it may be expected that simultaneous use of fluorides and laser irradiation result in less surface alterations than both treatments applied separately. Effects of laser irradiation on the pulp were not investigated in the present study; previous studies however suggested that the irradiation conditions applied here are safe.34,35 In the current investigation the following settings were used: laser light wavelength of 10.6 mm at an output of 2 W with an irradiation distance of 4 mm to the sample and an irradiation time of 15 s. Anic and co-workers demonstrated that the CO2 laser at 4 W continuous wave for 15 s caused a temperature raise of 3.5–4.1 8C in the pulp space. No change in temperature was recorded when 0.5–1 W was used for 10 s. Future studies should focus on combined laserfluoride treatment of incipient carious lesions to investigate whether lesion progression can be
640 influenced. Different fluoride preparations, concentrations and viscosities should also be tested in combination with different laser settings or types to evaluate and establish optimal prophylactic effects.
Conclusions The current study showed a beneficial effect of combining enamel laser irradiation with topically applied amine fluoride, resulting in high fluoride uptake and acid resistance, combined with decreased structural enamel disintegration. Laser irradiation of enamel through a topically applied amine fluoride solution may thus be useful and effective in the prophylaxis and management of patients at risk for dental caries. In vivo studies are necessary to confirm or disprove this hypothesis.
Acknowledgements The authors wish to thank Dr G. Menghini, Department of Preventive Dentistry and Oral Epidemiology, for performing the statistical analysis.
References 1. Stern RH, Sognnaes RF. Laser inhibition of dental caries suggested by first tests in vivo. Journal of the American Dental Association 1972;85:1087–90. 2. Yamamoto H, Ooya K. Potential of yttrium–aluminum–garnet laser in caries prevention. Journal of Oral Pathology 1974;3: 7–15. 3. Yamamoto H, Sato K. Prevention of dental caries by acoustooptically Q-switched Nd: YAG laser irradiation. Journal of Dental Research 1980;59:137. 4. Stern RH, Vahl J, Sognnaes RF. Lased enamel: ultrastructural observations of pulsed carbon dioxide laser effects. Journal of Dental Research 1972;51:455–60. 5. Nelson DG, Shariati M, Glena R, Shields CP, Featherstone JD. Effect of pulsed low energy infrared laser irradiation on artificial caries-like lesion formation. Caries Research 1986; 20:289–99. 6. Nelson DG, Wefel JS, Jongebloed WL, Featherstone JD. Morphology, histology and crystallography of human dental enamel treated with pulsed low-energy infrared laser radiation. Caries Research 1987;21:411–26. 7. Stern RH, Sognnaes RF, Goodman F. Laser effect on in vitro enamel permeability and solubility. Journal of the American Dental Association 1966;73:838–43. 8. Lenz P, Glide H, Walz R. Studies on enamel sealing with the ¨rztliche Zeitschrift 1982;37: CO2 laser. Deutsche Zahna 469–78. 9. Kuroda S, Fowler BO. Compositional, structural, and phase changes in in vitro laser-irradiated human tooth enamel. Calcified Tissue International 1984;36:361–9.
S.A. Tepper et al. 10. Fowler BO, Kuroda S. Changes in heated and in laserirradiated human tooth enamel and their probable effects on solubility. Calcified Tissue International 1986;38: 197–208. 11. Featherstone JD, Nelson DG. Laser effects on dental hard tissues. Advances in Dental Research 1987;1:21–6. 12. Fox JL, Yu D, Otsuka M, Higuchi WI, Wong J, Powell GL. Initial dissolution rate studies on dental enamel after CO2 laser irradiation. Journal of Dental Research 1992;71: 1389–98. 13. Tagomori S, Morioka T. Combined effects of laser and fluoride on acid resistance of human dental enamel. Caries Research 1989;23:225–31. 14. Hossain MM, Hossain M, Kimura Y, Kinoshita J, Yamada Y, Matsumoto K. Acquired acid resistance of enamel and dentin by CO2 laser irradiation with sodium fluoride solution. Journal of Clinical Laser Medicine and Surgery 2002;20: 77–82. 15. Goodman BD, Kaufman HW. Effects of an argon laser on the crystalline properties and rate of dissolution in acid of tooth enamel in the presence of sodium fluoride. Journal of Dental Research 1977;56:1201–7. 16. Bahar A, Tagomori S. The effect of normal pulsed Nd-YAG laser irradiation on pits and fissures in human teeth. Caries Research 1994;28:460–7. 17. Meurman JH, Hemmerle J, Voegel JC, Rauhamaa-Makinen R, Luomanen M. Transformation of hydroxyapatite to fluorapatite by irradiation with high-energy CO2 laser. Caries Research 1997;31:397–400. 18. McCormack SM, Fried D, Featherstone JD, Glena RE, Seka W. Scanning electron microscope observations of CO2 laser effects on dental enamel. Journal of Dental Research 1995; 74:1702–8. 19. Ferreira JM, Palamara J, Phakey PP, Rachinger WA, Orams HJ. Effects of continuous-wave CO2 laser on the ultrastructure of human dental enamel. Archives of Oral Biology 1989;34:551–62. 20. Kissa E. Determination of fluoride low concentrations with the ion selective electrode. Annals of Chemistry 1983;55: 1445–9. 21. Grobler SR, Senekal PJ, Laubscher JA. In vitro demineralization of enamel by orange juice, apple juice, Pepsi Cola and Diet Pepsi Cola. Clinical Preventive Dentistry 1990;12:5–9. 22. Fejerskov O, Clarkson BH. Dynamics of caries lesion formation. In: Fejerskov O, Ekstrand J, Burt BA, editors. Fluorides in dentistry. Copenhagen: Munksgaard; 1996. p. 187–214. 23. Bratthall D, Hansel-Petersson G, Sundberg H. Reasons for the caries decline: what do experts believe? European Journal of Oral Science 1996;104:416–22. 24. Clarkson JJ. International collaborative research on fluoride. Journal of Dental Research 2000;79:893–904. 25. Ripa LW. An evaluation of the use of professional (operatorapplied) topical fluorides. Journal of Dental Research 1990; 69:786–96. 26. Marks RG, Conti AJ, Moorhead JE, Cancro L, D’Agostino RB. Results from a three-year caries clinical trial comparing NaF and SMFP fluoride formulations. International Dental Journal 1994;44:275–85. 27. Chan JC, Hill FJ, Newman HN. Uptake of fluoride by sound and artificially carious enamel in vitro following application of topical sodium and amine fluorides. Journal of Dentistry 1991;19:110–5. 28. Flaitz CM, Hicks MJ, Westerman GH, Berg JH, Blankenau RJ, Powell GL. Argon laser irradiation and acidulated phosphate fluoride treatment in caries-like lesion formation in enamel: an in vitro study. Pediatric Dentistry 1995;17:31–5.
Increased fluoride uptake and acid resistance by CO2 laser-irradiation 29. Hsu J, Fox JL, Wang Z, Powell GL, Otsuka M, Higuchi WI. Combined effects of laser irradiation/solution fluoride ion on enamel demineralization. Journal of Clinical Laser Medicine and Surgery 1998;16:93–105. 30. Fox JL, Yu D, Otsuka M, Higuchi WI, Wong J, Powell G. Combined effects of laser irradiation and chemical inhibitors on the dissolution of dental enamel. Caries Research 1992; 26:333–9. 31. Fox JL, Yu D, Otsuka M, Higuchi WI, Wong J, Powell G. Initial dissolution rate studies on dental enamel after CO2 laser irradiation. Journal of Dental Research 1992;71: 1389–98.
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32. Robinson C, Kirkham J, Weatherell JA. Fluoride in teeth and bone. In: Fejerskov O, Ekstrand J, Burt BA, editors. Fluorides in dentistry. Copenhagen: Munksgaard; 1996. p. 69–87. 33. Chin-Ying SH, Xiaoli G, Jisheng P, Wefel JS. Effects of CO2 laser on fluoride uptake in enamel. Journal of Dentistry 2004;32:161–7. 34. Anic I, Vidovic D, Luic M, Tudja M. Laser induced molar tooth pulp chamber temperature changes. Caries Research 1992; 26:165–9. 35. Yu D, Powell GL, Higuchi WI, Fox JL. Comparison of three lasers on dental pulp chamber temperature change. Journal of Clinical Lasers Medicine and Surgery 1993;11:119–22.
www.intl.elsevierhealth.com/journals/jden
Increased fluoride uptake and acid resistance by CO2 laser-irradiation through topically applied fluoride on human enamel in vitro S.A. Teppera, M. Zehndera, G.F. Pajarolab, P.R. Schmidlina,* a
Department of Preventive Dentistry, Periodontology and Cariology, Dental Institute, University of Zurich, Plattenstrasse 11, Zurich 2028, Switzerland b Department of Oral and Maxillofacial Surgery, Dental Institute, University of Zurich, Plattenstrasse 11, Zurich 2028, Switzerland Received 16 September 2003; received in revised form 23 June 2004; accepted 24 June 2004
KEYWORDS CO2 laser; Fluoride uptake; Acid resistance; Enamel; SEM
Summary Objectives. The aim of the current in vitro study was to evaluate the effect of CO2-laser treatment immediately after applying amine fluoride solution on enamel. It was hypothesized that such a treatment would increase enamel fluoride uptake, and reduce dissolution rate and thermal surface alterations. Methods. Fluoride uptake was determined in 40 human enamel sections randomly assigned to four groups (nZ10), which were either left untreated (1), exposed to a 1% amine fluoride solution for 15 s without irradiation (2), irradiated for 15 s with a continuous-wave carbon dioxide laser (3), or laser-treated for 15 s through the amine fluoride solution applied immediately beforehand (4). Fluoride uptake was determined with an ion selective electrode after acid dissolution of the specimens (surface and subsurface layers). For the determination of acid resistance, another 40 enamel sections were treated according to the above protocol. Acid resistance was determined in surface and subsurface layers by measuring eluted calcium upon 3% lactic acid exposure with atomic absorption spectrometry. Enamel surface alterations after laser irradiation were monitored using scanning electron microscopy. Results. Laser irradiation through the fluoride solution led to significantly higher fluoride contents in the surface enamel layer compared to fluoride treatment alone or laser treatment alone (pZ0.002). Laser treatment with or without fluoride resulted in an increased acid resistance of enamel specimens. Fewer surface alterations were observed upon SEM examination of specimens irradiated through the amine fluoride solution compared to counterparts treated with laser only. Conclusions. CO2 laser light application through an amine fluoride solution may be useful and effective in the prevention of caries. q 2004 Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: C41-1-634-08-46; fax: C41-1-634-43-08. E-mail address: [email protected] (P.R. Schmidlin). 0300-5712/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2004.06.010
636
S.A. Tepper et al.
Introduction
Materials and methods
Many studies have been carried out since Stern and Sognnaes (1972) first suggested the use of laser irradiation to inhibit dental caries.1 Several investigations have demonstrated that treatment with various lasers can reduce the rate of subsurface demineralization in enamel.2–6 For this purpose, CO2 lasers appear to be most efficient.1 Different explanations for the increased acid-resistance of laser-treated enamel have been suggested, such as decreased enamel permeability,7,8 alterations in chemical composition,9–11 or a combination of both.6 CO2 laser irradiation of human dental enamel at 10.6 mm and continuous-wave mode led to significantly reduced initial dissolution rates.12 Similar results have been found when investigating the effect of additional fluoride application before or after laser treatment, leading to an increased fluoride uptake and decreased rate of dissolution in acidic solution.13,14 Various studies support findings of an increased fluoride uptake of lasertreated dental enamel.15,16 Instantaneous transformation of hydroxyapatite to fluorapatite by irradiation with a high-energy CO2 laser in the presence of amine fluoride has been demonstrated in vitro.17 While laser treatment in combination with fluoride application appears to have several advantages over fluoride application alone, some untoward effects are observed on enamel surfaces after laser treatment, such as surface cracking and melted areas.18,19 The aim of the present study was to evaluate the effect of high-energy CO 2 laser when irradiating through topically applied amine fluoride, in hope to find a regimen that would combine the advantages of both treatments and reduce untoward laser effects. Fluoride content of human enamel specimens irradiated with laser light through an amine fluoride solution was compared to enamel fluoride content after laser treatment alone or exposure to the amine fluoride solution alone. A second set of enamel specimens was used to test acid resistance of enamel after these treatments. In addition, surface alterations were studied using scanning electron microscopy. In contrast to other studies where laser irradiation was performed prior to or after topical fluoridation, the current study investigated a simultaneous application by irradiating through a layer of topically applied amine fluoride solution.
Tooth sample preparation and laser treatment Twenty extracted, non-carious human mandibular molars, stored in 0.1% thymol solution, were used for this study. All subjects enrolled in this research responded to an informed-consent protocol, which had been approved by the Ethics committee of the Dental Center of the University of Zu ¨rich. After removal of the root, teeth were cut in half longitudinally (disto-mesial) and in the bucco-oral direction. Four specimens were obtained from each tooth. These sections were comparable with respect to chemical composition of the enamel structure. Tooth sections were embedded in chemically-curing acrylic resin, and the enamel surface was cleaned by using a non-fluoridated polishing paste (Pellex, Hawe-Neos Dental, Bioggio, Switzerland) for 30 s with a standardized load of 200 g. Stereomicroscopic examination of the surface of all specimens was performed to ensure that there was no residual resin remaining on the surface area. The enamel specimens were randomly allocated to four test groups as follows: untreated control (Group 1), 15 s amine fluoride solution application alone (1%, Elmex fluid, GABA International AG, Basel, Switzerland) (Group 2), 15 s laser treatment alone (Group 3), and laser treatment performed through amine fluoride solution for 15 s (Group 4). Samples in Groups 3 and 4 were treated with a commercially available continuous-wave CO2 laser (Sharplan 15F, Lumenis Inc., Santa Clara, CA, USA). The laser light wavelength was 10.6 mm at an output of 2 W. The entire enamel surface of the samples was irradiated for 15 s by one calibrated dentist by moving the laser probe tip continuously at a standardized distance of 4 mm from the sample surface. Before and after laser treatment, a polyvinyl siloxane impression (President Plus Jet light body, Colte `ne AG, Altsta ¨tten, Switzerland) was made from each sample for SEM analysis of the enamel surface structure using a replica model (see below). Amine fluoride solution was applied immediately before starting the laser treatment. After treatment, all specimens were rinsed with deionized water for 30 min.
Evaluation of fluoride uptake Forty enamel specimens treated as described above (10 per group) were used for this experiment. For each of the 40 specimens, a circular enamel window with a diameter of 3 mm was created using
Increased fluoride uptake and acid resistance by CO2 laser-irradiation an acid-resistant adhesive tape. The adhesive tape was fixed to the specimen and its carrier with polyvinyl siloxane of low viscosity (President Plus Jet light body). For the determination of fluoride in the outer enamel surface, specimens were etched with 10 ml 2 M HCl for 5 s, and, in a second step, for another 10 s. This protocol allowed fluoride determination in the surface layer and in the underlying subsurface stratum. Acid and dissolved enamel components were collected using three blotting paper disks of standardized size, which were immediately transferred into 2 ml of TISAB solution (Total Ionic Strength Adjustment Buffer). For the determination of the fluoride concentration in these solutions, measurements were performed with an ion-selective electrode against blank measurements with paper disks only.20
Acid resistance Forty specimens (10 per group) treated as described above, were covered with adhesive tape with a 5 mm circular window. Samples were fixed on a shaker, and 120 ml of 0.005 M lactic acid, pH 3.0, was applied to each specimen. The lactic acid remained on the surface for 5 min under constant shaking. Subsequently, the acid and dissolved enamel components were removed with a pipette and collected in a test tube. Immediately after acid exposure, the sample surface was rinsed with 50 ml of deionized water, which was also added to the test tube, and the sample surface was dried.21 This procedure was repeated once to investigate enamel dissolution of two consecutive layers (surface and subsurface). Calcium content in these solutions was immediately measured using atomic absorption
637
spectroscopy at 422.7 nm. Phosphorous was masked with strontium chloride (0.25%).
Scanning electron microscopy (SEM) examination Using the polyvinyl siloxane impressions, replicas of the surfaces were cast and bonded to SEM mounts. The mounted replicas were gold-coated and analyzed using a scanning electron microscope (Amray 1810/T, Amray Inc. Bedford, USA) at low magnification (500!).
Statistical analysis For both test parameters, fluoride determination and evaluation of acid resistance, the subgroups were statistically analyzed using ANOVA. Individual post hoc comparisons were performed using a Student’s t-test followed by Bonferroni correction for multiple comparisons. Significance was set at 95% (p%0.05).
Results Fluoride uptake Determination of fluoride content of enamel surface layers revealed a statistically significant difference between the laser irradiation through amine fluoride solution regimen (Group 4) and the three other groups (pZ0.002, Fig. 1). No differences were found between the three controls (Groups 1–3). The fluoride content of the untreated control group was 531G131 ppm (mean valueG standard deviation), whereas laser treatment
Figure 1 Box plots depicting fluoride content in surface and subsurface layers after different treatments. (Box-plot explanation: horizontal bars: medians; boxes: inter-quartile areas; error bars: 10th and 90th percentile; dots: extreme values). NZ10 for each subgroup, ANOVA analysis and Bonferroni post hoc correction for multiple comparisons. Significant differences are marked with an asterisk.
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Figure 2 Determination of enamel dissolution after lactic acid exposure (pH 3) at 5 min per layer, expressed as micrograms of calcium. NZ10 for each subgroup, ANOVA analysis and Bonferroni post hoc correction for multiple comparisons. Significant differences are marked with an asterisk.
performed through amine fluoride solution resulted in 1064G198 ppm fluoride. A tendency of the laser combined with fluoride solution treatment to result in an elevated fluoride uptake compared to controls was also observed in the subsurface layer; however, these differences were not statistically significant.
cracking was markedly reduced. Few fused and melted enamel areas were observed, which were markedly smaller in diameter (Fig. 3).
Acid resistance
The current study suggested beneficial effects when enamel surfaces were wetted with a fluoride solution before they were laser-irradiated. The use of fluorides is one of the most effective methods in caries prophylaxis. Fluoride helps control decay by enhancing remineralization and altering the structure of the tooth, making the surface less soluble.22 Recent papers confirm that topical fluoride application is more effective in caries prevention than systemic fluoride application.23,24 A 20–40% caries-preventive effect of topical fluoride agents was shown in clinical studies.25 Several studies demonstrated the superiority of amine fluoride agents compared to inorganic fluoride solutions like sodium fluoride and SMFP.26 Chan and co-workers investigated the effect of fluoride uptake after topical fluoridation of sound enamel with an 1% amine fluoride solution in vitro.27 After an application time of 3 min, fluoride concentration reached a mean value of 1062G 59 ppm in the superficial 2.5 mm. Mean fluoride uptake was calculated to be 622G288 ppm within the most superficial 5 mm. These findings are in line with the results of the present study. The current study suggests that, with respect to the surface enamel layer, laser treatment significantly increases fluoride uptake when irradiated through topically applied amine fluoride. Several combinations of different laser types and topically applied fluorides have been tested in comparable laboratory studies.28,29 Irrespective of laser type and fluoride combination used, results showed an
Enamel acid resistance was measured by assaying the amount of dissolved calcium from enamel specimens upon exposure to lactic acid. In the surface layer, a statistically significant difference between untreated controls (9.6G1.3 mg of dissolved calcium per specimen) and the laser-treated groups, with or without topical amine fluoride application (7.9G1.0 and 7.0G1.1 mg, respectively) was observed (pZ0.002, Fig. 2). Fluoride treatment alone also significantly (pZ0.004) increased acid resistance. No significant differences were found between test groups (Group 2–4). Results of the subsurface layer revealed statistically significant differences in mean values between both laser-treated test groups and untreated controls (pZ0.002). In this layer, Fluoride treatment alone did not significantly increase acid-resistance (pZ0.012).
Scanning electron microscopy (SEM) examination Evaluation of micromorphological surface changes after laser treatment at a magnification of 500! showed a pattern of microscopic mosaic cracks and isolated spots where the enamel was melted, in contrast to the smooth and sound enamel surfaces in the control groups without laser irradiation. When amine fluoride solution was applied immediately prior to laser treatment, enamel surface
Discussion
Increased fluoride uptake and acid resistance by CO2 laser-irradiation
639
Figure 3 Representative SEM micrographs at a magnification of 500!, showing enamel surface alterations after different treatment modalities. Panel A: untreated smooth surface (Group 1). Panels B and C: laser irradiation for 15 s (Group 3) resulting in a pattern of mosaic cracks and extensive melting areas. Panel D: laser-treatment through amine fluoride solution for 15 s, showing less surface cracks and smaller melted areas (arrow) compared to laser treatment of dry surface (Panels B, C).
increased resistance against caries formation when compared to laser irradiation alone. Crystalline phase transformation,17 other changes in chemical composition,10,12 and reduced acid permeability caused by surface alterations (i.e. fusion and melting of crystallites)4,6 are discussed as fundamental principles for reduced acid solubility of dental enamel after laser treatment. Laboratory studies have shown that the critical pH for enamel dissolution (pH 5.5) is lowered considerably by laser irradiation (pH 4.8). In the presence of as little as 0.1 ppm fluoride, lased enamel will not undergo dissolution until a critical pH of 4.3 is reached.30,31 A significant synergism between laser and fluoride in the reduction of enamel solubility was confirmed in the present study. Using synthetic hydroxyapatite, it has been shown that CO2 laser treatment could transform hydroxyapatite into fluorapatite in the presence of fluoride.17 However, it remains uncertain as to whether the firmly-bound or the loosely-bound fluoride plays the major role in the laser-induced increase of fluoride uptake. Superficially located fluoride may be lost again in vivo by back exchange, back diffusion, and migration from the mineral to the surrounding tissue fluid, saliva, or plaque fluid;32 such possible in vivo phenomena could not be evaluated in the present study. Surface cracking and melted areas after laser treatment, as observed by SEM in the current investigation, correlate well with findings by other
authors.18,19 The laser-treated specimens that received irradiation through amine fluoride solution showed less surface cracking on SEM images. This may be caused by a cooling effect of the amine fluoride solution due to the heat absorbing capacity of liquids. In another in vitro study, enamel was treated with a 2% NaF topical fluoride.33 After 4 min, fluoride was wiped off with tissue papers and teeth were irradiated with a CO2 laser. This combination of laser and fluoride treatment was found to induce more surface alterations than the laser alone. While not tested in the present study, it may be expected that simultaneous use of fluorides and laser irradiation result in less surface alterations than both treatments applied separately. Effects of laser irradiation on the pulp were not investigated in the present study; previous studies however suggested that the irradiation conditions applied here are safe.34,35 In the current investigation the following settings were used: laser light wavelength of 10.6 mm at an output of 2 W with an irradiation distance of 4 mm to the sample and an irradiation time of 15 s. Anic and co-workers demonstrated that the CO2 laser at 4 W continuous wave for 15 s caused a temperature raise of 3.5–4.1 8C in the pulp space. No change in temperature was recorded when 0.5–1 W was used for 10 s. Future studies should focus on combined laserfluoride treatment of incipient carious lesions to investigate whether lesion progression can be
640 influenced. Different fluoride preparations, concentrations and viscosities should also be tested in combination with different laser settings or types to evaluate and establish optimal prophylactic effects.
Conclusions The current study showed a beneficial effect of combining enamel laser irradiation with topically applied amine fluoride, resulting in high fluoride uptake and acid resistance, combined with decreased structural enamel disintegration. Laser irradiation of enamel through a topically applied amine fluoride solution may thus be useful and effective in the prophylaxis and management of patients at risk for dental caries. In vivo studies are necessary to confirm or disprove this hypothesis.
Acknowledgements The authors wish to thank Dr G. Menghini, Department of Preventive Dentistry and Oral Epidemiology, for performing the statistical analysis.
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