Effects of experimental bleaching agents on the mineral content of sound and demineralized enamels

Abstract High concentrations of hydrogen peroxide can cause adverse effects on composition and structure of teeth. However, the addition of calcium and fluoride in bleaching agents may reduce enamel demineralization. Objective: To evaluate chemical changes of sound and demineralized enamels submitted to high concentrations of hydrogen peroxide containing fluoride (F) or calcium (Ca). Material and Methods: Enamel blocks of bovine incisors with standard dimensions were obtained and half of them were submitted to pH-cycling to promote initial enamel caries lesions. Sound and demineralized enamel samples were divided into (n=10): (C) Control (no whitening treatment); (HP) 35% hydrogen peroxide; and two experimental groups: (HPF) 35% HP+0.2% F and (HPC) 35% HP+0.2% Ca. Experimental groups were submitted to two in-office bleaching sessions and agents were applied 3 times for 15 min to each session. The control group was kept in remineralizing solution at 37°C during the bleaching treatment. The surface mineral content of sound and demineralized enamels was determined through Fourier Transform Raman spectroscopy (FT-Raman), Energy dispersive Micro X-ray fluorescence spectroscopy (μ-EDXRF); and the subsurface, through cross-sectional microhardness (CSMH). In addition, polarized light microscopy (PLM) images of enamel subsurface were observed. Results: According to three-way (FT-Raman and μ-EDXRF analyses) or two-way analysis of variance (ANOVA) (CSMH) and Tukey test (α=5%), the calcium or fluoride added to high-concentrated bleaching agents increased phosphate and carbonate concentrations on sound and demineralized enamels (p<0.05). However, HPC and HPF were unable to completely reverse the subsurface mineral loss promoted by bleaching on sound and demineralized enamels. The calcium/ phosphate (Ca/P) ratio of sound enamel decreased after HP treatment (p<0.001). Conclusion: Even though experimental bleaching agents with Ca or F reduced mineral loss for both sound and demineralized enamel surfaces, these agents were unable to reverse the enamel subsurface demineralization.


Introduction
In-office bleaching has been extensively performed clinically due to the fast whitening promoted by the higher concentrations of hydrogen peroxide (HP) in one single application. 1 However, low and high concentrations of HP can uphold adverse effects on enamel composition and structure. 2,3 Among these alterations, the decrease of enamel inorganic content and enamel cohesive strength 4 was observed. In addition, in vitro studies have demonstrated increase in surface roughness after bleaching. 5,6 High concentrations of HP can modify calcium/ phosphate (Ca/P) ratio 7 and the loss of mineral content decreases microhardness, which might increase enamel susceptibility to demineralization. 8 Such considerations are concerning since bleaching could interfere in the development of early caries lesions that are not properly detected. [9][10][11] The mechanism of action of the agents may be responsible for the structural enamel changes after bleaching. 6 It is believed that the active compound H 2 O 2 penetrates enamel and dentin and, due to its chemical instability, it is decomposed into different active oxygen species under specific temperature, pH and light conditions. 12 The free radicals formed, as hydroxyl, may oxidize the conjugated chain of organic compounds, the chromophores. 12 The oxidation or cleavage of organic molecules that occur during bleaching possibly promotes morphological changes of the enamel. 13 On the other hand, fluoride has been extensively proven efficient to increase F concentration in enamel and reduce enamel demineralization. 14 Due to the effectiveness of re-hardening on demineralized enamel, 14 fluoride has been added to bleaching agents to minimize the effects of bleaching on enamel. 7,15,16 It is believed that high concentrations of fluoride and calcium added to the bleaching agents will control mineral loss, even if the agent has low pH, in view of the fact that the bleaching will be saturated with ions. 15,16 Yet, studies have observed that home-applied bleaching agents (10% carbamide peroxide), enhanced with high concentrations of fluoride (0.2%) or calcium (0.2%) applied 6 h a day for 14 days, minimized enamel mineral loss. 7,16 Therefore, it is possible that the addition of fluoride or calcium to high-concentrated bleaching agents (35% hydrogen peroxide) could prevent mineral loss. This possibility is particularly interesting to avoid further mineral loss in patients with early caries lesions. 9,10 Although the effectiveness of the addition of fluoride and calcium to bleaching agents has been evaluated, 7,17 the concentrations of both agents vary considerably. Previous studies attest that high concentrations of calcium (0.2%) and fluoride (0.2%) are required to prevent demineralization of bleached enamel. 7,16 However, these concentrations have been tested mainly in low-concentrated bleaching gels. 7,16 Therefore, the aim of this study was to determine mineral loss of sound and demineralized enamels submitted to experimental in-office bleaching agents containing 0.2% calcium or 0.2% fluoride. The surface and subsurface of enamel were evaluated to assess the extension of demineralization. Therefore, the tested hypotheses were that (1) the 35% hydrogen peroxide bleaching agent containing either calcium or fluoride would change the surface mineral content of sound or demineralized enamels and that (2) the addition of calcium or fluoride to 35% hydrogen peroxide would increase mineralization of enamel subsurface.

Experimental design
Eighty dental enamel blocks obtained from bovine incisors were used in this randomized in vitro study and half of them (40) were submitted to pH-cycling to promote enamel demineralization. Thereafter, sound and demineralized enamel were assigned to four treatments (n=10) as follows:

Specimen preparation
This study was analyzed and approved by the   Micro energy dispersive X-ray fluorescence spectrometer (μ-EDXRF 1300, Shimadzu, Kyoto, Japan) was used to map the surface area to measure the weight of Ca/P ratios on sound and demineralized enamel surfaces before and after bleaching treatments.
The selection of parameters used for the μ-EDXRF and subsequent data analyses were performed according to the method described by Paula et al. 19 (2010).

Cross-sectional microhardness analysis (CSMH)
Specimens were cut longitudinally through the center of the exposed enamel area and two halves were obtained. One of the enamel segments was embedded in acrylic resin and the inner exposed surface was

Polarized light microscopy (PLM)
The other half of the specimen was polished to  In addition, the phosphate concentration (ν 2 and ν 4 ) of groups C, HPF and HPC increased after bleaching (p<0.0001, Table 1: Table 2: ν 4 , Figure 1).  Figure 2). Phosphate ν 4 concentrations of HPF and HPC groups were similar (p=0.0675) but higher than the ones of C and HP groups, after treatments (p<0.001, Table   2, Figure 2).

μ-EDXRF
Sound enamel: At baseline, sound enamel showed similar Ca/P ratios for all groups tested (p=0.177, Table 4). After bleaching, the Ca/P ratio of HP-treated group decreased (p<0.0001), HPF-and HPC-treated groups increased (p<0.001) and the mineral content of the C group was similar to baseline (p=0.069).
HPF-and HPC-treated groups had the highest Ca/P ratio among groups at T b as the HP group had the lowest (p<0.001).
Demineralized enamel: At baseline, all groups had similar Ca/P ratios (p=0.188, Table 4). After bleaching, the Ca/P ratio of HP group was similar to T 0 but significantly increased for C-, HPF-and HPCtreated groups (p<0.0001). After bleaching, groups C, HPF and HPC had higher Ca/P ratios than HP group (p<0.001).
Sound versus demineralized enamel: At baseline, the demineralized enamel had lower Ca/P ratio than that of sound enamel (p<0.001). After treatments, demineralized enamel submitted to HPF and HPC treatments showed lower Ca/P ratios than that of sound enamel (p<0.001).

Cross-sectional microhardness analysis (CSMH)
Sound enamel: The mineral loss area (∆Z) of sound C group was significantly lower than HP, HPF and HPC (p<0.001). No differences were observed among HP,        However, 35% hydrogen peroxide with F (HPF) or Ca (HPC) increased phosphate (ν 2 and ν 4 ) and carbonate (ν 3 ) concentrations after bleaching and the mineral content was higher than that of HP-treated group (Tables 1, 2 and 3). Nevertheless, μ-EDXRF analysis showed that HP bleaching reduced the Ca/P ratio on sound enamel (Table 4), which indicates that HP alone could compromise enamel inorganic concentration.
According to this scenario, F and Ca ions possibly Although this is an in vitro evaluation and we cannot extrapolate to a clinical condition, these results signalize that HP could promote demineralization, as well as F and Ca could remineralize enamel surface after bleaching.
A previous investigation reported that the addition of 0.2% F or 0.2% Ca to home-applied bleaching agents controlled enamel inorganic loss on intact enamel surfaces compared to conventional 10% carbamide peroxide gels. 16 According to these findings, the effectiveness of enhanced bleaching agents is  Ca/P ratio, the interaction of calcium chloride and free radicals was not chemically evaluated.
The phosphate (ν 2 and ν 4 ), carbonate (ν 3 ), and Ca/P concentrations of demineralized enamel increased after treatments, except for the Ca/P concentration of the HP group, which remained with similar mineral concentration after bleaching ( Table 4). The response of demineralized enamel toward treatments (HPF and HPC) relies on the action of F and Ca deposition on the undersaturated enamel surface and the action of the remineralizing solution, since C group also had increase in the inorganic concentration. It must be noted that F and Ca uptake of demineralized enamel was higher than sound enamel, because in the first condition the enamel was undersaturated regarding the ionic concentration, which balanced the content of the parts involved (substrate, remineralizing solution and/ or bleaching agent). 7 Therefore, the first hypothesis can be accepted, as the 35% hydrogen peroxide agent containing calcium or fluoride increased the mineral content of sound and demineralized enamels.
CSMH and PLM provided evidence of enamel subsurface demineralization. In this specific region, demineralization could be noted for both sound and demineralized enamels. However, the mean for the mineral loss of sound enamel of the control group was lower compared to the other treatments. As expected, samples submitted to previous demineralization showed subsurface demineralization, but control group had lower demineralization area, followed by samples submitted to HPF and HPC and finally, HP bleaching agent, which presented the greatest mineral loss. A similar finding 16 showed that enamel submitted to 10% carbamide peroxide (CP) had enamel subsurface demineralization, which was significantly greater and deeper than that observed for the placebo group (without CP), followed by 10% CP with F or Ca.
Although F or Ca were unable to completely reverse the inner demineralization, the agents could control it in that report, 6 different from the present study. Therefore, the second hypothesis was rejected, as the addition of Ca or F to the 35% HP bleaching agent was unable to increase mineral concentration of enamel subsurface.
Based on the exposed, the addition of F and Ca to the 35% HP agent could keep and increase the mineral content of enamel surface for both sound and demineralized substrates, but could not reverse subsurface enamel demineralization. This outcome confirms that the F and/or Ca associated with bleaching decrease enamel mineral loss of the enamel surface. 16,30 In addition, previous findings observed that F could not only control the mineral loss, but also did not compromise the whitening efficacy. 31,32 One might say that enamel mineral loss observed after bleaching may not be threatening to dental hard tissues, since it has been shown that enamel exposed to acidic beverages for some minutes promote similar alterations on enamel surface. 33 However, if a patient with early caries lesions undergoes bleaching therapy, its consequences could be intensified. For those patients, the addition of F or Ca could be an interesting therapy choice. Besides, it should be kept in mind that only two bleaching sessions were performed and, possibly, more bleaching sessions would increase the severity of enamel mineral loss.

Conclusion
The addition of Ca and F to experimental bleaching agents could control the decrease of phosphate and carbonates for both sound and enamel surfaces. On the other hand, experimental bleaching agents were unable to reverse the subsurface demineralization of sound and demineralized enamels.