Effect of Bleaching on Sound Enamel and with Early Artificial Caries Lesions Using Confocal Laser Microscopy

Vital tooth bleaching has gained popularity in recent years as an easy, affordable, and conservative way of treating discolored teeth. Since its introduction, bleaching has been accepted as an efficient and simple aesthetic procedure for removing intrinsic and extrinsic stains from teeth (1,2). In the last decade, many products with varying concentrations and techniques have been indicated for in-office use or mouthguard bleaching (3). Bleaching occurs due to the decomposition of peroxide into free radicals, which subsequently react Effect of Bleaching on Sound Enamel and with Early Artificial Caries Lesions Using Confocal Laser Microscopy


INTRODUCTION
Vital tooth bleaching has gained popularity in recent years as an easy, affordable, and conservative way of treating discolored teeth.Since its introduction, bleaching has been accepted as an efficient and simple aesthetic procedure for removing intrinsic and extrinsic stains from teeth (1,2).In the last decade, many products with varying concentrations and techniques have been indicated for in-office use or mouthguard bleaching (3).
Bleaching occurs due to the decomposition of peroxide into free radicals, which subsequently react with the large pigment molecules, transforming them into smaller, less pigment molecules and so the hydrogen peroxide (HP) oxidizes a wide variety of organic compounds (1,2).Some studies have found mineral loss, increased susceptibility to erosion or caries, increased surface roughness, reduced enamel tensile strength, reduced fracture stability or a decrease in abrasion resistance of bleached dental hard tissues (4,5).
In an attempt to decrease tooth sensitivity, reestablish surface hardness and support remineralization of initial white spot lesions, some manufacturers have incorporated fluorides into their bleaching gel formulations (6,7).More recently, amorphous calcium phosphate (ACP) has become available in tooth whitening products; however, the effects of ACP on enamel remineralization have rarely been analyzed (8).Calcium (Ca) ions have also been added in the bleaching agent's composition, however, little is known about the effectiveness of the ACP's components (9).
This study used confocal laser scanning microscopy (CLSM) to evaluate the effect of bleaching agents containing Ca on sound enamel and with early artificial caries lesions.The null hypothesis was that the Ca-containing bleaching agents would not effect the demineralization of sound enamel and promote remineralization of early artificial caries lesions.

Specimen Preparation
Twenty extracted sound bovine incisors stored in 0.1% thymol solution were used within 1 month of extraction.The roots were separated from the crowns with a water cooled low-speed diamond saw (Buehler-Series 15LC Diamond; Buehler, Lake Bluff, IL, USA).Crowns were sectioned mesio-distally and buccal-lingually to obtain 4 dental blocks (4 mm long x 3 mm wide x 3 mm thick) from each crown.Except for the buccal surfaces, all other surfaces of the dental blocks were sealed with acid-resistant nail polish (Revlon Corp., New York, NY, USA).Eighty dental blocks were randomly assigned to 8 groups (n=10).

Induction of Early Artificial Caries Lesions (pH Cycling) and Bleaching
Forty samples were subjected to a pH cycling model to induce early artificial carious lesions, while other 40 enamel samples remained sound.All dental blocks were submitted to a 8-day pH-cycling regimen, simulating daily acid challenge and bleaching treatments.Each cycle consisted of an individual immersion in a demineralization solution (3.12 mL/mm 2 ; composition: 1.4 mM Ca, 0.9 mM P, 0.05 M acetate buffer, pH 5.0) and remineralization solution (1.56 mL/mm 2 ; composition: 1.5 mM Ca, 0.9 mM P, 0.1 M Tris buffer, pH 7.0) for 8 and 16 h, respectively.Between immersions, and at the end of the cycle, the samples were rinsed with distilled and deionized water and gently dried with absorbent paper (10).Immediately after pH cycling, samples were subject to bleaching treatments described in Table 1.The enamel blocks were daily exposed to bleaching agent with 0.05 mL of artificial saliva and covered with an individual tray (10).

Post-Treatment Analysis
Samples were sectioned longitudinally through the treated area with a low speed water-cooled diamond saw (Isomet 1000; Buehler) and embedded perpendicular to the demineralized surface in epoxy resin (Buehler).Next, the samples were polished on a water-cooled polishing unit (EcoMet 3000; Buehler) with abrasive paper (400-, 600-and 1200-grit) and 0.3 and 0.1 μm diamond mask alumina suspension (Metaldi Supreme; Buehler).The polished samples were cleaned ultrasonically in deionized water for 15 min to remove the residues from polishing procedure.

CLSM
The samples were immersed in a fresh 0.1 mM rhodamine B solution (Aldrich Chem.Co., Milwaukee, WI, USA) for 1 h, without further water-rinsing (11,12).Samples were analyzed with CLSM (Zeiss LSM 510; Carl Zeiss, Inc., Germany), using an argon laser with a 529-nm excitation wavelength.Areas were scanned between 10 and 50 μm below the treated surface to reduce the influence of the smear layer created during the cutting and polishing procedure (11,12).The quantification of fluorescence demineralization area was analyzed with an image-analysis system (ImageJ 1.41; Wayne Rasband, National of Institutes of Health, Bethesda, MD, USA).

Statistical Analysis
Data were analyzed statistically by two-way ANOVA (factors: bleaching treatments x enamel conditions) at a 95% confidence interval and Fisher's PLSD test.

ResUlTs
The total fluorescence demineralization area (in μm 2 ) and standard deviations (S.D.) are shown in Table 2. Two-way ANOVA revealed that there was statistically significant difference for both factors "enamel" (p<0.0001) and "bleaching treatment" (p=0.0189).However, the interaction between these factors was not statistically significant (p=0.1510).
Fisher's PLSD test showed that sound enamel presented increase of demineralization area after bleaching treatments when compared to a control group (p≤0.0004).The fluorescence area in sound enamel for each bleaching treatment is depicted in Figure 1.The results of fluorescence area (demineralization) in the groups with early artificial caries lesions showed no significant difference among bleaching treatments (p=0.4795).The bleached groups were also statistically similar to the control group (p>0.05).Figure 2 illustrates the fluorescence areas of the groups with early artificial caries lesions.Comparison between the enamel condition (sound versus early artificial caries lesions) showed statistically significant difference (p<0.0207).

DIsCUssION
The first use of CLSM in dental research was proposed by Watson in 1989 (13).The objective of that study was to evaluate the morphology of the tooth-composite interfaces.González-Cabezas et al. (11) reported that CLSM could be used with thin enamel sections or half a tooth stained with a 0.1 mM solution of Rhodamine B. This method can replace or supplement microradiography analysis that quantify the demineralization and analyze the remineralization phenomenon.Few studies have used CSLM to evaluate the effect of bleaching agents on tooth structure (14,15), therefore, this study compared the effect of different formulations of bleaching agents on enamel demineralization of sound enamel and with early artificial caries lesions by quantifying the fluorescence areas.The bleaching products tested in this study contained as active ingredient the 4% and 7.5% HP and were applied for 8 h daily or for 30 min twice daily, respectively.One was a commercial product (with ACP) and the other two were experimental bleaching agents that contained only 7.5% HP or the addition of 0.05% Ca.All bleached groups were compared with unbleached groups and the results of this in vitro study demonstrated that a 14-day regimen of whitening using different formulations of bleaching agents produced different results on sound enamel and enamel with early artificial caries lesion.Sound enamel was more resistant to demineralization than carious enamel, which showed larger fluorescence demineralization area.
Moreover, the bleached groups showed similar demineralization fluorescence areas to unbleached groups, regardless of enamel conditions.Thus, the null hypothesis of this study was accept, since that the addition of Ca or ACP did not reduce the demineralization area on sound enamel and did not promote remineralization of the enamel with early artificial lesions.Tschoppe et al. ( 6) also evaluated the effect of fluoridated and ACP containing bleaching gels on remineralization of predemineralized bovine enamel and did not observed mineral uptake, which support the results of the present study.
A previous study evaluated some commercial bleaching agents containing HP or carbamide peroxide (CP) and did not found alterations on surface morphology after bleaching (4).However, other studies reported that bleaching agents produced mineral loss and formation of porosities on enamel surface (3,(16)(17)(18).The surface and subsurface changes correspond to the areas with significant dye penetration (Rhodamine B), as seen in Figures 1 and 2  enamel after treatments with 10% CP (8 h daily for 2 weeks) or 5.3% HP (1 h daily for 2 weeks) and found lesion depths of 4.8 μm and 1.6 μm, respectively.Attin et al. (21) showed that the reduction in hardness of enamel was confined to superficial layers, confirming the demineralization in depth.In this current study the demineralization depth caused by pH cycling was 135 μm, which corresponded to the control group (unbleached).The bleaching treatments did not increase the demineralization depth for the pH-cycled groups that ranged from 166 (4% HP + 0.05% Ca or 4% HP) to 168 μm (7.5% HP + ACP).The whitening treatment of sound bleached, resulted in a demineralization depth, which ranged from 31 (4% HP) to 52 μm (7.5% HP + ACP).
pH cycling was used because it simulates the dynamic process of caries lesion development, alternating demineralization and remineralization periods (10).The role of Ca and phosphate ions in demineralization and remineralization process is clearly established, but the influence of organic content of saliva should also be considered.This is a limitation of an in vitro study that cannot reproduce all variables acting in in situ or in vivo studies.
Increase of dye penetration was more intense for the enamel with artificial caries lesions (Fig. 2) than sound bleached enamel due to the mineral loss induced by pH cycling.Figures 2B and 2D are representatives CSLM images of groups submitted to bleaching agents containing Ca ions and ACP, respectively.Their demineralization fluorescence areas were similar to that observed for 4% HP (Fig. 2C) and control group (Fig. 2A).Thus, the induction of artificial caries lesions for pH cycling was the determinant factor for dye penetration and not the bleaching treatments.Furthermore, the bleaching composition had no influence on demineralization of sound enamel or enamel with early artificial lesions.
The bleaching treatments promoted increased sound enamel demineralization, while the addition of Ca ions or ACP did not prevent/reverse the effects caused by the bleaching treatment in both conditions of the enamel.Early artificial caries induced by pH cycling model were not affected by the bleaching treatment, regardless of the type of bleaching agent.

Table 1 .
Material used in the study.

Table 2 .
Total area of demineralization (in μm 2 ) and standard deviation in the groups measured by fluorescent dye using confocal laser scanning microscopy.
Different uppercase letters in columns and lowercase letters in rows indicate statistically significant differences among groups (Fisher's test, p<0.05).HP: hydrogen peroxide; ACP : amorphous calcium phosphate; EACL: early artificial caries lesions.