ADRF RESEARCH REPORT Australian Dental Journal 2007;52:(2):101-105 Laser-activated fluoride treatment of enamel against an artificial caries challenge: comparison of five wavelengths J Vlacic,* IA Meyers, J Kim, LJ Walsh Abstract Background: Laser-activated fluoride (LAF) therapy with 488nm laser energy has been shown previously to increase the resistance of human enamel and dentine to acid dissolution in laboratory models of dental caries. The aims of this study were to examine whether LAF therapy, conducted using a range of wavelengths in the visible and near infrared regions, can protect human dental enamel from an artificial cariogenic challenge. Materials and methods: Buccal and lingual surfaces of extracted sound, molar and premolar teeth were used to prepare matched pairs of enamel slabs (N=10 per group). After application of neutral sodium fluoride gel (12300ppm F ion), slab surfaces were lased (energy density 15 J/cm 2 ; spot size 5mm, wavelength 532, 633, 670, 830 or 1064nm), then exposed to an artificial cariogenic challenge for a period of seven days. The Vicker s hardness number (VHN) was recorded before and after laser treatment and again following the cariogenic challenge. Negative controls did not receive laser exposure. Results: All wavelengths of laser light examined provided an effective LAF effect, compared with the unlased negative control surfaces. Conclusion: Using this in vitro model, we conclude that the action spectrum of the LAF effect extends across the visible and near-infrared regions of the spectrum. Key words: Caries, enamel, laser, fluoride. Abbreviations and acronyms: LAF = laser-activated fluoride; VHN = Vicker s hardness number. (Accepted for publication 6 September 2006.) *PhD Scholar, School of Dentistry, The University of Queensland, Brisbane. Professor of General Dental Practice, School of Dentistry, The University of Queensland, Brisbane. School of Dentistry, The University of Queensland, Brisbane. Professor of Dental Science, School of Dentistry, and Programme Director, Centre for Biophotonics and Laser Science, The University of Queensland, Brisbane. INTRODUCTION Dental caries can be defined as the localized destruction of tissues of the tooth by bacterial fermentation of dietary carbohydrates. 1 The process of fermentation by cariogenic micro-organisms generates lactic, pyruvic, formic, acetic and other organic acids which demineralize enamel (and subsequently dentine) during the caries process. Since Sognnaes and Stern 2 first demonstrated the effect of laser irradiation on enamel resistance to demineralization, numerous studies have examined the process by which laser energy, either alone or in combination with topical fluoride therapies, can increase the resistance of tooth structure to mineral loss from the organic acids involved in dental caries. 3-17 Two key points have been established by this body of research work. First, the protection provided is against both caries initiation and caries progression. 18 Second, the greatest protection is provided through the combination of laser irradiation of enamel in combination with topical fluoride application, a technique termed laser-activated fluoride (LAF) therapy. Laser irradiation alone has been shown to reduce the critical ph at which enamel dissolution occurs from 5.5 to 4.31. However, this critical ph is further reduced in the presence of fluoride in concentrations as low as 0.01 ppm. 14 Through such a reduction in the critical ph of enamel, LAF therapy may protect tooth structure from cariogenic challenges. 3,5,9,13,15,16,19-22 Previous studies of LAF have employed either the argon ion, carbon dioxide or Nd:YAG lasers, of which the 488nm laser wavelength from the argon ion laser appears to be the most effective. The aims of this study were to examine the action spectrum of the LAF effect using a more extensive range of laser wavelengths across the visible and near infrared regions, in conjunction with an in vitro model of dental caries. As softening of enamel is a clinical feature of active caries, we used microhardness measurements to assess the extent of protection afforded by LAF therapy. Australian Dental Journal 2007;52:2. 101
Table 1. Laser equipment and parameters Configuration Model and manufacturer Wavelength Power Exposure Power density (nm) (mw) time (sec) (mw/cm 2 ) KTP (Frequency doubled Nd:YAG) Nuvolase Smartbleach, ARC, Belgium 532 100 30 510 InGaAsP diode laser SaveDent, Denfotex, UK 633 50 60 255 InGaAs diode laser Omnilase, Laserdyne, Australia 670 10 300 51 GaAs diode laser Omnilase, Laserdyne, Australia 830 60 50 36 Nd:YAG dlase300, Sunrise Technologies, USA 1064 30 10 153 MATERIALS AND METHODS Enamel slabs were prepared from sound extracted human molar and premolar teeth. After debridement of gingival soft tissue remnants and prophylaxis with a fluoride-free paste, the lingual and buccal surfaces of the teeth were sectioned into slabs using a diamond saw. The surfaces of the slabs were polished with 1200 grit silicon carbide paper, and the prepared samples stored in a humidor at room temperature until used. The slabs were allocated randomly into treatment groups (N=10 per group, with the group size determined by power analysis) and each divided into two halves to provide matched pairs. The baseline Vicker s hardness number (VHN) of each surface was determined using a mini-load hardness tester (Ernst Leitz). The test and control surfaces of every slab in every group were measured twice to determine an average. To ensure protection from fluoride exposure, the control and the treatment sides were separated by a clear nail varnish, and the control covered with an opaque shield to prevent exposure to laser light. The test side was covered with 100µL of neutral sodium fluoride gel (Colgate NeutraFluor; 12300ppm F ion) followed immediately by laser activation of the fluoride gel (energy density 15 J/cm 2 for all samples). Past studies of LAF have used either the APF gel or neutral sodium fluoride gel, generating similar results. However, for the present study neutral sodium fluoride gel was chosen to avoid possible effects of APF gel on enamel microhardness measurements. Table 1 specifies the various parameters used with each laser, such as wavelength, power, exposure time and power density. An energy density of 15 J/cm 2 was used with all wavelengths since this was shown to be the optimal value for laser fluence for activation of fluoride in previous studies. 3-6,8 The spot size used was 5mm for all lasers. This was established for each laser system using a custom-made holder to ensure a constant distance from the distal end of the optical fibre to the target area on the slab, to achieve this constant spot size. All lasers were used in continuous wave mode except the Nd:YAG laser, which could only be operated in free-running pulsed mode. Immediately after laser treatment, the fluoride gel was rinsed from the enamel surfaces with deionized water and the surface hardness of all treated surface remeasured twice with the mini-load hardness tester. The whole slab was placed in a 70mL sterile specimen container and immersed under 40mL of acetate demineralizing buffer. 23 The containers were then placed in an incubator at 37 C for a period of seven days, with no disturbance to the solution. A pilot study showed that an incubation period of seven days was optimal for the creation of a clinically visible incipient caries-like (white spot) lesion. An incubation period of less than seven days was not sufficient to create a visible lesion, while a period of more than seven days resulted in extensive damage to the tooth surface. Following the seven-day incubation period, the VHN of both the control and the test surfaces, of every slab for all the groups, was re-measured twice and the average determined. Means were calculated, and differences between groups assessed using one sample paired t-tests, since a normal distribution for each of the data sets was established on the basis of the skewness normality, the kurtosis normality and omnibus normality. To compare the effects of different wavelengths (baseline versus final hardness after acetate buffer challenge), one-way ANOVA was used with the Tukey-Kramer method for multiple comparisons. In all cases, both variance and normality assumptions were met. A value of = 0.05 was used as the level for significance. RESULTS Control groups The control slabs subjected to the demineralizing solution without protection from LAF therapy demonstrated, as expected, a marked reduction in hardness (Table 2, Figs 1-5). Effect of LAF prior to demineralization challenge When comparing the VHN measurements before and after lasing (Table 3), only the 830nm wavelength showed a small (but statistically significant) change in enamel surface hardness (P=0.015). In vitro demineralization challenge Prior to LAF treatment and the demineralization challenge there was no statistically significant Table 2. Reduction in Vicker s hardness values for the five control groups Laser type P value (one-tail t-test) KTP 0.000169 InGaAsP diode laser 0.000367 InGaAs diode laser 0.000072 GaAs diode laser 0.003365 Nd:YAG 0.000744 102 Australian Dental Journal 2007;52:2.
Fig 1. Data for 532nm (KTP laser) control and treatment surfaces. Datasets for each slab are, in sequence, control surface at baseline, control surface after demineralization challenge, test surface at baseline, test surface after LAF and test surface after demineralization challenge. The same sequence is used in the subsequent figures. Fig 4. Data for 830nm (GaAs diode laser) control and treatment Fig 5. Data for 1064nm (Nd:YAG laser) control and treatment Fig 2. Data for 633nm (InGaAsP diode laser) control and treatment Table 3. Statistical analysis of Vicker s hardness before and immediately after LAF treatment Laser type Wavelength (nm) Probability level KTP 532 0.130 (NS) InGaAsP diode laser 633 0.311 (NS) InGaAs diode laser 670 0.544 (NS) GaAs diode laser 830 0.015 (significant) Nd:YAG 1064 0.095 (NS) NS = not significant. P values shown are for 2 tailed tests. Fig 3. Data for 670nm (InGaAs diode laser) control and treatment difference between the means of the matched control and the test slabs. However, following demineralization, there was a statistically significant difference for all laser wavelengths between the matched test and control slabs (Table 4, Figs 1-5). While the control groups exhibited a significant reduction in their VHN values, all of the LAF treatment groups showed a protective effect. Comparing the effect of the different wavelengths, the mean effect for the GaAs diode laser was significantly different to the means of Nd:YAG and KTP respectively. Although both Nd:YAG and KTP were significantly different to the GaAs diode laser, they were not significantly different from each other. To rank order the laser wavelengths in terms of their overall efficacy, the effect size was used, comparing the treatment groups and their matching control groups. This method gave the following ranking: Nd:YAG> GaAs diode laser > KTP> InGaAsP diode laser> InGaAs diode laser. DISCUSSION The present study examined the successfulness of LAF therapy using five different laser wavelengths, for increasing micro-hardness and potentially conferring Australian Dental Journal 2007;52:2. 103
Table 4. Analysis of control versus matched test slabs Laser type Wavelength Prior to LAF After (nm) treatment demineralization KTP 532 0.432 (NS) 0.002921 InGaAsP diode laser 633 0.621 (NS) 0.002428 InGaAs diode laser 670 0.103 (NS) 0.019531 GaAs diode laser 830 0.202 (NS) 0.000848 Nd:YAG 1064 0.504 (NS) 0.000005 NS = not significant. P values shown are for 2 tailed tests. protection to tooth structure from an artificial carieslike challenge. The results extend previous studies on LAF by demonstrating that the effect can be obtained with a number of laser wavelengths across the visible and near infrared regions of the spectrum, and is also obtained using neutral sodium fluoride gel at the same irradiance shown previously to be optimal for the argon laser. While the caries-like challenge employed in this study did not allow for the possibility of repair from salivary minerals (as may occur in vivo), previous studies using the argon laser by Powell and colleagues have shown a strong correlation between LAF effects using demineralizing gels in vitro and clinical protection from carious attack in vivo. 4-6,8,10,24 Recognizing that dental caries is a process based on cycles of demineralization and remineralization, it is likely that the effects from LAF therapy, demonstrated in this laboratory study, extend to the clinical setting. Numerous in vitro and in vivo studies 4,6-8,10-13,24 have demonstrated that irradiation of enamel with certain wavelengths of laser energy provides increased resistance to cariogenic attacks, with a corresponding reduction in lesion depth ranging from 15 to 46 per cent. 3-6 The combination of argon laser energy (488nm, 10 12 J/cm 2, 10 seconds exposure time) with fluoride results in even greater lesion depth reduction than either laser or fluoride used alone, 3,14 with Hicks et al. 15 and Flaitz et al. 5 recording greater than 50 per cent and 51 55 per cent reduction, respectively. It is clear that the synergistic effect of laser irradiation and topical fluoride on tooth surface is not confined to the argon laser. The benefits of LAF with the CO 2 laser in terms of increased caries resistance of enamel, has been reported by a number of studies. 18,25-27 An important potential difference between the argon and CO 2 laser is the mechanism by which the change in enamel is achieved. Greater fluence and higher power outputs are used with the CO 2 laser than with the argon laser to achieve comparable results (45-170 vs. 10-12 J/cm 2 ; 1-2W vs. 0.23-0.25W, respectively). The mechanisms by which the LAF protective effect is achieved has been the subject of much conjecture in the literature. Several physicochemical changes have been suggested to occur during LAF treatment, including deposition of calcium fluoride, 19 formation of microspaces in the dental hard tissue, 20,21 formation of tri-calcium phosphate, 22 and phase transformation of hydroxyapatite to fluorapatite. 16 With the far infrared wavelengths of the carbon dioxide laser, photothermal effects will dominate the interaction, leading to phase changes in the enamel as well as physical alterations. The low photon energies in this region exclude the possibility of pure photochemical effects. In contrast, the high photon energies in the visible region offer the potential for photochemical as well as photothermal effects, such as the replacement of carbonate or hydroxyl within the apatitic structure, with fluoride, since this will give a more stable (lower energy) molecular configuration. The concept that fluoride may be incorporated into the enamel mineral is seen as important to the LAF effect since fluorapatite is more resistant to both strong (corrosive/erosive) acids as well as to weaker acids than apatite with carbonate or hydroxyl ions incorporated into its structure. Fox et al. 14 have shown that the enamel dissolution rate is influenced by laser treatment, with a corresponding reduction in the ph required to induce dissolution of the enamel. This notion of reduction of enamel dissolution is also supported by Goodman and Kaufman 28 who concluded that laser irradiation of enamel results in superficial melting or dissolution of crystals, followed by cooling and recrystallization and incorporation of fluoride to form less soluble fluorapatite. Tagomori and Morioka 29 have reported the enhanced uptake of fluoride after laser irradiation, and more recently Nammour et al. 24 have demonstrated that in vivo use of argon laser irradiation allowed greater retention of fluoride compared to non-lased enamel as a result of structural changes that create a reservoir. The same concept is also supported by the SEM studies of Westerman et al. 30 which showed changes in surface morphology. In the present study, changes in the enamel hardness were not seen immediately after lasing, except in the GaAs diode laser (830nm) group. Further studies to characterize the surface changes which occur with LAF are justified, particularly at the visible red and green wavelengths. CONCLUSION The results of this study show that the action spectrum of the LAF effect is relatively broad and extends across the visible and near-infrared wavelengths of laser light. The laser fluence parameters employed to achieve the changes in microhardness are relatively low and are identical to that used previously for the argon laser. Further studies should be conducted to establish whether such treatments may also have beneficial effects on enamel challenged with a corrosive acid assault (dental erosion). ACKNOWLEDGEMENTS This study was supported in part by the National Health and Medical Research Council of Australia and the Australian Dental Research Foundation Inc. REFERENCES 1. Marsh P, Martin MV. Dental Caries. Oral Microbiology. Edinburgh: Wright, 1999:82-96. 104 Australian Dental Journal 2007;52:2.
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Caries Res 1989;23:225-231. 30. Westerman GH, Hicks MJ, Flaitz CM, Powell GL, Blankenau RJ. Surface morphology of sound enamel after argon laser irradiation: an in vitro scanning electron microscopic study. J Clin Pediatr Dent 1996;21:55-59. Address for correspondence/reprints: Professor LJ Walsh School of Dentistry The University of Queensland 200 Turbot Street Brisbane, Queensland 4000 Email: l.walsh@uq.edu.au Australian Dental Journal 2007;52:2. 105