Lasers Med Sci (2014) 29:525–535 DOI 10.1007/s10103-012-1259-0 ORIGINAL ARTICLE Effect of CO2 laser on root caries inhibition around composite restorations: an in vitro study Jociana Bandeira de Melo & Fernando Seishim Hanashiro & Washington Steagall Jr. & Miriam Lacalle Turbino & Marinês Nobre-dos-Santos & Michel Nicolau Youssef & Wanessa Christine de Souza-Zaroni Received: 28 February 2012 / Accepted: 26 December 2012 / Published online: 5 January 2013 # Springer-Verlag London 2013 Abstract The aim of the present study was to investigate the in vitro effect of CO2 laser on the inhibition of root surface demineralization around composite resin restorations. For this purpose, 30 blocks obtained from human molar roots were divided into three groups: group 1 (negative control), cavity prepared with cylindrical diamond bur + acid etching + adhesive + composite resin restoration; group 2, cavity prepared with cylindrical diamond bur + CO2 laser (5.0 J/cm2) + acid etching + adhesive + composite resin; and group 3, cavity prepared with cylindrical diamond bur + CO2 laser (6.0 J/cm2) + acid etching + adhesive + composite resin. After this procedure, the blocks were submitted to thermal and pH cycling. Root surface demineralization around the restorations was measured by microhardness analysis. The hardness results of the longitudinally sectioned root surface were converted into percentage of mineral volume, which was used to calculate the mineral loss delta Z (ΔZ). The percentage of mineral volume, ΔZ, and the percentage of demineralization inhibition of the groups J. B. de Melo : M. N. Youssef : W. C. de Souza-Zaroni (*) School of Dentistry, Cruzeiro do Sul University (UNICSUL), Av. Dr. Ussiel Cirilo, 225, 08060-070 São Paulo, Brazil e-mail: wansouzazaroni@gmail.com F. S. Hanashiro : W. Steagall Jr. : M. L. Turbino : M. N. Youssef Department of Restorative Dentistry, School of Dentistry, University of São Paulo (USP), Av. Prof. Lineu Prestes, 2227, Cidade Universitária, 05508-900 São Paulo, Brazil M. Nobre-dos-Santos Department of Pediatric Dentistry, Faculty of Dentistry of Piracicaba, State University of Campinas (UNICAMP), Av. Limeira, 901, P.O. Box 52, 13414-903 Piracicaba, São Paulo, Brazil were statistically analyzed by using analysis of variance and Tukey–Kramer test. The percentage of mineral volume was higher in the irradiated groups up to 80 μm deep. The ΔZ was significantly lower in the irradiated groups than in the control group. The percentage of reduction in demineralization ranged from 19.73 to 29.21 in position 1 (50 μm), and from 24.76 to 26.73 in position 2 (100 μm), when using 6 and 5 J/cm2, respectively. The CO2 laser was effective in inhibiting root demineralization around composite resin restorations. Keywords Carbon dioxide laser . Root caries . Composite resin restorations Introduction There have been constant advancements in Dentistry, seeking the development of new techniques and materials, which offer characteristics closer to those of natural teeth, are more resistant to caries development, and are capable of restoring patients’ lost tooth structure. However, this improvement in techniques and materials has not resulted in a reduction of recurrent caries at the tooth/restoration interface. Clinical studies have shown that secondary caries lesions are the most common cause of replacing restorations [1–4]. This fact is even more severe at the root surface owing to the clinical limitations of restorative treatment because of difficult access to the lesion and isolation of the operative field [5]. Thus, new methodologies have been studied in an endeavor to control recurrent caries in this region. Root lesions are more frequent in elderly patients [6–8] and are usually related to factors such as reduced salivary flow, reduced manual dexterity, and motor coordination 526 which frequently prevent correct cleaning. The root surface is rougher than enamel, which facilitates plaque formation in the absence of adequate oral hygiene. In addition to this, caries lesions may progress rapidly on this surface due to the differences in the chemical and structural composition of the mineral tissues in the teeth, so that the critical pH for dissolution of the cementum and dentin is approximately 6.7 [9], whereas for enamel, it is 5.5. Therefore, when considering the phenomenon of world population aging, an increase in prevalence and incidence of root lesions becomes predictable [8, 10]. In this context, the laser has been presented as an alternative for preventive Dentistry. Much research has been conducted with the use of several different types of lasers such as Nd:YAG [11, 12], Er:YAG [13], and CO2 [14–17] in the enamel and dentine of human teeth, demonstrating an increase in demineralization inhibition of these substrates. The study developed by Konish et al. [18] showed that caries removal using CO2 laser produced cavity walls that were more resistant to caries around restorations, when compared with conventional mechanical removal. Thus, irradiation with CO2 laser on the surfaces adjacent to the restoration seems to be an alternative way to reduce enamel demineralization around the restorations, without risk of pulp damage. In addition, Klein et al. [19] showed that CO2 laser irradiation of cavosurface margins of enamel cavities was able to inhibit enamel demineralization around composite restorations. However, it is observed that the existent studies in the literature do not focus on the effect of the CO2 laser on the prevention of secondary caries lesions on the root surface, and there is no consensus between the different authors about which parameters would cause inhibition of demineralization. Both increase and decrease in the dentine acid dissolution rate have been observed in different investigations [20, 21]. Continuous CO2 laser (λ=10.6 μm) irradiation of dentine with 1 W caused a significant decrease in calcium acid solubility in the study of Hossain et al. [22], and in a contradictory way, the study of Featherstone et al. [21] observed an increase in acid dissolution, using the same power and the same laser. Additionally, over half of the published studies that used the 10.6-μm CO2 laser in dentine were performed using the continuous wave emission mode [21–24]. As it is known that continuous irradiation significantly increases the chances of thermal damage to the hard and soft dental tissues, this irradiation mode has been not recommended for clinical treatment [25, 26]. Considering that pulsed irradiation might be more indicated for a future clinical trial, the present research aimed to investigate, in vitro, the effect of a CO2 laser (1=10.6 μm) emitting pulses of 10 ms, at two energy densities, on root demineralization inhibition around composite restoration. Lasers Med Sci (2014) 29:525–535 Material and methods This study was conducted after the being approved by the Research Ethics Committee (CEP) of Cruzeiro do Sul University. The factor under study was the laser energy density used to irradiate the cavosurface margins of the root cavities. In group 1, ten tooth blocks with cavity preparation were performed with diamond bur no. 2294 (KG Sorensen, Medical Burs Ind. e Com. de Pontas e Brocas Cirúrgicas Ltda., Barueri, São Paulo, Brazil) in a highspeed turbine, submitted to phosphoric acid etching and adhesive system application, and restored with resin composite (negative control). In group 2, ten tooth blocks with cavity preparation were performed with a diamond bur mounted in a high-speed turbine; submitted to the surface treatment of the cavosurface margins with the CO2 laser, energy density of 5 J/cm2, phosphoric acid etching, and adhesive system application; and restored with resin composite. In group 3, ten tooth blocks with cavity preparation were performed with a diamond bur mounted in a high-speed turbine; submitted to the surface treatment of the cavosurface margins with the CO2 laser, energy density of 6 J/cm2, phosphoric acid etching, and adhesive system application; and restored with resin composite. Preparation of specimens The teeth were stored in 0.1 % thymol solution for a minimum of 30 days after cleaning with periodontal curettes, followed by prophylaxis with a Robinson brush at low speed and aluminum paste (5 μm) for 30 s and two ultrasound baths of 30 s each. Afterwards, the teeth were examined under a stereoscopic loupe at ×10 magnification; those with cracks or structural abnormalities were discarded, and the remainders were kept in humid medium at 4 °C until they were used. In order to obtain the root surface specimens, the teeth were fixed in a cutting machine to remove the coronal portion 1 mm beyond the enamel–cement junction, which was discarded (Fig. 1a). The root portion of each tooth was longitudinally sectioned in order to obtain two specimens (Fig. 1b, c). The sections were identified so that the two specimens from the same tooth were not used in the same group. Cavity preparation A no. 2294 cylindrical diamond bur (KG Sorensen, Barueri, Brazil) in a high-speed turbine with air–water spray was used. A standard cavity was prepared with diameter 1.7± 1 mm and depth 1.5 mm. Lasers Med Sci (2014) 29:525–535 527 Fig. 1 Sections of root surface specimens. a Teeth were fixed in a cutting machine to remove the coronal portion 1 mm beyond the enamel–cement junction, which was discarded. b, c The root portion of each tooth was longitudinally sectioned in order to obtain two specimens. d The specimens were sectioned in the center of the window of exposed dentin, using a precision cutting machine with a diamond disk with thickness of 0.3 mm, undercooling. e One half of each specimen was selected and positioned in the center of a semirigid plastic tube, leaving the internal (sectioned) part of the specimen exposed. f The indentations were located at 50 and 100 μm distance from the tooth/restoration interface, and 20, 40, 60, 80, 100, 140, and 220 μm in depth from the margin of the cavosurface margins (tooth external surface), in the direction of the pulp tissue Cavosurface angle treatment with pulsed CO2 laser Irradiation of the cavosurface margins of the root surface preparations was performed using a pulsed CO2 laser with a wavelength of 10.6 μm (UM-L30, Union Medical Engineering Co., Yangju-si, Gyeongii-Do, Korea—PROJETO FAPESP CEPID/CEPOF, processo no. 98/14270-8). The selection of irradiation parameters was based on a previous study by Souza-Zaroni et al. [27], which was also conducted on root surfaces. The specimens were irradiated at a fixed repetition rate of 50 Hz, pulse duration of 10 ms, 10 ms of time off, and beam diameter of 0.3 mm, and the power used for groups G2 and G3 was 0.7 and 0.8 W, respectively. The power set on the appliance was confirmed by measurement with a power meter (Scientech 373 Model–37-3002, Scientech, Inc., Boulder, CO, USA), so that the mean powers measured for these groups were 0.37 and 0.42 W, respectively. The energy densities under these irradiation conditions were approximately 5 J/cm2 (G2) and 6 J/cm2 (G3). Irradiation was performed with the active point of the laser perpendicular to the tooth surface at a distance of 1 cm from it for a period of 10 s, and the speed of lasing movement was 2 mm/s. Restorative procedure After performing the cavity preparations in all the dental root blocks, prophylaxis was performed with pumice stone 528 Lasers Med Sci (2014) 29:525–535 paste (SS White) and distilled and deionized water. The cavities were then washed with water and air spray and were air dried afterwards. The preparations were restored with resin composite (Filtek Z 250, 3M ESPE, St. Paul, MN, USA), using 37 % phosphoric acid etching and Single Bond 2 adhesive (3M ESPE, St. Paul, MN, USA) before inserting the resin composite. The materials were manipulated, and the cavities were restored in accordance with the manufacturer’s instructions. When the restorative procedure was concluded, the dental blocks were stored for 24 h at 37 °C in a 100 % humid ambient and were then polished with a sequence of aluminum oxide disks (Sof-Lex, 3M ESPE, St. Paul, MN, USA). level of saturation of the apatite minerals found in saliva, being similar to the one used by Ten Cate and Duijsters [29]. Both solutions contained thymol crystals to prevent bacterial growth, and they were prepared using the same reagents as sources of calcium and phosphate. The quantities of 6.25 and 3.12 mL/mm2 of de- and remineralizing solutions were used, respectively, per treatment area. During the entire process, the specimens remained in an oven set at 37 °C, except in the intervals of washing and alternating the solutions. When the pH cycling was concluded, specimens were washed with jets of distilled and deionized water for 10 s, dried with absorbent paper, and kept in a closed and humid ambient, undercooling until preparation for the microhardness readouts. Thermal cycling Cross-sectional microhardness analysis The main objective of this procedure was to expose the restorative material to a high thermal challenge. In the thermal cycling process, all the groups were stored in tulle bundles, each containing a group, which were submitted to 1,000 cycles. Each cycle consisted of the group being immersed in distilled and deionized water for 60 s at a temperature of 5±1 °C, and 60 s at a temperature of 55±1 °C, with temperatures in the thermal cycling machine kept constant. After concluding the pH cycling, the specimens were sectioned in the center of the window of exposed dentin, using a precision cutting machine (Labcut, 1010, Extec, USA) with a diamond disk with thickness of 0.3 mm, undercooling (Fig. 1d). Afterwards, one half of each specimen was selected (Fig. 1e) and positioned in the center of a semirigid plastic tube (sample cups), so that it would be embedded in the acrylic resin that was poured over it, leaving the internal (sectioned) part of the specimen exposed. After resin polymerization, the plastic tubes were removed, and the specimens were polished with water abrasive papers in decreasing order of grit nos. 600 and 1000 for 1 and 5 min, respectively. Felt disks with 6 μm diamond paste (2 min) and 3 μm (4 min) were also used after polishing with the abrasive papers. Between the changes of abrasive papers, all specimens were submitted to an ultrasound bath with distilled and deionized water for 3 min, and between the pastes/suspension with common liquid detergent diluted in water, for 5 min. Next, they were washed abundantly under distilled and deionized water and stored in a closed and humid ambient, undercooling until they were used. The microhardness test has been performed in order to verify the presence or absence of demineralization on the root surface adjacent to the tooth/restoration interface. The blocks were visualized with the aid of a monitor. Fourteen indentations were made on each dental block, only on the root surface adjacent to the tooth/restoration interface of each tooth, at different points, however, at a standardized distance for all the restorations evaluated. The analyses were performed using the microhardness tester HMV-2000 (Shimadzu Corporation, Kyoto, Japan) and the Knoop-type penetrator with a 5-g load and duration of application of 15 s. The indentations were made longitudinally on the cut faces, with the long axis The pH cycling After thermal cycling, the dental root area that would be exposed to the de- and remineralizing solutions during the pH cycling was delimited. Each specimen was completely covered with acid resistant varnish (red cosmetic nail polish), leaving an area measuring 4×4 mm (window) of the root exposed. In order to perform pH cycling, we used the model described by Kawasaki and Featherstone [28] and modified by Souza-Zaroni et al. [27]. The pH cycling was performed for 5 days in which the root specimens were immersed for 4 h/day in the demineralizing solution and for approximately 20 h/day in the remineralizing solution. During this period, the specimens were washed with deionized water twice a day (before and after immersion in the demineralizing solution) for 10 s and dried with absorbent paper. After the fifth day, the specimens remained in the remineralizing solution for 2 days (corresponding to the days of the weekend). The specimens were kept individually in the demineralizing solution containing 2.0 mmol/L Ca and 2.0 mmol/L P in 75 mmol/L acetate buffer, pH4.8. The remineralizing solution used contained 1.5 mmol/L Ca, 0.9 mmol/L P, and 150 mmol/L KCl in 20 mmol/L Tris buffer, pH7.0. The chemical composition of this solution is close to the Lasers Med Sci (2014) 29:525–535 529 of the diamond indenter parallel to the external portion of the root surface. The indentations were located at 50 and 100 μm distance from the tooth/restoration interface, and 20, 40, 60, 80, 100, 140, and 220 μm in depth from the margin of the cavosurface margins (tooth external surface) (Fig. 1f), in the direction of the pulp tissue (adapted from Klein et al. [19]). Determining the mineral profile The hardness results of the longitudinally sectioned root surface were converted into percentage of mineral volume by the following formula: 4.3×(HNK1/2)+11.3 [30]. After calculating this value at all depths assessed, the integrated area under the mineral volume profile curve was calculated in percentages of mineral volume per micrometer (area formed between the axes y and x), using the trapezoidal rule. Therefore, from each dental block, a hardness mean was obtained in the healthy region (100, 140, and 220 μm from the surface), which was projected as the percentage of normal mineral content of each block. The value of mineral volume in each block treated was measured at the depths of 20, 40, 60, 80, and 100 μm from the external surface. Delta Z (ΔZ) of each block was calculated by integrating the area between the mineral profile of the lesion and the mean percentage of mineral volume. ΔZ was obtained by subtracting the integrated area found in the treated block from the integrated area of the projection for the intact root surface [27, 30]. This procedure allowed the parameter ΔZ to be calculated and defined as follows: ΔZ (mineral volume percent×micrometer)=difference between Z for the intact root surface and Z for the root surface after undergoing several treatments. Based on this parameter, the percentage of inhibition of mineral loss was calculated for each group according to the following formula: percentage of inhibition = ΔZ (control) − ΔZ (treatment)×100/ΔZ (control). Table 1 Split plot three-way ANOVA for the microhardness data MS mean square, OP observed power a Computed using alpha=0.05 Statistical analysis The response variable analyzed was the percentage of mineral volume at different depths from the tooth external surface and distances from the tooth/restoration interface and the laser energy density used to irradiate the cavosurface margins of the root cavities as a source of variation. The SPSS® Program was used for data analysis. The presuppositions necessary for the analysis of variance (ANOVA) were verified. The data with reference to the response variables showed normal distribution and homogeneity of variances. After ANOVA, multiple comparisons were made among the means, using the Tukey–Kramer test at a level of significance of 5 %. Results The variables, percentage of mineral volume, and percentage of mineral loss (ΔZ) were assessed in this research. The percentage of mineral volume was assessed at seven depths and in two positions, according to the above-mentioned microhardness scheme. Three-way ANOVA (Table 1) has been used in this experimental design, since there was one independent variable, the treatments received by the cavosurface margins (laser energy density used to irradiate the margins) of the root cavities, and two dependent variables, the depths of microhardness indentations from the outer surface and distances from cavosurface margins. Percentage of mineral content The ANOVA results showed that the main factors, depths [F(6, 162) =1.32E-26] and treatments F(2, 27) =7.03E-06, were significant, whereas distance [F(1, 27) =0.33] was not. The two-way interactions depth×treatments [F(12, 162) =2.26E22] and distance×treatments [F(2, 27) =0.01] were significant, Source of variation MS Depths Depths×treatments Error (depths) Distance Distance×treatments Error (distance) Depths×distance Depths×distance×treatments Error (depths×distance) Intercept Treatments Error (teatments) 165.44 78.53 4.81 9.63 54.06 9.98 5.71 1.84 3.66 445,489.35 866.41 45.57 Fa Significance F critical OP 1.32E-26 2.26E-22 2.15 1.81 1.00 1.00 0.96 5.42 0.33 0.01 4.21 3.35 0.16 0.80 1.56 0.50 0.16 0.91 2.15 1.81 0.59 0.28 4.20E-36 7.03E-06 4.21 3.35 1.00 1.00 34.43 16.34 9,775.20 19.01 530 whereas depths × distance [F(6, 162) = 0.16] was not. The three-way interaction depths ×distance ×treatments [F(12, 162) =0.91] was not significant. Since there were two significant interactions, depths× treatments and distances × treatments, these interactions were further explored to investigate the statistical nature of their interaction. The statistical significance of an interaction indicates that the levels of a main factor behave differently regarding the levels of another main factor. Tukey–Kramer pairwise multiple comparison method was utilized to compare the means of these two interactions. This method employed pairwise comparison, between all the means, adjusting the degrees of freedom and, therefore, the error, for each comparison, through the Satterthwaite estimate adjustment. The statistical behavior for distances×treatments multiple comparisons is displayed at Fig. 2, and the data, at Table 2. The data at Table 2 and Fig. 2 showed that the control group presented the lower values of microhardness, regardless the distance from tooth/restoration interface. The group irradiated with laser 5 J also showed no statistical difference between the 50 and 100-μm distances but showed higher microhardness values in comparison to the control group. The group irradiated with laser 6 J showed statistical Fig. 2 The statistical behavior for distances×treatments multiple comparisons Lasers Med Sci (2014) 29:525–535 differences between the two distances, being the microhardness values of the 50 μm greater than the values of 100 μm. Finally, the microhardness values of laser 5 J group at 100μm distance from tooth/restoration interface were similar to the values of the both distances of laser 6 J group. The statistical behavior for depths×treatments multiple comparisons is displayed at Fig. 3, and the data, at Table 3. The data at Table 3 and Fig. 3 showed that the control group presented the lower values of microhardness for the first four depths, 20, 40, 60, and 80 μm, being all these values statistically similar. The last three depths, 100, 140, and 220 μm, were also statistically similar and presented higher microhardness values in comparison to the first four depths. These last three values of microhardness were also statistically similar to the two laser groups’ values at the same depths. Additionally, the laser groups did not show statistically significant differences between the microhardness values for all the depths. Mineral loss (ΔZ) and percentage of demineralization inhibition Table 4 shows the means and standard deviations of the effect of CO2 laser on the reduction of mineral loss from the Lasers Med Sci (2014) 29:525–535 Table 2 Descriptive statistics and Tukey–Kramer multiple comparisons for the distances× treatments interaction Bold means followed by different letters differ statistically among them by the Tukey test at a level of significance of 5 % 531 Distances Treatments Mean Standard deviation 50 μm Control Laser 5 J Laser 6 J Control Laser 5 J Laser 6 J 29.84 32.77 35.55 29.84 33.53 33.88 1.43 0.99 3.34 0.95 1.56 2.50 100 μm root surface. The results indicated that the loss of mineral content from the root surface in the groups treated with CO2 laser was significantly lower when compared with that of the control group (without laser), irrespective of the energy density used (5 or 6 J/cm2). However, mineral loss did not differ among the groups treated with laser. Comparison among the results of the loss of mineral content (ΔZ) of control group with the laser treatments (5 and 6 J/cm2) was performed with the aim of establishing the whether or not irradiation with CO2 laser acted on the human tooth root surface, after pH cycling. Thus, by means of the mathematical calculation previously described, it was possible to determine the percentage of root demineralization inhibition after laser application. The percentage of Fig. 3 The statistical behavior for depths×treatments multiple comparisons Tukey–Kramer A B C A B, C B inhibition ranged from 19.73 to 29.21 in distance 1 (50 μm) and from 24.76 to 26.73 in distance 2 (100 μm), when the energy densities 6 and 5 J/cm2, respectively, were used, as shown in Table 4. Discussion In the literature, there are a large number of studies that report the action of CO2 laser on preventing caries in enamel on free flat surfaces [31–39], as well as in enamel adjacent to restorations that had cavity preparations performed with this laser [18] or only irradiation of their cavosurface angles [19]. CO2 laser has four main wavelengths in the infrared 532 Lasers Med Sci (2014) 29:525–535 Table 3 Descriptive statistics and Tukey–Kramer multiple comparisons for the distances× treatments interaction Depths Treatments Mean Standard deviation 20 μm Control Laser 5 J Laser 6 J Control Laser 5 J Laser 6 J Control Laser 5 J Laser 6 J Control Laser 5 J Laser 6 J Control Laser 5 J 27.12 33.02 33.88 26.34 32.76 33.90 26.56 33.23 34.63 26.79 32.36 34.34 33.92 33.73 0.89 1.89 3.91 1.73 1.11 2.61 1.34 1.32 3.12 1.48 1.04 2.61 2.11 1.89 A B B A B B A B B A B B B B Laser 6 J Control Laser 5 J Laser 6 J Control Laser 5 J Laser 6 J 35.17 34.16 33.55 35.37 33.99 33.45 35.71 2.78 2.79 1.46 3.29 2.99 1.21 3.27 B B B B B B B 40 μm 60 μm 80 μm 100 μm 140 μm Bold means followed by different letters differ statistically among them by the Tukey test at a level of significance of 5 % 220 μm spectrum: 9.3, 9.6, 10.3, and 10.6 μm. Among these, the most indicated for use in caries prevention, because of being better absorbed by hydroxyapatite and promoting efficient and rapid heating of this mineral, are the wavelengths 9.3 and 9.6 μm. However, up to now, there is no laser commercially available that can produce these conditions [30], so that the researches that have been conducted with these parameters used prototypes. Studies that used CO2 laser with wavelength of 10.6 μm and energy densities from 0.3 to 12.5 J/cm2 have found a significant reduction of mineral loss [19, 35, 40–42]. These results, in addition to the possibility of using this technology available on the market to prevent caries disease, have stimulated the development of new research with the 1 10.6 μm. Tukey–Kramer However, when one refers to dentin and/or cement, the tissues of which the root surface is composed, few studies [16, 27, 43] have been conducted with the aim of demonstrating the effect of CO2 laser on reducing demineralization of these substrates. Significantly different from the composition of human enamel, dentin is composed of 70 wt% hydroxyapatite, 18 wt% collagen, and 10 wt% water [44]. The composition dissimilarity of dentin gives it different thermal properties from those of enamel. Therefore, knowing that for similar irradiation intensities, the temperatures produced in dentin are two times higher than they are in enamel, and theoretically, only half of the amount of an energy density, successfully tested in enamel, would be necessary to cause the same effects in dentin [26]. Table 4 Means, standard deviations, and percentage of caries inhibition in distance 1 (50 μm) and distance 2 (100 μm) Groups Distance from interface (μm) 50 μm Control Laser 5 J/cm2 Laser 6 J/cm2 ANOVA p value 100 μm ΔZ % caries inhibition ΔZ % caries inhibition 1,990.65 (442.89) a 1,409.03 (240.98) b 1,597.93 (308.42) b 0.0028 – 29.21 19.73 1,902.55 (345.76) a 1,394.01 (116.12) b 1,431.54 (258.05) b 0.0003 – 26.73 24.76 Means followed by different letters differ statistically among them by the Tukey test at a level of significance of 5 % Lasers Med Sci (2014) 29:525–535 In this study, both parameters (5 and 6 J/cm2) used were previously tested by Souza-Zaroni et al. [27], who verified a significant reduction on root demineralization and also used a thermographic analysis to demonstrate that these parameters promoted a small variation in temperature inside the pulp chamber (around 0.9 °C) during irradiation of the root surface, even when the highest energy density, 6 J/cm2, was used. In the present study, these laser parameters were also capable of promoting a significant reduction in demineralization of the root surface adjacent to resin composite restorations when compared with the control group, which was not submitted to any preventive treatment. However, although no thermal signs of carbonization were verified on the irradiated root surfaces, and the only visible change observed was a whitish appearance, probably indicating water loss, further studies are necessary to evaluate the thermal effects of the parameters used in the present study, since they are quite different from those employed by Souza-Zaroni et al. [27], who used the same pulsed mode of irradiation (10 ms pulse duration, with 10 ms time off and 50 Hz), but with the half speed of movement. The percentage of demineralization reduction observed in this study ranged from 19.73 to 29.21 in position 1 (50 μm) and from 24.76 to 26.73 in position 2 (100 μm), obtained in specimens irradiated with energy densities of 6 and 5 J/cm2, respectively. In addition, it was observed that at both distances from the tooth/restoration interface (50 or 100 μm), the mineral content of the root surfaces irradiated with CO2 laser, irrespective of the energy density used, was higher than that of nonirradiated surfaces up to a depth of 80 μm from the external dental root surface. At this depth, the action of CO2 laser on the root tissues differs from that found in enamel, as was shown in the study of Klein et al. [19], in which specimens of human tooth enamel irradiated with CO2 laser using energy densities of 8 or 16 J/cm2 showed higher mineral content than the nonirradiated specimens, only up to depth of 40 μm from the external surface of enamel at a distance of 50 μm from the tooth/ restoration interface. The probable deeper action of laser in dentin may be explained due to the different properties of such tissue and the different laser parameters used. Fried et al. [26] have observed that the surface temperatures of dentin were markedly higher than those measured on enamel for similar irradiation intensities due to the lower reflectance losses of dentin and the lower thermal diffusivity of dentin at the same laser wavelengths. Previous studies have also observed that dentin has longer thermal relaxation time (210 μs) than enamel (90 μs) [26, 45]. Considering these characteristics of this tissue, one may explain the fact that laser energy may affect deeper layers in dentin than in enamel. Furthermore, 533 the present study employed a shorter pulse (10 ms), and the repetition rate was a factor of five higher than the repetition used in the study of Klein et al. [19]. Some researchers have shown the protective effect of CO2 laser on dentin and/or on the root surface submitted to in vitro cariogenic challenges [23, 46]. However, it is important to emphasize that it is difficult to make a direct comparison of the results of this study with those reported in the literature, because the irradiation conditions used are quite different, with different energy densities, pulse durations, repetition rates, etc. On the other hand, it is verified that the findings of this study are closer to those reported by Gao et al. [43], in which there was 29.8 % reduction in root surface demineralization with the use of the CO2 λ 10.6 μm laser and energy density of 1.14 J/cm2. As also stated in the reports of Souza-Zaroni et al. [27], who found reductions of 17.05 and 20.59 % in root surface demineralization with the use of the CO2 λ 10.6 μm laser and energy densities of 5 and 6 J/cm2, respectively. Additionally, it should be considered that although the parameters used by the present study were the same of Souza-Zaroni et al. [27], the methodology condition of both studies is strongly different, since the irradiated area was modified, and the simulation of root demineralization adjacent to restorations involves the fact that the tooth structure immediately adjacent to restorations is more susceptible to caries progression, owing to the imperfect adaptation of restorative materials and subsequent microleakage. As regards the mechanism through which laser irradiation interacts with dental tissues, it is mostly related to the temperature increase caused after absorption. In general, dentin irradiation with a CO2 laser causes changes both to the mineral and to the organic matrix. Depending on the energy applied, carbonate can be reduced or eliminated, and crystallinity can be increased [26, 47]. Reduction of collagen content, loss of water, and formation of amorphous carbon bands have also been observed [17, 48]. It is, though, specially the reduction of carbonate and hydroxyapatite phase changes that happen between 600 and 900 8 C that have shown to be related to a decrease of tooth solubility after laser irradiation [26, 47, 49]. Therefore, considering the results of this study, it is suggested that future studies be conducted with the aim of comparing the effects of CO2 laser with established methods of caries prevention, as is the case of fluoride. The use of fluorides has also been shown to be efficient in inhibiting secondary caries when it is released by the restorative material [50] or as pretreatment of the cavity walls [51]. Nevertheless, the effect of fluoride is limited over time, while the duration of the effect of the laser remains unknown. In addition, there are many possibilities with regard to the combination of laser treatments and fluoride-based treatments, or restorative materials that release fluorides, which 534 may promote synergic effects on the resistance to the initiation and development of secondary caries. In conclusion, the findings of the present investigation suggest that the irradiation of dental cavities by using a CO2 laser reduces root surface demineralization around composite restoration, but it is necessary to use in situ and in vivo models to provide complementary data for a broader assessment of the behavior of the root surface adjacent to restorations when irradiated with CO2 laser in the view of cariogenic challenges that are a characteristic of these conditions. References 1. Elderton RJ, Nuttal NM (1983) Variation among dentists in planning treatment. Br Dent J 12:201–206 2. 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