INTRODUCTION

Some studies have demonstrated that tooth bleaching agents can produce changes in the enamel surface properties,^1-6 while others showed no alterations.^7,8 The changes were attributed to the low pH of bleaching gels,^2,3,9 but other studies reported that even neutral pH bleaching gels can cause morphological alterations in the enamel surface.^1,10 These changes were characterized by increased surface porosity, degradation of the organic matrix, and loss of calcium and phosphate, causing reduction in surface microhardness.^1,4-6 This reduction may increase enamel wear produced by tooth brushing. The intensity of this wear depends on the pH of the bleaching gels, the abrasive potential of the toothpaste, and the timing of tooth brushing after bleaching.^11-13 All these factors lead to increased surface roughness and staining susceptibility.¹⁴

With the intention of reversing the demineralizing effects of bleaching agents on dental enamel, several studies have investigated the effects of treatment using remineralizing gels after bleaching to recover enamel surface microhardness. Many substances were tested, such as sodium fluoride,^15,16 nanohydroxyapatite, hydroxylated apatite, and casein phosphopeptide-amorphous calcium phosphate.¹⁷ Fluoride is widely used in postbleaching treatment, since fluoride-containing agents promote the deposition of calcium fluoride on the enamel surface.¹⁸ This increases the incorporation of fluoride ions into the demineralized area in the form of fluorapatite or fluoridated hydroxyapatite.^19,20

Other remineralizing substances have been added to bleaching gels in an attempt to protect the enamel surface. Borges and others¹³ added 0.5% calcium gluconate to the bleaching gel and concluded that this supplementation reduced surface wear by abrasion. In addition, da Costa and Mazur²¹ tested different carbamide peroxide bleaching gels containing 0.11% sodium fluoride or amorphous calcium phosphate (ACP), as well the effect of subsequent application of 1.23% acidulated phosphate fluoride gel for 5 minutes after tooth bleaching. They concluded that fluoride or ACP in the bleaching agent did not prevent reduction in microhardness after bleaching. However, the final application of a fluoride gel led to significant microhardness recovery.

Recently, an enamel remineralizing system has been described, indicated for treatment of dental erosion. It consists of a specific association of calcium silicate and sodium phosphate salts with fluoride ions. The same active ingredients are available as a gel (boosting serum) and as a remineralizing toothpaste.^22-24 Previous investigators, using this system and comparing it with the use of regular fluoride toothpaste after erosive cycles, have concluded that gel application followed by the use of remineralizing toothpaste produced a significantly higher enamel remineralization than regular toothpaste alone.^22-24 It was also shown that this system formed hydroxyapatite on the surface of the demineralized enamel.²⁵ Considering the remineralizing potential of this product, it can be speculated that the application of this remineralizing gel immediately before or after dental bleaching may be favorable for protecting or remineralizing.

Considering that bleaching may promote demineralization and reduction of enamel microhardness, thus increasing its abrasion susceptibility,^1,4-6 and that some postbleaching treatments may remineralize the enamel surface, the incorporation of this step in the bleaching protocol seems highly advisable.^15,16 In this sense, the use of fluoride or other remineralizing agents that induce deposition of hydroxyapatite on the demineralized surface is quite promising.^15,16

The aim of this study was to evaluate the protective and remineralizing potential of the new calcium silicate/phosphate system and the 2% sodium fluoride gel on bleached enamel microhardness and wear with different toothpastes. The interference of remineralizing agents on the bleaching efficacy was also investigated. The null hypothesis tested was that treatment with remineralizing gels would not result in significant differences in relation to enamel microhardness reduction, color change (ΔE), or wear after bleaching and that the kind of toothpaste would not influence abrasive wear.

METHODS AND MATERIALS

Distribution of Experimental Groups

According to the baseline microhardness values, specimens were stratified into six groups (n=40) to avoid initial differences among the groups. The groups were NC (negative control) no treatment performed; BL (positive control) application of 40% hydrogen peroxide (H₂O₂) bleaching gel (Opalescence boost, Ultradent, South Jordan, UT, USA); BL/Rs bleaching gel, followed by the application of calcium silicate/phosphate fluoride gel Regenerate Boosting Serum (Unilever, Le Meux, France); Rs/BL application of the calcium silicate/phosphate fluoride gel followed by bleaching; Rs/BL/Rs application of calcium silicate/phosphate fluoride gel followed by bleaching and a new application of calcium silicate/phosphate fluoride gel; and BL/F bleaching followed by application of 2% sodium fluoride gel. Additional information about the products tested are shown in Table 1.

Table 1 Composition, Manufacturer, and Batch Number of the Products Used

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Abrasive Wear

After the final microhardness and color readings, the specimens were submitted to abrasive wear. For this, each group was randomly divided into two subgroups (n=20). In the first subgroup (Rt), the remineralizing toothpaste Regenerate (Unilever, Le Meux, France) was used, while in the second subgroup (Cp) a regular fluoride-containing toothpaste, Close Up Bioactive protection (Unilever, Ipojuca, PE, Brazil), was used. The specimens were placed on a holder with a metal perforated mask protecting the acrylic resin and the scratches of the abrasion, exposing only the enamel area. They were placed into a brushing machine (MEV-2T, Odeme, Luzerna, Santa Catarina, Brazil) and underwent 100,000 brushing strokes¹² receiving 50,000 strokes for two consecutive days, with a load of 200 g, according to ISO/TS 14569-1.³¹ During brushing, the samples were immersed in a toothpaste slurry, prepared with toothpaste diluted in artificial saliva (1:2 w/w).¹² The specimens were stored in a closed container at 100% relative humidity overnight.

In order to obtain a homogeneous wear, the toothbrushes were fixed in an automatic brushing machine so that their long axes were at an angle of 12° to the brushing direction. This avoided the formation of nonbrushed tracks induced by the bristles on the enamel surface.³² In addition, the brushing direction was perpendicular to the reading direction of the profilometer.

After tooth brushing, the final profile reading of each sample was performed. The final profiles were superimposed on the corresponding baseline ones, using dedicated software for contour evaluation (MarSurf XCR 20, 4.50-07 SP3, Mahr, Göttingen, Germany). The parallel scratches were used as a reference for profile superimposition. For each pair of superimposed profiles in the area corresponding to the wear, regression lines were calculated in both the baseline and final profiles. The distance between these lines was measured in micrometers by the same software (Figure 1). Three values were obtained for each specimen, and the mean was calculated.

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RESULTS

There were no statistically significant differences among the groups in relation to the baseline microhardness (p=0.1119) or color coordinates (p>0.05 for L*, a*, and b*). After the treatments, an ANOVA test showed statistically significant differences for absolute microhardness values (p=0.0001), percentage of surface microhardness change (p=0.0001), and ΔE (p=0.0001) among the groups. All groups that were bleached showed a significant reduction of final microhardness in relation to the negative control (Table 2). However, all groups that received the treatment with remineralizing gels showed a significantly smaller percentage of microhardness reduction than the positive control, which received only the bleaching gel. Regarding color difference, the bleached groups showed a significantly higher ΔE than did the negative control group. The ΔE was produced by the reduction of values on the b* coordinate and an increase on the L* coordinate, which means a reduction of chroma and an increase of lightness. No significant differences in ΔE were found among bleached groups, regardless of whether remineralizing gels were used (Table 2).

Table 2 Means (Standard Deviation) of Baseline and Final Microhardness, Percentage of Surface Microhardness Change (SMC), Color Difference, and Wear

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Results of the two-way ANOVA test concerning abrasive wear showed significant differences among the treatments (p=0.0001) and between toothpastes (p=0.0001). No interactions were observed between the two factors (p=0.4860). Results of the Tukey test for the factor treatment showed that the groups treated with remineralizing gels had significantly less wear than did the group only bleached. However, nonsignificant differences were observed among them. Only the positive control, just bleached, showed greater wear than did the negative control group (Table 2). In relation to the factor type of toothpaste, the groups brushed with Regenerate showed significantly less wear (2.04±0.80 μm) than those that brushed with the regular toothpaste (2.92 ±0.88 μm). The means (μm) of surface loss for each group and toothpaste are shown in Figure 2.

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DISCUSSION

In the present study, dental bleaching caused a significant reduction in enamel microhardness of about 23.16%. Similar percentages were found in other studies, ranging from 11.12% to 25%, increasing with application time.^10,33 The literature is inconsistent as to which percentage of enamel microhardness reduction is considered clinically relevant. Acid etching of enamel surfaces for adhesive purposes, a common clinical procedure in daily dental practice, effects a microhardness reduction of 73.78%.³⁴ In a previous in vitro study, the correlation between microhardness of eroded enamel surfaces with toothbrush wear was investigated. The specimens were immersed in carbonated beverage (pH=2.91) for 1, 5, or 15 minutes before brushing (8000 strokes).³⁵ The percentage of microhardness reduction and wear after treatments were, respectively, 11.52% (0.63μm), 14.18% (1.19 μm), and 30.69% (2.22 μm). Regression analysis showed a significant negative linear correlation between hardness and abrasive wear. Therefore, even a microhardness reduction of only 11% can significantly influence enamel wear.

Another study showed a positive correlation between peroxide concentration and microhardness reduction.³⁶ We tested the most concentrated product on the market, containing 40% H₂O_2, which is a nonspecific oxidizing agent that acts not only on the decomposition of chromophore molecules but also on tooth structure.² H₂O₂ can penetrate enamel structure mainly via proteins (amelogenin and enamelin), causing fragmentation of the polypeptide chain due to cleavage of the peptide bonds.³⁷ As the organic content also affects enamel integrity, its changes by oxidization may also indirectly alter the mineral content.²

It has been reported that bleaching gels with an acidic pH cause more demineralization and reduction of enamel surface microhardness than those with a higher pH.^2,3,10 However, some bleaching agents with a pH close to neutral also cause some changes in the enamel by decreasing its surface microhardness.^1,10 In the present study, Opalescent Boost bleaching gel has a pH of 7.4, yet it reduced enamel microhardness. It is speculated that, besides the nonspecific oxidative effect, the low concentration of calcium and phosphate in the whitening gel formulation makes it undersaturated compared with hydroxyapatite, promoting its demineralization regardless of the pH.¹⁰

When a solid is immersed in a solution, the interface between the two phases becomes a site for continuous exchange of the components from solid (in the case of enamel, Ca²⁺, PO₄^3-, and OH^- ions).³⁸ The ions are released from the solid surface and dissolve in the solution (dissolution) and, in the same way, other ions leave the solution to attach to the solid surface (crystal growth). When the rate at which ions enter the solution equals that at which they attach to the crystal surface, the solid and solution are in a state of equilibrium and there is no net change in the solid. At equilibrium, the solution is said to be saturated with respect to the solid.³⁸ If the solution is undersaturated, the solid loses ions to the solution until a balance is reached, and if the solution is supersaturated, the solution loses ions to the solid until, again, equilibrium is reached.³⁸ Remarkably, this balance varies according to the pH and background ion concentration.³⁸ Lussi and Hellwig³⁹ studied the effects of various beverages on promoting enamel softening during the erosive process and observed that it depends not only on the pH of the substances, but also on the concentration of Ca²⁺ ions in their composition.

Studies have found that remineralization by saliva can recover the microhardness reduced by the bleaching procedure, repairing the lost calcium and phosphate ions.⁴⁰ However, this process can take up to 15 days before the surface microhardness returns to baselines values.⁴¹ During this period in which the enamel is not completely repaired, teeth would be more susceptible to loss of structure due to abrasive wear, such as during toothbrushing.^12,13,42 The abrasive wear can be intensified, for example, if highly abrasive toothpastes and strong brushing forces are used. A study suggested delaying tooth brushing at least 1 hour after bleaching in order to reduce enamel wear.¹³ However, more studies are needed to confirm the effectiveness of brushing delay.

The application of fluoridated agents in a gel or solution on the enamel surface after bleaching is being recommended for remineralization and increasing microhardness.^{15,21,33,43-45} This procedure can promote formation of calcium fluoride deposits on the enamel surface.¹⁸ When the local pH decreases, F⁻ ions are released from calcium fluoride deposits; this release is accompanied by Ca²⁺ ion release from the tooth surface due to demineralization. The presence of these ions changes the fluoride content into hydroxyapatite molecules by replacing OH⁻ ions for F⁻ ions, forming fluorapatite or fluoridated hydroxyapatite.⁴⁶ Fluorapatite is formed when all hydroxyl ions of the hydroxyapatite molecule are replaced by fluoride, while fluoridated hydroxyapatite is formed when this replacement by fluoride is partial.⁴⁷ Fluorapatite is less soluble and more acid resistant than fluoridated hydroxyapatite.⁴⁷ These ionic changes lead to a greater tendency for the ions to join the structure, repair the enamel surface, and produce an increase in microhardness.^19,20 Lewinstein and others³³ observed that exposure, even to a low concentration of fluoride, can restore surface microhardness after bleaching. However, China and others¹⁵ concluded that only a neutral fluoride increases the microhardness of enamel bleached with 35% H₂O₂, while the application of acidulated fluoride causes an even greater reduction. In the present study, the effect of a neutral 2% fluoride gel³³ (9000 ppm fluoride ions) on the surface of the bleached enamel was tested. It is a highly concentrated fluoride compound commonly applied in-office for caries prevention. This postbleaching application increased the surface microhardness compared with the positive control group, but the surface did not return to its baseline surface microhardness values.

In an attempt to protect the enamel during bleaching, 1.1% of sodium fluoride (4950 ppm of fluoride ions) was incorporated in the bleaching gel analyzed in this study. However, it did not prevent enamel surface demineralization, indicating that something was interfering with its protective effect, possibly an interaction with H₂O₂ or other ingredients of the formulation.

The calcium silicate/phosphate fluoride gel was tested as a protective agent when applied before bleaching, as a remineralizing agent when applied after, and with both actions when applied before and after the bleaching procedure. This double application was tested in an attempt to intensify its effect on the enamel microhardness. In all cases, its application resulted in statistically higher final values of microhardness compared with the group that was only bleached. Based on this, in relation to microhardness, the null hypothesis was rejected. The use of this gel before bleaching significantly reduced the demineralizing effect of the H₂O₂ gel, while its use afterward promoted enamel remineralization. However, the use prior and after did not show any significant differences in relation to when it was used only once.

The remineralization process using this system starts when calcium silicate is deposited onto the enamel surface, causing an exchange of calcium ions with the hydrogen ions in the oral fluid, leading to the formation of silanol groups (Si-OH) in the surface layer of deposited calcium silicate. This causes an increase in the local pH, generating a negatively charged surface with the formation of silicon oxide (Si-O) groups. The calcium ions in the oral fluid are attracted to the surface of the calcium silicate layer. Consequently, the concentration of calcium and phosphate ions in the oral fluid is high enough to nucleate hydroxyapatite on the calcium silicate surface.^20,25,48 The formation of hydroxyapatite over the enamel using this system has been demonstrated in both in vitro and in situ studies.^25,49

The mode of action of calcium silicate/phosphate fluoride formulations on enamel remineralization by the deposition of hydroxyapatite has been investigated. This effect was observed after the use of toothpaste alone^24,25 and after the use of toothpaste followed by the boosting gel.^22,23 Both the gel and the toothpaste have fluoride in their composition, which may allow an additional mode of action for repair and protection by promoting the formation of calcium fluoride deposits on the enamel surface.¹⁸ A previous study found that the minimum concentration of fluoride necessary for the formation of calcium fluoride deposits on the enamel surface is 0.1%, and the first globules will be precipitated in the first 20 seconds.⁴⁹ These two mechanisms acting together may increase remineralization of the enamel surface. It must be noted that applying calcium silicate/phosphate fluoride gel over the bleached enamel, even though containing only 1450 ppm of fluoride ions, did not show statistically different mineralization effects in relation to the neutral 2% fluoride gel containing 9000 ppm of fluoride ions (Table 1). This might be related to the synergistic action of the hydroxyapatite formation with the fluoride mineralization mechanism of the boosting serum gel.

It could be supposed that the mineralizing potential of boost serum gel, applied before bleaching, could interfere with tooth enamel permeability, or even prevent the penetration of H₂O₂ into the tooth structure and reduce its bleaching effect. Additionally, when applied after bleaching, some ingredient in both gels' formulation could produce some kind of tooth staining, impairing the whitening results. In the present study, the previous application of calcium silicate/phosphate fluoride gel did not interfere with the ΔE promoted by bleaching. Studies evaluating the use of desensitizing agents containing 2% sodium fluoride before bleaching with 35% H₂O₂ also showed no interference with the ΔE.⁵⁰ However, no previous studies have analyzed the interference of the calcium silicate/phosphate fluoride gel on the bleaching effect. It has been observed that the application of both remineralizing gels postbleaching did not interfere with the ΔE of the tooth structure. Sodium fluoride gel is colorless, while calcium/phosphate silicate gel is slightly milky white. All bleached groups, despite the remineralizing treatments, presented statistically similar ΔE overall. The bleaching effect was related to a reduction in the b* coordinate and an increase in the L* coordinate. The a* value was not significantly influenced by the bleaching procedure. Thus, in relation to ΔE, the null hypothesis was accepted.

Regarding abrasive wear, the group that received only bleaching treatment showed a significantly higher wear than the nonbleached one (Table 2). A negative correlation between hardness and wear was observed, since the reduction of surface microhardness caused by bleaching increases the enamel loss by toothbrush abrasion. Other studies showing the relation between enamel bleaching and wear have obtained similar results.^11-13 In addition, the bleached groups in which the remineralizing gels were applied showed less wear than did the group that was only bleached, indicating that this procedure could offer some clinical benefits to the patients subjected to this kind of treatment. Thus, in relation to wear, the null hypothesis was rejected.

Another important aspect in relation to abrasive wear is that the groups that were bleached and received the remineralizing gels showed less wear than did the negative control group (Table 2). This might be due to the fact that after bleaching treatments and before the color reading, the specimens were immersed in artificial saliva for 7 days. The artificial saliva may have interacted with any calcium fluoride deposited on the enamel, forming fluorapatite or fluoridated hydroxyapatite, or continued to promote hydroxyapatite formation/deposition when the calcium silicate/phosphate fluoride gel was used.^40,41 Both these processes may have further increased remineralization and enamel surface microhardness, leading to less enamel wear than the bleached as well as the nonbleached groups.

Significant differences were observed in the wear caused by the toothpastes tested. The groups that were brushed with Regenerate showed less wear than those brushed with Close Up. The use of a less abrasive toothpaste after bleaching might be clinically relevant, avoiding additional tooth wear on those patients receiving this kind of treatment, although this should be confirmed in controlled clinical trials. Thus, in relation to the kind of toothpaste, the null hypothesis was rejected. Two suppositions could be made about this result. The first is that the remineralizing effect of Regenerate could protect the surface from abrasion by hydroxyapatite formation,^25,48 increasing enamel microhardness.³³ The second is probably related to lower abrasivity of the abrasive particles in this toothpaste.

One factor that impacts toothpaste abrasivity is the relative hardness of the abrasive particles. Regenerate contains hydrated silica as an abrasive, while the Close Up used in this study has a combination of hydrated silica and calcium carbonate. Moh's hardness is used to measure material hardness on a scale of 0 to 10, wherein 10 represents the hardness of a diamond. In this scale, the hydrated silica has a hardness of 2.5 and the calcium carbonate, 3.⁵¹ However, previous studies have tested various toothpastes having calcium carbonate and hydrated silica alone or in combination, but no statistically significant differences between the toothpastes in terms of enamel wear were found.^52-54 In contrast, the relation between toothpaste abrasiveness and wear was evaluated by Borges and others,¹³ who subjected bleached enamel and dentin to brushing abrasion with slurries having different degrees of abrasivity; they observed that the higher the abrasiveness, the greater the tooth wear. However, it should be noted that the relationship between toothpaste abrasivity and dental wear is complex; it depends on the concentration and size of the abrasive particles.⁵³ In addition, the presence of the acquired pellicle has been shown to reduce enamel wear from toothpaste abrasion.⁵⁴

It should be emphasized that this is an in vitro study, which has limitations in reproducing conditions found in the oral cavity, eg, the presence of saliva. The acquired pellicle and its action, together with bleaching and remineralization treatment, may modify the results of the current study such that complementary investigations are necessary to confirm their impact. The importance of clinical studies to evaluate the performance of calcium silicate/phosphate fluoride formulations or fluoride gels for longer periods of use after the bleaching treatment is worthy of further study.

CONCLUSIONS

Considering the limitations of this in vitro study, we concluded that

1. Bleaching reduced enamel microhardness and increased abrasion suitability.

2. Although the remineralizing gels tested did not prevent microhardness reduction and wear of bleached enamel, they did bring about their decrease without interfering with the bleaching efficacy.

3. Regenerate toothpaste resulted in less enamel abrasive wear than did Close Up.

4. There were no significant differences among the remineralizing gels in their effect on microhardness, color, or wear.