Effect of Er,Cr:YSGG Laser and Professional Fluoride Application on Enamel Demineralization and on Fluoride Retention

Original Paper Received: April 20, 2012 Accepted: April 24, 2012 Published online: June 27, 2012

Caries Res 2012;46:441–451 DOI: 10.1159/000333603

Effect of Er,Cr:YSGG Laser and Professional Fluoride Application on Enamel Demineralization and on Fluoride Retention P.A. Ana a C.P.M. Tabchoury b J.A. Cury b D.M. Zezell c a Universidade Federal do ABC, UFABC, Santo André, b Piracicaba Dental School, University of Campinas, Piracicaba, and c Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, São Paulo, Brazil

Key Words Demineralization ⴢ Enamel caries ⴢ Fluoride uptake, in vitro ⴢ Lasers ⴢ

Abstract This study evaluated the effect of Er,Cr:YSGG laser irradiation and professional fluoride application on enamel demineralization and on fluoride formation and retention. In a blind in vitro study, 264 human enamel slabs were distributed into 8 groups: G1 – untreated; G2 – treated with acidulated phosphate fluoride gel (APF gel, 1.23% F) for 4 min; G3, G4 and G5 – irradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively; G6, G7 and G8 – preirradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively, and subjected to APF gel application. Twenty slabs of each group were submitted to a pH-cycling regimen, and enamel demineralization was evaluated in 10 slabs of each group. In the other 10 slabs, CaF2like material was determined. To evaluate F formed, 10 additional slabs of each group, not subjected to the pH cycling, were submitted to analysis of CaF2-like material and fluorapatite, while the other 3 slabs of each group were evaluated by scanning electron microscopy. The F content was also measured in all pH-cycling solutions. Laser at 8.5 J/cm2 and APF treatment reduced enamel demineralization compared to the control (p ! 0.05), but the combination of these treat-

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ments was not more efficient than their isolated effect. A higher concentration of retained CaF2-like material was found in laser groups followed by APF in comparison with the APF gel treatment group. The findings suggest that laser treatment at 8.5 J/cm2 was able to decrease hardness loss, even though no additive effect with APF was observed. In addition, laser treatment increased the formation and retention of CaF2 on dental enamel. Copyright © 2012 S. Karger AG, Basel

The Er,Cr:YSGG laser, with a wavelength of 2.78 ␮m, is widely used in dentistry for cavity preparation, caries removal, surgeries, endodontics and other applications, and is commercially available, which offers a great advantage for clinicians. Due to this fact, this wavelength has also been studied for caries prevention, because of its higher absorption by water and OH– contents of hydroxyapatite [Fried et al., 1996; Seka et al., 1996]; hence, Er,Cr:YSGG laser irradiation promotes surface temperatures up to 800 ° C at the ablation threshold [Fried et al., 1996]. The higher absorption of laser energy in the enamel surface can lead to changes in the crystalographic structure of enamel as a result of the surface temperature increase during irradiation, and it depends on the fluence applied [Apel et al., 2002].  

 

Denise Maria Zezell Centro de Lasers e Aplicações, Laboratório de Biofotônica Instituto de Pesquisas Energéticas e Nucleares, Av. Prof. Lineu Prestes 2242 Cidade Universitária, São Paulo, SP 05508-000 (Brasil) Tel. +55 11 3133 9370, E-Mail zezell @ usp.br

The effects of laser irradiation associated with fluoride application have been reported using other wavelengths, such as CO2, Nd:YAG, Argon and Er:YAG, showing a significant synergism on reducing enamel demineralization and increasing fluoride retention. Several studies revealed the benefits of laser irradiation on preventive dentistry, and some contradictory observations concerning the Er,Cr:YSGG laser [Fried et al., 1996; Hossain et al., 2001; Apel et al., 2002, 2004] were reported. However, there is no consensus in the literature about the parameters of the Er,Cr:YSGG laser that could be used for this application, and there is no report which explains the interaction of this wavelength with the use of professional fluoride application either. Therefore, the purpose of this study was to investigate the parameters of the Er,Cr:YSGG laser, which could improve the resistance of enamel to demineralization, and to verify the action of laser associated with professional fluoride application in order to confirm the potential of this association to prevent dental caries on enamel.

Material and Methods Experimental Design A blind in vitro study was conducted and 264 sound human enamel slabs were randomized in 8 treatment groups of 33 specimens each: G1 – untreated enamel surface; G2 – treated with acidulated phosphate fluoride gel 1.23% F (APF gel), pH 3.6–3.9, for 4 min; G3, G4 and G5 – irradiated with Er,Cr:YSGG laser at 2.8, 5.6 and 8.5 J/cm2, respectively; G6, G7 and G8 – combination of preirradiation with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively, followed by APF gel application. Twenty slabs of all groups were further submitted to a pH cycling [Argenta et al., 2003] and after that each group was randomized in 2 groups of 10 specimens each. Enamel demineralization was evaluated by cross-sectional microhardness in 10 slabs, and in the other 10 slabs the CaF2-like material retained was determined. The total content of enamel fluoride formed (expressed by the analysis of CaF2-like material and fluorapatite, FA) was also determined in 10 additional slabs of each group, which were not subjected to the pH-cycling regimen. The remaining 3 slabs of each group, not submitted to pH cycling, were examined by scanning electron microscopy (SEM). For the statistical analysis, each treatment was considered a separate block and the experimental unit was the slab (n = 10). The sampling size of 10 enamel slabs/treatment was established in our previous studies and confirmed by a pilot study, which showed that for a power of 95% (␤ = 0.05) to detect a difference among treatments at the 5% level (␣ = 0.05) in terms of enamel demineralization and fluoride uptake, 8 and 4 slabs would be enough, respectively. Preparation of the Specimens and Treatments The enamel slabs (4 ! 4 ! 2 mm) were obtained from smooth surfaces of 132 third human molar teeth, obtained from the Hu-

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man Teeth Bank of the University of Sao Paulo after approval from the Human Ethics Committee of the Nuclear and Energy Research Institute (Proj. CEP-IPEN 094-2004). The slabs were obtained from the central area of proximal surfaces of all teeth. After examination under a stereomicroscope to verify the absence of cracks and white spots, the selected slabs were cleaned with pumice during 10 s, and kept in a humid environment under refrigeration until the beginning of the experiments. The laser irradiation was performed using an Er,Cr:YSGG hydrokinetic laser device (Millenium, Biolase Inc., San Clemente, Calif., USA), which operates at a wavelength of 2.78 ␮m, pulse width of 140 ␮s, repetition rate of 20 Hz and a power output ranging from 0 to 6 W. The energy is delivered through a fiberoptic system with 430 ␮m of spot size ended by a sapphire terminal of 750 ␮m diameter and 6 mm long (S75 tip), bathed in an adjustable air and water spray. All samples of this study were irradiated without air and water spray [Bevilácqua et al., 2008], and were immobilized in X-Y-Z optical supports. The laser handpiece was coupled to a computer managed motion control system (Newport, Irvine, Calif., USA) adjusted to a speed of 4 mm/s in order to avoid unlased areas or overlapping of focused areas. The laser tip was kept at a standardized distance of 1 mm from the enamel surface, and laser irradiation was done scanning all areas of enamel surface. The spot size on the sample surface is 0.376 mm at 1/e2 energy density. APF gel (1.23% F–, 0.1 M of phosphoric acid, pH 3.6–3.9 – NuproGel쏐, Dentsply Industries, Petropolis, R.J., Brazil) was applied to the enamel slabs with a cotton swab for 4 min, followed by washing with distilled and deionized water during 1 min and drying with absorbent paper [Delbem and Cury, 2002]. pH Cycling After treatments, all surfaces of specimens were protected with an acid-resistant varnish, except for a circular area [Delbem and Cury, 2002] of 3.14 mm2 of enamel, where the treatments were applied. Twenty slabs of each group were subjected to a 10-day in vitro pH cycling validated protocol [Argenta et al., 2003] that showed a dose-response effect to F treatment. The slabs were kept individually in a demineralizing solution for 3 h (6.37 ml/mm2); after that, the slabs were immerged in a remineralizing solution for 20 h (3.18 ml/mm2) each day, at 37 ° C. At the time of changing solutions, all slabs were individually washed with distilled and deionized water for 15 s and dried with absorbent paper and, then, returned to the same solution of the previous day. The solutions were changed only on the 5th day of the experiment in order to avoid an excessive release of CaF2-like material formed. The demineralizing solutions were composed of 2.0 mM calcium, 2.0 mM phosphate, 0.03 ppm F– in 75 mM acetate buffer, pH 4.3, and the remineralizing solutions were composed of 1.5 mM calcium, 0.9 mM phosphate, 150 mM of KCl, 0.05 ppm F– in 20 mM cacodylic buffer, pH 7.4. All solutions contained thymol (0.64 g/l) to avoid fungal growth and were prepared at the same time in order to avoid differences in their composition.  

 

Enamel Demineralization Assessment After pH cycling, 10 slabs of each group were longitudinally sectioned through the center of the exposed enamel and embedded in acrylic resin so that the cut section could be exposed. A FMARS FM 7386 (Future Tech, Kawasaki, Japan) microhardness tester with a Knoop diamond with 25 g load for 5 s was used [Ar-

Ana /Tabchoury /Cury /Zezell  

 

 

 

genta et al., 2003]. Three rows of 12 indentations each were made, one in the central region of the dental enamel exposed and the other two 100 ␮m below and above this; the indentations were made at 10 ␮m from the outer enamel surface up to 160 ␮m. The mean values at all measuring points at each distance from the surface were then averaged and expressed as Knoop hardness number (kg/mm2), since there is a discrepancy in the literature regarding conversion of hardness to mineral concentration [Featherstone et al., 1983; Kielbassa et al., 1999]. The area of hardness loss was calculated by numerical integration using a trapezoidal rule by the difference between the area under the curve (kg/ mm2 ! ␮m) of the sound enamel minus the area of the demineralized one. Fluoride Determination in Enamel After demineralization, the other 10 samples of each group were protected with wax No. 7 and individually immersed in plastic tubes containing 0.5 ml of 1.0 M KOH to extract the CaF2-like material retained in enamel after the pH cycling [Caslavska et al., 1975]. After 24 h, the extracts were neutralized with 0.5 ml of TISAB II containing 1.0 M HCl. These analyses were also made in the remaining 10 additional slabs of each group, which were not subjected to the pH-cycling regimen. In these same samples, firmly bound fluoride (FA) found in enamel was also extracted by acid etching. Three layers of enamel were sequentially removed by immersion of each slab in 0.25 ml of 0.5 M hydrochloric acid for 15, 30 and 60 s under constant agitation. An equal volume of TISAB II, pH 5.0, modified with 20 g NaOH/l, was added to each solution containing the dissolved enamel layer [Paes Leme et al., 2003]. All fluoride measurements were performed using an ion-selective electrode (Orion 96-09, Orion Research, Boston, Mass., USA) and an ion analyzer (Orion 720 A+, Orion Research) previously calibrated with proper F standard solutions for the analysis of CaF2-like material and FA. The concentration of CaF2-like material was expressed in micrograms F per square centimeter; the amount of FA found in each layer of enamel removed was summed and the results were also expressed in micrograms F per square centimeter. Fluoride Determination on pH-Cycling Solutions The fluoride analysis was performed in all pH-cycling solutions (demineralizing and remineralizing ones) used in the present study at three different times: before pH cycling, after 5 days and after 10 days of pH cycling. For that, samples were prepared with TISAB III. An ion-selective electrode (Orion 96-09, Orion Research) and an ion analyzer (Orion 720 A+, Orion Research) were used, previously calibrated with proper F standard solutions. The concentration of fluoride in each pH-cycling solution was expressed in micrograms F per square centimeter. Morphological Analysis Three samples of each experimental group, which were not subjected to the pH-cycling regimen, were half fractured and attached to aluminum stubs with a self-adhesive carbon conductive tape. Samples were examined by a scanning electron microscope (JEOL 6460LV, Tokyo, Japan), under low vacuum (150 Pa), using 30 kV accelerating voltage. SEM micrographs were made on treated enamel surfaces and on fractured ones, mounted with the fractured surface normal to the incident laser beam [Nelson et al.,

Effects of Er,Cr:YSGG Laser and APF on Demineralization

1983]. For the evaluation of the elemental composition of the surfaces, an energy-dispersive spectrometer system (EDS; Noran System Six) attached to the SEM device was used. The elemental microanalysis was performed under 15 kV accelerating voltage. Statistical Analysis Before the statistical analysis, independence, homogeneity and normality of variances of experimental data were tested. The independence was assured by the way the experiments were conducted. The homogeneity was evaluated by Levene’s test and the normality was evaluated by the Shapiro-Wilks test (all tests performed at a significance level of 5%). After the confirmation of all these requirements, all results were analyzed by ANOVA (2-factor with replication) followed by Tukey’s test. Only the results of the CaF2-like material analysis were transformed by log10 before ANOVA. For all analyses, 5% was considered the limit of significance, and the software SPSS 13.0 for Windows (SPSS Inc., Chicago, Ill., USA) was used.

Results

The isolated effect of the treatments (table 1) showed that the applications of APF gel (G2) or laser at 8.5 J/cm2 alone (G5) were efficacious to reduce enamel demineralization in comparison with the untreated control group (G1; p = 0.0001). It was also evidenced that the use of laser alone, at any energy density, was not able to reduce the loss of hardness when compared to the APF group. In addition, although laser at lower energy densities (G3 and G4 groups) was not able to reduce demineralization compared with the control (p = 0.0839), the combination with APF gel was more efficacious (p ! 0.0001). In fact, the combination resulted in a significant reduction in hardness loss for all energy densities when compared to the use of laser alone (p ! 0.0001). However, no additive effect was found when laser at the highest energy density tested and APF gel were combined, because the reduction of enamel demineralization was not greater than the isolated effect of these treatments (p = 0.1446). These data are illustrated in figure 1, showing that the caries lesion depth extended up to 100 ␮m from the surface. Regarding CaF2-like material found in enamel after the pH cycling (table 2), the results showed that although Er,Cr:YSGG laser alone, at any energy density, did not promote an increase in CaF2-like material formation immediately after irradiation, this laser promoted a significant increase in CaF2-like material retention when compared to the untreated group (p = 0.0119), independently from the energy density used. When combined with APF gel application, laser irradiation at 8.5 J/cm2 promoted a significant augmentation of CaF2-like material formation (p = 0.0002) when compared to the positive control Caries Res 2012;46:441–451

443

450 400

Knoop hardness (kg/mm2)

350 300 250

Control APF

200

Laser 2.8 J/cm2 Laser 2.8 J/cm2 + APF

150

Laser 5.6 J/cm2 Laser 5.6 J/cm2 + APF

100

Laser 8.5 J/cm2 50

Fig. 1. Means of enamel Knoop hardness

(kg/mm2) according to treatments and the distance (␮m) from the surface (bars denote standard errors; n = 10).

Laser 8.5 J/cm2 + APF

0 0

10

20

Table 1. Means 8 SD (n = 10) of area of enamel hardness loss (⌬S)

according to the treatments Treatments

⌬S, kg/mm2 ! ␮m

Untreated APF Laser 2.8 J/cm2 Laser 2.8 J/cm2 + APF Laser 5.6 J/cm2 Laser 5.6 J/cm2 + APF Laser 8.5 J/cm2 Laser 8.5 J/cm2 + APF

11,338.982,759.6a 5,954.582,588.2c 9,283.783,735.1a, b 5,943.983,485.2c 11,321.383,084.3a 4,178.482,094.8c 7,226.883,838.5b, c 4,275.482,544.2c

Treatments followed by distinct superscript letters differ statistically by the Tukey test (p < 0.05).

group (APF). Also, it is possible to observe that there is a significant increase in CaF2-like material retention after pH cycling (p = 0.0112), and this fact is observed with laser energy densities of 5.6 and 8.5 J/cm2 used in combination with APF application. The initial content of F on enamel structure (FA), only determined in treated samples, is shown in table 2. In all groups with APF application, a very high amount of F was deposited within the enamel. Data collected also show that the laser irradiation was not able to increase concen444

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30

40

50

60

70

80

90 100 110 120 130 140 150 160

Surface distance (μm)

trations of F in enamel in comparison with the control groups (p = 0.2521). In this way, no interaction of laser irradiation with APF application on FA formation was statistically evidenced. The analysis of fluoride on pH-cycling solutions showed a reduction of fluoride content on both demineralizing and remineralizing solutions in the control group after 5 and 10 days of pH cycling (fig. 2); however, in the APF group, an increase in fluoride concentration was observed in remineralizing solutions mainly after 10 days of pH cycling. An important fluoride release was noted in remineralizing solutions of groups treated with laser 5.6 J/cm2 + APF and laser 8.5 J/cm2 + APF, which was indicated by the significant increase in fluoride concentration in pH-cycling solutions of these groups. The morphological analysis by SEM evidenced the slightly rough surfaces promoted by laser irradiation used at the conditions of this study (fig. 3–5), which depended on the energy density applied. The surface morphology of enamel irradiated at 2.8 J/cm2 presented an increased roughness (fig. 3a) when compared to untreated samples (fig. 6a), while samples irradiated at 5.6 and 8.5 J/cm2 also presented slight cavities, typical of microablation areas, with fissures and conical craters with sharp enamel projections (fig.  4a, 5a). In samples with APF application, the formation of a uniformly thick surface coating, with the formation of some globules, was Ana /Tabchoury /Cury /Zezell  

 

 

 

0.16

De. 5 days De. 10 days

F– concentration (μg F–/ml)

0.14

Re. 5 days Re. 10 days

0.12 0.10 0.08 0.06 0.04 0.02 0 Initial

Control

APF

2.8 J/cm2

2.8 J/cm2 + APF

5.6 J/cm2

5.6 J/cm2 + APF

8.5 J/cm2

8.5 J/cm2 + APF

Fig. 2. Means of fluoride concentration (␮g F–/ml) on demineralizing (De.) and remineralizing (Re.) solutions

at the initial time (initial), after 5 days and after 10 days of pH cycling. Bars denote standard errors.

Table 2. Means 8 SD (n = 10) of CaF2-like material concentration found on enamel before (formed) and after

pH cycling (retained) and means 8 SD (n = 10) of FA concentration found on enamel before pH cycling, according to the treatments Treatment groups

Untreated APF Laser 2.8 J/cm2 Laser 2.8 J/cm2 + APF Laser 5.6 J/cm2 Laser 5.6 J/cm2 + APF Laser 8.5 J/cm2 Laser 8.5 J/cm2 + APF

CaF2-like material, ␮g F–/cm2 formed (before demineralization)

retained (after demineralization)

2.581.0a 115.8838.8b 1.780.3a 123.6852.9b 2.880.7a 177.6857.4b 2.680.5a 216.3851.6c

0.680.2d 24.4817.6f 3.783.4e 25.189.6f 2.480.9e 44.889.8g 1.981.3e 50.0822.8g

FA, ␮g F–/cm2 formed (before demineralization) 1.980.8a 8.382.2b 2.480.7a 5.281.9b 2.982.3a 4.382.1b 2.880.9a 5.281.6b

Treatments followed by distinct superscript letters differ statistically by the Tukey test (p < 0.05).

observed (fig. 6b). In irradiated surfaces, in addition to the surface coating, it is possible to note the tendency to form agglomerates of particles in the irregularities promoted by laser irradiation (fig. 3b, 4b, 5b). Figures 3d, 4d and 5d show details of CaF2-like material globules highly agglomerated in depressions formed on enamel after laser irradiation, evidencing particles of up to 1 ␮m in diameter. Figure 7 shows the result of the EDS microanaly-

sis, evidencing the increase in fluoride ion content in the region with the higher concentration of globules. Figure 8 shows the surface coating in APF (fig. 8a) and laser + APF samples (fig. 8b–d), where the formation of smaller globules on APF-treated enamel can be noted, while higher globule concentrations can be found on enamel irradiated at 5.6 and 8.5 J/cm2.

Effects of Er,Cr:YSGG Laser and APF on Demineralization

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Fig. 3. SEM images of enamel surfaces irradiated at 2.8 J/cm2 (a) and at 2.8 J/cm2 + APF (b–d). a Rough surfaces promoted by laser irradiation, which can retain the CaF2-like material globules as it can be seen in b. c Fractured cross-section, evidencing the retention of CaF2-like material globules (arrows). d Higher magnification of b, where the higher concentration of CaF2-like material globules on rough surfaces can be seen. Original magnification: a, b !1,000; c !2,000; d !5,000.

a

b

c

d

a

b

c

d

Fig. 4. SEM images of enamel surfaces irradiated at 5.6 J/cm2 (a) and at 5.6 J/cm2 + APF (b–d). a The typical microablation

pattern promoted by laser irradiation, which can retain the CaF2-like material globules as it can be seen in b (arrows). c A fractured cross-section, evidencing the retention of CaF2-like material globules (arrows). d Higher magnification of b, where the higher concentration of CaF2-like material globules near rough surfaces can be seen. Original magnification: a, b !1,000; c !2,000; d !5,000.

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Ana /Tabchoury /Cury /Zezell  

 

 

 

a

b

c

d

a

b

c

d

Fig. 5. SEM images of enamel surfaces irradiated at 8.5 J/cm2 (a) and at 8.5 J/cm2 + APF (b–d). a The typical microablation

pattern promoted by laser irradiation, which can retain the CaF2-like material globules as it can be seen in b (arrows). c A fractured cross-section, evidencing the retention of CaF2-like material globules (arrows). d Higher magnification of the treated surface, where the higher concentration of CaF2-like material formation can be seen. Original magnification: a, b !1,000; c !2,000; d !5,000.

Fig. 6. SEM images of enamel surfaces untreated (a) and treated with APF gel (b), evidencing the presence of CaF2-like material globules on APF-treated samples. c, d SEM images of fractured cross-sections of enamel untreated (c) and treated with APF (d), also evidencing the presence of CaF2-like material globules (arrows). Original magnification: a !1,000; b !10,000; c, d !2,000.

Effects of Er,Cr:YSGG Laser and APF on Demineralization

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447

×2,000 30347

65535

504 Counts

Group laser 2.8 J/cm2 + APF X: 583 Y: 18 I: 37827

OK: 169 FK: 52 CaK: 581

336

168

a

5 34

.9

7

9 .1

.0 31

0 27

23

.3

2

4 .5

.4 19

.6

5 15

88

77 7.

11

10 μM

3.

0.

00

0

Size (μm)

b

Fig. 7. EDS analysis of the sample of figure 3b, evidencing the increase in the fluoride signal (white line) in the center of the higher globule concentration (a). This observation suggests that these globules could be globules of CaF2-like material (b).

Discussion

The use of laser irradiation for preventing dental caries is based on the chemical, physical and crystalline changes induced on enamel due to the heating of the surface. In fact, the heat generated in depth by laser irradiation could be responsible for a long-lasting preventive effect together with fluoride, which is reported in the literature [Westermann et al., 2003; Tepper et al., 2004; Demortier and Nammour, 2008; Zezell et al., 2009a. The results of the present study (table  1) show that, regarding laser irradiation alone, only the condition of 8.5 J/cm2 was able to reduce loss of hardness when compared to the untreated group, which confirms that higher temperatures are necessary to change the chemical properties of enamel. In fact, previous studies showed that Er,Cr:YSGG laser irradiation, at similar parameters than those used in the present study, is able to promote crystalline changes on enamel, promoting the formation of new crystallographic phases that can interfere in enamel resistance to demineralization [Bachmann et al., 2009]. The loss of hardness in lased groups decreased with the increase in the laser energy density, and this fact suggests that the interaction of laser irradiation with enamel has a direct relation with the energy density [Apel et al., 2002]. The data are also in agreement with Fried et al. [1996], who described a decrease of 30% in mineral loss of samples irradiated with Er,Cr:YSGG laser at 9.6 J/cm2. Later, Apel et al. [2004] reported that the energy density of 8 J/cm2 was not able to alter the surface microhardness 448

Caries Res 2012;46:441–451

after an in situ cariogenic challenge when compared to the untreated group using a similar but different laser equipment, in which the volunteer sampling was small, they were subjected to a low cariogenic challenge and for a short period of time. Differences in the sample characteristics such as hardness, total mass, thickness and hydration can lead to different results when the sample interacts with lasers or even with a drill. Other important factors are laser fluence (effective beam diameter on the sample), pulse width and repetition rate. To predict laser effects on enamel, besides sample characteristics, the laser equipment/manufacturer and irradiation conditions must also be considered. In fact, previous studies have shown that Er,Cr:YSGG laser irradiation, at the same energy densities as those of the present paper, are able to promote significant chemical changes, such as loss of carbonate, organic matter and water [Zezell et al., 2009b. Also, it was evidenced that laser irradiation promotes crystallographic changes on enamel, including the formation of tricalcium phosphate in the alpha phase, tricalcium phosphate in the beta phase and tetracalcium phosphate [Bachmann et al., 2009]. These changes can be responsible for the improved overall resistance of irradiated enamel to demineralization reported by previous studies [Fried et al., 1996; Apel et al., 2002, 2004]. Still concerning enamel hardness, the benefits obtained with laser irradiation alone are not superior to those obtained with topical APF treatment (table 1), but had similar effects to APF when the laser was applied at 8.5 J/cm2. An interesting fact is that, although Er,Cr:YSGG Ana /Tabchoury /Cury /Zezell  

 

 

 

Fig. 8. SEM images of enamel surface treated with APF (a), irradiated at 2.5 J/ cm2 + APF (b), irradiated at 5.6 J/cm2 + APF (c) and irradiated at 8.5 J/cm2 + APF (d), evidencing the differences in CaF2like material layers formed after irradiation. Original magnification !20,000.

a

b

c

d

laser irradiation has a depth penetration of about 2 ␮m in enamel [Seka et al., 1996], the laser energy density of 8.5 J/cm2 reduced the surface solubility and permeability so that the effect on reducing loss of hardness was detected up to 40 ␮m, similar to that obtained by APF application (fig.  1). The effect of the Er,Cr:YSGG laser in depth agrees with that reported by Fried et al. [1996], even using a different demineralization model. In this work, the enamel treatment with the association of laser and fluoride resulted in the reduction of hardness loss when compared to only irradiated groups, with 63% of reduction considering the condition of 5.6 J/ cm2 + APF (table 1). However, the effects of an association of laser and fluoride, in any laser parameter used, were not higher than the effect of topical APF application alone. Although there is no data reporting the use of Er,Cr:YSGG with APF in the literature, these findings are consistent with the ones obtained with CO2 [Santos et al., 2002] and Er:YAG [Delbem et al., 2003] lasers, which reported a reduction of 20% in hardness loss when comparing APF + laser and only-laser groups. On the other hand, taking into account the higher formation and retention of CaF2-like material shown in laser + APF groups in the present study (table 2), it is possible to suppose that the effect of the association of treatments on reducing the hardness loss could be demonstrated using a longer demineralization model.

Concerning the formation and retention of CaF2-like material, laser irradiation increased the CaF2-like material formation at the condition of 8.5 J/cm2 + APF (table 2), and this increase also had a positive relation with the energy density applied. This fact could be due to the ablation sites promoted by Er,Cr:YSGG laser irradiation [Ana et al., 2007], exposing a higher number of hydroxyapatite crystals that reacted with APF, and, as a consequence, a significantly higher quantity of CaF2-like material was produced. It is reported that the thermal effect of laser is responsible for promoting enamel uptake; however, the results of the present study confirm the hypothesis that laser-induced surface alterations, such as an increase in roughness, may also be a factor for enhancing fluoride formation and retention [Zhang et al., 1996; Chin-Ying et al., 2004]. The minimal surface changes promoted by laser irradiation are acceptable for a future clinical application mainly due to the benefits promoted in both chemical composition of enamel and calcium fluoride-like material formation and retention. The higher formation of CaF2-like material can be seen in SEM pictures (fig. 3–5, 8) and by EDS microanalysis (fig. 7), which confirmed the higher quantity of CaF2-like material globule formation on lased enamel, mainly near the ablation sites (fig. 3d, 4d, 5d). SEM micrographs also suggest that the globules formed after laser irradiation at 5.6 and 8.5 J/cm2 are also higher in size when compared to

Effects of Er,Cr:YSGG Laser and APF on Demineralization

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the globules formed in the APF group (fig. 8c, d). These findings imply a higher thickness of the surface layer deposited, which propitiates the remaining of CaF2-like material for relatively long periods of time, confirming the hypothesis of Nelson et al. [1983]. However, further studies are necessary to evaluate the long-time effect of this higher CaF2-like material globule formation on laserirradiated enamel. It is possible to note that, in the combined groups of laser 5.6 J/cm2 + APF and 8.5 J/cm2 + APF, a higher content of CaF2-like material after demineralization was found on enamel when compared to the APF group, which indicates that laser irradiation also increases fluoride retention, which can be a result of higher fluoride formation after treatments. Several literature studies also showed an enhanced fluoride adsorption [Tagomori and Morioka, 1989], uptake [Chin-Ying et al., 2004; Tepper et al., 2004; Schmidlin et al., 2007] and retention [Nammour et al., 2003; Demortier and Nammour, 2008] due to laser irradiation. Although several mechanisms were suggested to explain the increase in fluoride retention after laser irradiation, it seems that the surface roughness plays a role in increasing fluoride retention. The analysis of table  2 also shows that Er,Cr:YSGG laser irradiation alone increases the fluoride retention in comparison with the untreated group, which suggests that lased enamel could be more reactive to fluoride [Hossain et al., 2002], probably increasing its adhesion to the underlying surface of enamel, and then propitiating retention of fluoride presented in pH-cycling solutions. Even at low concentration (less than 0.2 ppm in some cases; fig. 2), the available fluoride in these solutions could have attracted the calcium ions and could have been incorporated into the hydroxyapatite [Featherstone, 1999], forming an FA-like layer that was extracted by Caslavska’s method [Caslavska et al., 1975]. Table 2 also shows that APF forms a higher quantity of FA in enamel relative to the untreated group, confirming data from the literature [Paes Leme et al., 2003]. However, the analysis of FA revealed that laser irradiation was not able to increase the FA amount formed on enamel, either when applied alone or before APF application, at any laser parameter. In fact, the formation of FA due to laser irradiation was demonstrated only with the use of CO2 lasers [Meurman et al., 1997; Phan et al., 1999], which promoted temperature increments of about 1,200 ° C on enamel and has an optical absorption coefficient 13 times higher than Er,Cr:YSGG laser on enamel [Featherstone, 2000]. In this way, it seems that the FA formation after laser irradiation is also dependent on the temperature, and the tempera 

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ture increments promoted by Er,Cr:YSGG laser irradiation are insufficient to promote such a kind of formation in the present study. Subsequently, at the irradiation conditions of this study, the Er,Cr:YSGG laser did not promote an increase in fluoride uptake on enamel. In the pH-cycling solutions, a significant increase in fluoride content was observed in remineralization solutions of groups treated with laser 5.6 J/cm2 + APF and 8.5 J/cm2 + APF, which suggests a fluoride release from enamel (fig. 2). The results agree with the observed higher formation of CaF2-like material in these groups (table 2), implying that laser irradiation can enlarge the effect of the APF gel. Although this increment of fluoride concentration was detected in remineralizing solutions, F concentrations were not superior to 0.15 ppm at any time; in this way, the rate of demineralization could not be inhibited by fluoride in pH-cycling solutions, since the F concentration was not higher than 1 ppm [Margolis et al., 1986]. These results support the hypothesis that the effects observed on demineralization resulted from a reduced solubility promoted by laser + APF treatments and were not due to a topical effect of F dissolved in the demineralizing solutions. In conclusion, the effects on loss of hardness seem to suggest that there is no advantage in using laser alone or as a pretreatment for topical APF over just using topical APF. However, the most promissory effect of Er,Cr:YSGG laser for caries prevention seems to be the enhancement of the CaF2-like material formation and retention on enamel after APF application, which could possibly maintain the cariostatic effect of APF for a longer period of time even with a single application. Thus, the energy density of 8.5 J/cm2 is indicated, because it is the parameter which also reduced loss of enamel hardness, with similar effects than topical APF treatment.

Acknowledgments The manuscript was based on a thesis submitted by the first author to the Instituto de Pesquisas Energéticas e Nucleares, S.P., Brazil, in partial fulfillment of the requirements of the PhD Program in Sciences. The authors would like to thank FAPESP (Proc. 2004/02229-6), PROCAD-CAPES (Proc. 0349/05-4) and CEPOFFAPESP (Proc. 05/51689-2) for giving financial support for this investigation.

 

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Disclosure Statement There are no conflicts of interest.

Ana /Tabchoury /Cury /Zezell  

 

 

 

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