Original Article
Photomedicine and Laser Surgery Volume 27, Number 4, 2009 ª Mary Ann Liebert, Inc. Pp. 585–590 DOI: 10.1089=pho.2008.2311
Physical and Compositional Changes on Demineralized Primary Enamel Induced by CO2 Laser Elaine Pereira da Silva Tagliaferro, D.D.S., Ph.D.,1 Lidiany Karla Azevedo Rodrigues, D.D.S., Ph.D.,2 Luı´s Eduardo Silva Soares, D.D.S., Ph.D.,3 Airton Abraha˜o Martin, Phys., Ph.D.,4 and Marineˆs Nobre-dos-Santos, D.D.S., Ph.D.1
Abstract
Objective: This in vitro study aimed to evaluate the physical and chemical changes promoted by a CO2 laser at 10.6-mm wavelength on primary dental enamel with artificial caries-like lesions. Background Data: Several previous investigations have shown that enamel can be modified by CO2 laser to obtain a caries-preventive effect, but the specific mechanism remains uncertain. Materials and Methods: Twenty-seven primary molars were randomly assigned to three groups as follows: control, carious, and laser (n ¼ 9). The specimens from the carious and laser groups were demineralized and treated with or without CO2 laser, according to the group. Enamel surface changes after treatments were monitored using Fourier transform Raman spectroscopy and scanning electron microscopy (SEM). Results: The Raman spectra showed a statistically significant reduction of mineral content in carious and laser groups when compared to control group. Additionally, carbonate content was reduced in irradiated specimens when compared to the other groups. No physical change was observed in specimens evaluated by SEM. Conclusion: The results suggest that CO2 laser irradiation may reduce the carbonate content of enamel, which is likely to make this substrate more acid-resistant.
Introduction
D
uring the past several decades, it has been demonstrated that CO2 lasers can increase acid resistance of enamel by altering its physical or compositional characteristics.1–3 In addition, the efficacy of CO2 laser irradiation combined with fluoride in caries inhibition has been demonstrated by several studies,4–7 including studies performed in deciduous teeth.6,7 Consequently, the use of this laser with or without fluoride for young children at high caries risk might be a promising method to prevent and control the disease. The use of fluoridated vehicles, which deliver fluoride to the oral cavity, has contributed substantially to the widespread decline in caries incidence in some western countries.8,9 However, there is evidence that the anticaries effect of fluoride is related to its sustained presence in the oral environment,10 making the effect dependent on the patient’s
oral hygiene habits. For effective prevention, therapies not dependent on the patient’s compliance would be more advantageous for young children with high caries risk. Thus, use of a pulsed CO2 laser at 10.6 mm might be a good alternative for these patients. Also, for preventive purposes, laser treatment may be performed in a unique section being more comfortable for using in children. However, few reports about the caries-preventive effect of CO2 laser with or without fluoride on carious primary dental enamel are found in the scientific literature.6,7 The studies that have been done have focused on the mechanism of action for CO2 lasers in demineralization inhibition in primary enamel. One might consider that there are differences in the pattern of caries development and prevention between permanent and primary teeth.11 Another important aspect to consider is that there is no consensus about the exact mechanism of action of CO2 lasers in inhibiting enamel demineralization. Most theories focus
1
Faculty of Dentistry of Piracicaba, State University of Campinas, Piracicaba, SP, Brazil. Faculty of Pharmacy, Dentistry and Nursing, Federal University of Ceara´, Fortaleza, Ce, Brazil. 3 Faculty of Dentistry, Dental Materials and Operative Dentistry Departments, UniVap, Sa˜o Jose´ dos Campos, SP, Brazil. 4 Laboratory of Biomedical Vibrational Spectroscopy, Research and Development Institute, IP&D, UniVap, Sa˜o Jose´ dos Campos, SP, Brazil. 2
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586 on enamel mineral phase changes, such as surface melting and hydroxyapatite crystal fusion. On the other hand, Kantorowitz et al.12 and Hsu et al.13 showed no conclusive evidence that such physical changes, frequently shown by scanning electron microscopy (SEM), are necessary to increase enamel resistance to demineralization. Moreover, Hsu et al.13 have shown in both irradiated and nonirradiated enamel that the organic matrix plays an important role in caries inhibition. In this context, a more sensitive analysis should be performed to clarify the enamel modifications induced by laser irradiation, with particular attention to the chemical aspect. Raman spectroscopy is a nondestructive, information-rich, and highly selective technique for investigating molecular species14 that can be applied to almost any biomolecule. The Raman spectrum of any mineral structure, and especially of human teeth, can reveal the chemical composition and structure of mineral and organic contents. Spectra of irradiated enamel and dentin have been recorded to evaluate the compositional changes after Nd:YAG, Er:YAG, and CO2 laser irradiation.3,15–22 However, the effect of CO2 laser on chemical composition of primary enamel has not been investigated using Fourier transform (FT) Raman spectroscopy. Thus, this in vitro study evaluated the physical and chemical changes promoted by CO2 laser at 10.6 mm wavelength on demineralized primary dental enamel. Materials and Methods Tooth selection and sample preparation The 27 primary teeth used in this investigation were collected from 27 children living in Piracicaba, Brazil, in conformity with the rules of the Research and Ethics Committee of Faculty of Dentistry of Piracicaba (Process No. 146=2001). Primary molars stored in 0.1% supersaturated thymol solution were free of apparent enamel defects and caries; specimens containing macroscopic cracks, abrasions, or staining, as assessed by visual examination, were eliminated. After selection, molars were sectioned mesiodistally (without being embedded) using a water-cooled diamond saw as well as a cutting machine (Isomet, Buehler, Lake Bluff, IL, USA). Only the buccal surface of each tooth was used and the specimen halves were coated with an acid-resistant varnish leaving a window (4 mm2) of exposed enamel in the middle third of buccal surfaces. Caries-like lesion formation and grouping In order to obtain information about the demineralization process, nine sound specimens were reserved (control group) and the caries-like lesion formation was performed in 18 specimens according to Paes Leme et al.23 Early caries lesions were produced in these specimens by individual immersions in an acetate buffer (6.25 mL of solution=mm2 of exposed enamel): 0.05 mol=L, pH 5.0, 50% saturated with hydroxyapatite (Gen-phos HA Hospita´lia Ciru´rgica Catarinense Ltda., Floriano´polis, SC, Brazil) for 48 h at 378C. The demineralized enamel specimens were randomly distributed between the two groups: the carious enamel and irradiated groups. Raman spectroscopy and SEM were performed on the specimens of each group.
TAGLIAFERRO ET AL. Laser treatment A pulsed CO2 laser at a 10.6 mm wavelength (OpusTM 20, Lumenis, Yokneam, Israel) was used with the following parameters: 1 W, 50 ms pulse duration, 1 Hz repetition rate, and a beam diameter of 0.8 mm.6 For these conditions, a power meter (Model 201; Coherent Radiation, Palo Alto, CA, USA) indicated a 0.5 W peak power, thus determining an incident fluency of 5 J=cm2 per pulse. A 5 mm distance from the tip of the handpiece to the specimen was maintained during irradiation using a device made with orthodontic wire and fixed to the laser tip. Laser irradiation was carried out by scanning the exposed enamel of each specimen for approximately 30 s by moving the laser tip manually. Raman spectroscopy and SEM In order to assess the chemical and physical changes promoted by the treatments, the specimens of each group were analyzed by FT Raman spectroscopy followed by SEM. For the Raman spectroscopy, the specimens were placed in the sample-holder and the IR354 lens collected radiation scattered over 1808. Spectra of the specimens were obtained using an FT Raman spectrometer (RFS 100=S; Bruker Inc., Karlsruhe, Germany) with one Ge diode detector cooled by liquid N2. To excite the spectra, the focused l ¼ 1064.1 nm line of an air-cooled Nd:YAG laser source was used. The maximum laser power incident on the sample surface was about 77 mW, and the spectrum resolution was 4 cm1. The FT Raman spectra were obtained using 150 scans. The explored frequency ranged from 300 to 4000 cm1 and allowed both mineral and organic constituents to be characterized. The mean spectrum from each specimen was obtained from the whole isolated zone since three points of analysis were chosen; one central and the others on the right and left sides of the exposed enamel. After Raman analysis, the specimens were frozen in liquid nitrogen and freeze-dried at 15,466103 PSI and 508C, for 18 h (Modulyo 4K Freeze Dryer; Edwards High Vacuum International, West Sussex, UK). The freeze-dried specimens were first fractured to expose the interior structure, and then affixed to aluminum SEM stubs using double-sided tape. Then the prepared specimens were coated with gold using a sputter coater (Denton Vacuum Desk II; Moorestown, NJ, USA). The coating was achieved by applying a vacuum of 0.005 mbar and a current of 15 mA for 215 s, resulting in an approximately 10–12 nm thick coating. The coated specimens were examined with a scanning electron microscope ( JSM5600 LV; JEOL, Tokyo, Japan) operated at an accelerating voltage of 10–15 kV at pertinent magnification (up to 2500). Statistical analysis For Raman spectroscopy data, for the purpose of normalizing measurements and allowing their comparison, we used the band surface parameter, which corresponds to the area under the curve for the ‘‘n’’ analyzed bands.24 Thus, all bands in the spectrum were analyzed. Averages of the Raman spectra were obtained from all groups (n ¼ 9). The spectra fluorescence was removed with a polynomial fitting from the spectra, with varying degrees in the Microcal Origin5.0 software (Microcal Software, Northampton, MA). Relative areas of the peaks were calcu-
CO2 LASER IRRADIATION ON ENAMEL
FIG. 1. Average Raman spectra of experimental groups in the 300–3100 cm1 range, after normalization.
lated by the Microcal Origin5.0 software. The changes in mineral and organic structures were evaluated by comparing the relative intensity of the peaks. First, the data were evaluated to check the equality of variances and normal distribution of errors, and then those that did not achieve these requests were transformed to square root. Statistical analysis of the Raman results was performed by ANOVA followed by Tukey–Kramer test for differences between means at a 95% level of confidence using the BioStat Professional 2007 3.9.5 software, (Analyst Soft Robust Business Solutions, Vancouver, BC, Canada) in order to assess the significance of the evaluation of the relative area between the normal, carious, and irradiated enamel data. The Kolmorogov–Smirnov test verified the normal distribution of the Raman data.
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FIG. 2. Average Raman spectra of sound, carious, and laser groups in the 350–1150 cm1 range, after normalization, showing the components associated with the mineral sample content. of the organic bands in this range can be clearly observed in carious and irradiated enamel when compared to the sound enamel (Fig. 3). Table 1 shows the mean value for the area under each peak for sound, carious, and irradiated groups. For all bands, although not always statistically significant, sound enamel presents more evident bands than those found for carious or irradiated enamel. The demineralization process significantly affected enamel spectra at 612 and 960 cm1 (mineral phase). Furthermore, the mineral part of the enamel spectrum was affected by laser irradiation when compared to carious enamel at 450 and 1072 cm1 (Table 1).
Results Average Raman spectra of all groups are shown in Figs. 1–3. The raw Raman spectra of all experimental groups are shown in Fig. 1. Spectral analysis showed two characteristic parts: first, a region spanning from 300 to 3,100 cm1, characteristic of phosphate groupings and representative of the mineral phase of the enamel (Fig. 2); another region, representative of the collagen phase, shows organic grouping vibration modes (amide and CH) in the 1,200–3,000 cm1 region (Fig. 3). The FT Raman bands at v2 (430–450 cm1), v4 (585–612 cm1), v1 (960 cm1), and v3 (1026–1072 cm1) represent the phosphate vibrations in hydroxyapatite (Fig. 1). The band in the range of 1026–1072 cm1 can also represent the v1 carbonate vibration (type B carbonate). All the spectra of lased surfaces appear very similar to those that were carious or untreated. However, in Figs. 2 and 3, averaged Raman spectra of sound, carious, and irradiated enamel are presented and the differences can be seen more clearly. In Fig. 2, a mineral content decrease can be observed in carious and irradiated specimens. Figure 3 shows no difference between carious and irradiated enamel in the range of 1200–3100 cm1. On the other hand, a decrease in intensity
FIG. 3. Average Raman spectra of sound, carious and laser groups in the 1200–3100 cm1 range, after normalization, showing the components associated with the organic materials.
0.19 0.06a 0.15 0.04ab 0.10 0.03b
FIG. 4. Representative scanning electron micrograph of the surface morphology of nonirradiated carious primary enamel.
The SEM observations showed no evidence of melting, fusion, or other physical changes in specimens treated with CO2 laser compared to the nonirradiated specimens (Figs. 4, 5).
Means followed by the same letters are not significantly different by LSD test ( p < 0.05).
Discussion
a
0.20 0=03a 0.19 0.04a 0.17 0.03a 0.04 0.02a 0.03 0.02a 0.03 0.01a 0.32 0.03a 0.31 0.05a 0.25 0.04b 0.29 0.07a 0.27 0.07a 0.21 0.03b Sound Carious Irradiated
0.42 0.05a 0.37 0.09ab 0.31 0.04b
0.37 0.05a 0.39 0.12a 0.35 0.05a
0.10 0.03a 0.07 0.02b 0.05 0.01b
2.17 0.34a 1.69 0.30b 1.71 0.20b
0.16 0.06a 0.19 0.07a 0.17 0.03a
1665 1450 1243 1072 1044 960 612 585 450 430 Groups
Raman bands (cm1)a
Table 1. Areas Under the Peak Bands (Mean SD; n ¼ 9) for Sound, Carious, and Irradiated Groups
0.88 0.24a 0.74 0.20a 0.75 0.23a
TAGLIAFERRO ET AL.
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The spectra of lased surfaces appeared very similar to those of the untreated enamel, showing that low-energy laser treatment only slightly affected the enamel apatite and caused no structural damage, which is confirmed by the SEM data. Thus, as previously demonstrated in other studies, pulsed CO2 laser at 10.6 mm using fluencies lower than 12 J=cm2 induced little or no morphologic change,12 and no melting13,14 or formation of craters in enamel surfaces was observed with SEM.13 The SEM data in this study, with similar or even greater magnification than used by other authors,12,25,26 showed that there was no surface melting or fusion with the
FIG. 5. Representative scanning electron micrograph of the surface morphology of carious primary enamel irradiated with a CO2 laser.
CO2 LASER IRRADIATION ON ENAMEL fluency used (5 J=cm2). However, to our knowledge, no other studies have evaluated the morphological or chemical composition of deciduous teeth irradiated by CO2 lasers. In the FT Raman spectroscopy results, the strongest bands of phosphate v1 and carbonate v1 modes, which have been previously reported,20,27–30 were immediately identified. However, the organic phase (Fig. 2), which could be systematically observed in the dentin spectrum but not in the enamel,24 was observed in this study, similar to the results found in the recent study conducted by Liu and Hsu20 and Steiner-Oliveira et al.3 These different spectral results could be explained due to the method used. FT Raman spectroscopy presents a reduction in the fluorescent background, making it possible to identify bands in that region, even in the enamel spectrum, although enamel is a substrate that has a small organic component content. In addition, our results are in agreement with another recent study that showed Raman spectra of enamel samples presenting the structured bands within the 1100–1700 cm1 spectral range due to the presence of organic materials.30 The bands at around 1243 and 1665 cm1 can be ascribed to amide III and amide I, and the band around 1450 cm1 to CH2 and NH modes. It is worth recalling that, due to the vibrational modes of the organic peptide group, the amide bands (–CONH–) are associated with the C–O stretching mode (amide I), the N–H bending mode (amide II), and a combination of N–H bending and C–N stretching modes (amide III). Figure 2 shows a decrease in irradiated enamel of the band around 450 cm1, ascribed to phosphate vibrations in hydroxyapatite. This effect, a reduction in the mineral content of irradiated enamel, may be attributed to the lower degree of enamel crystallinity after irradiation. Our results are in line with those reported by Stern et al.,31 Kantola et al.,32 and Ferreira et al.,33 which demonstrated ultrastructural crystallographic effects, such as apatite crystals with a different shape and larger size, and a loss of prismatic structure in irradiated enamel. It is possible that these effects and the reduction in the carbonate content, as previously reported by Zuerlein et al.34 and Steiner-Oliveira et al.,3 were responsible for the increased enamel acid resistance demonstrated by Tagliaferro et al.6 using the same irradiation parameters. Carbonate fits less well in the lattice, causing distortions in the hydroxyapatite structure, generating a less stable and more acid-soluble apatite phase.35,36 In addition, the substantial caries-preventive results found by Tagliaferro et al.6 may be partially explained by the higher carbonate content in deciduous teeth when compared to the permanent enamel. Furthermore, the deciduous enamel contains significantly more type A carbonate (carbonate in the hydroxide positions) than permanent enamel. It is possible that the carbonate ion in the hydroxide position will distort the lattice more than in the phosphate position (type B carbonate).37 In this way, these facts might have made the caries-preventive effects of the CO2 laser more evident in primary enamel. With regard to the demineralization process, based on the concept that the intensity of the Raman bands are related to the amount of each component in the analyzed substrate, it seems reasonable to assume that the bands’ intensity reduction, observed after caries-like lesion production, is due to a caries process. These results are in agreement with Tramini et al.,14 who have shown decreases in band intensity
589 after dentin and enamel were submitted to acid challenge. However, the same authors did not find any visible difference in the enamel spectrum after 7 d of lactic acid action. This could be explained by the different in vitro acid penetration in their research, since enamel specimens were cut in longitudinal sections and it is known that in vitro acid penetration may be less efficient in this manner as compared to that utilizing transverse sections.14 Thus, since phosphate vibration modes (v4 [585–612 cm1] and v1 [960 cm1]) changed after enamel demineralization, the sensitivity of the phosphate vibration bands to changes in enamel structure, especially in these regions, may make it possible to follow the early stages of tooth mineralization28 and quantify enamel components. Under the conditions of this study and considering the results, it can be concluded that the CO2 laser irradiation at l ¼ 10.6 mm may provide caries-preventive effects on enamel through reduction of carbonate content. This effect may be especially important considering groups of young children with limited access to or low compliance with prophylactic measures, such as the use of fluoridated products. Acknowledgments This research was supported by FAPESP, grants 00=097028 and 01=14384-8 and by CNPq grant 302393=2003-0. The authors would especially like to thank the laboratory assistance given by Evelyn Alvarez Vidigal and Marcela Cristina de Souza. We also wish to thank Paulo Ricardo S. Wagner for his valuable help with the laser irradiation, and the LELOFOUSP for the use of their CO2 laser. The first author received a scholarship from CNPq-UNICAMP during her Master’s course in dentistry. This paper was based on a thesis submitted by the first author to the Faculty of Dentistry of Piracicaba, University of Campinas, in partial fulfillment of the requirements for an M.S. degree in Dentistry (cariology area). Disclosure Statement No competing financial interests exist. References 1. Featherstone, J.D.B., Barrett-Vespone, N.A., Fried, D., Kantorowitz, Z., and Seka, W. (1998). CO2 laser inhibition of artificial caries-like lesion progression in dental enamel. J. Dent. Res. 77, 1397–1403. 2. Klein, A.L., Rodrigues, L.K., Eduardo, C.P., Nobre dos Santos, M., and Cury, J.A. (2005). Caries inhibition around composite restorations by pulsed carbon dioxide laser application. Eur. J. Oral Sci. 113, 239–244. 3. Steiner-Oliveira, C., Rodrigues, L.K., Soares, L.E.S., Martin, A.A., Zezell, D.M., and Nobre-dos-Santos, M. (2006). Chemical, morphological and thermal effects of 10.6-micron CO2 laser on the inhibition of enamel demineralization. Dent. Mater. J. 25, 455–462. 4. Hsu, C.Y.S., Jordan, T.H., Dederich, D.N., and Wefel, J.S. (2001). Laser-matrix-fluoride effects on enamel demineralization. J. Dent. Res. 80, 1797–1801. 5. Rodrigues, L.K., Nobre dos Santos, M., and Featherstone, J.D. (2006). In situ mineral loss inhibition by CO2 laser and fluoride. J. Dent. Res. 85, 617–621.
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Address correspondence to: Luı´s Eduardo Silva Soares, D.D.S., Ph.D. Universidade do Vale do Paraı´ba, UNIVAP Instituto de Pesquisa e Desenvolvimento, IP&D Laborato´rio de Espectroscopia Vibracional Biome´dica, LEVB Av. Shishima Hifumi, 2911, Urbanova, 12244-000 Sa˜o Jose´ dos Campos, SP Brazil E-mail:
[email protected]
