Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel

Abstract Objective This study was designed for the chemical activation of a 35% hydrogen peroxide (H2O2) bleaching gel to increase its whitening effectiveness and reduce its toxicity. Methodology First, the bleaching gel - associated or not with ferrous sulfate (FS), manganese chloride (MC), peroxidase (PR), or catalase (CT) - was applied (3x 15 min) to enamel/dentin discs adapted to artificial pulp chambers. Then, odontoblast-like MDPC-23 cells were exposed for 1 h to the extracts (culture medium + components released from the product), for the assessment of viability (MTT assay) and oxidative stress (H2DCFDA). Residual H2O2 and bleaching effectiveness (DE) were also evaluated. Data were analyzed with one-way ANOVA complemented with Tukey’s test (n=8. p<0.05). Results All chemically activated groups minimized MDPC-23 oxidative stress generation; however, significantly higher cell viability was detected for MC, PR, and CT than for plain 35% H2O2 gel. Nevertheless, FS, MC, PR, and CT reduced the amount of residual H2O2 and increased bleaching effectiveness. Conclusion Chemical activation of 35% H2O2 gel with MC, PR, and CT minimized residual H2O2 and pulp cell toxicity; but PR duplicated the whitening potential of the bleaching gel after a single 45-minute session.


Introduction
Hydrogen peroxide (H 2 O 2 ) is found in high concentrations in bleaching gels widely used for inoffice tooth-bleaching therapies. 1Whitening outcome is believed to be a consequence of H 2 O 2 decomposition, which generates free radicals that interact with chromophores present in dentin substrate. 2Instead of being considered a strong oxidant agent, H 2 O 2 is the reactive oxygen species (ROS) with the lowest oxidative potential 3 .Therefore, to achieve effective dental color alteration in short periods, highly concentrated H 2 O 2 bleaching gels (35-40%) have been traditionally used for professional tooth-bleaching. 1 However, many studies have shown that such esthetic therapy allows diffusion of high amounts of H 2 O 2 through enamel and dentin, 4,5 causing in vitro [6][7][8][9][10][11] and in vivo [12][13][14][15] toxicity to pulp cells.Therefore, the "non-reacted H 2 O 2 " has been considered the main pathway for bleaching-induced tooth sensitivity, claimed by 80-100% of patients undergoing professional tooth-bleaching performed with high-concentrated gels. 16,17e cell toxicity mechanism mediated by bleaching gels on pulp cells has been correlated with two main pathways: (1) H 2 O 2 arising in the pulp chamber during the bleaching procedure can diffuse through the cell membrane, followed by dissociation into free radicals on cytoplasm, causing a pathologic stress oxidative condition, lipid peroxidation, and necrosis; and (2) free radicals in the extracellular environment after H 2 O 2 dissociation cause direct damage to cell membranes, leading to cell death by necrosis. 18[15][19][20][21] Recent demonstrations show H 2 O 2 from bleaching gels induces pro-inflammatory cytokine release by pulp cells, negatively influencing their long-term regenerative potential. 8,11,14,15,22erefore, many alternative therapies have been proposed to increase the biocompatibility of in-office bleaching therapy with the pulp-dentin complex, mainly aimed at reducing the amount of H 2 O 2 capable of reaching pulp cells. 18][13][14][15]22 However, additional sessions are needed to achieve bleaching outcomes similar to those of traditional therapies. 7,10,235][26][27][28][29] Indeed, when manganese and iron-rich molecules are used to induce H 2 O 2 decomposition into free radicals with Fenton and Fenton-like reactions, the H 2 O 2 amount that reaches the pulp chamber in vitro is intensely reduced 24,27

Methodology
Enamel/dentin discs Enamel/dentin discs (from 24-to 30-monthold bullocks), measuring 5.6 mm in diameter and 3.5 mm in thickness, were obtained as previously described. 29Enamel surface was cleaned with pumice stone solution under low speed handpiece (Dabi Atlante, Ribeirão Preto, SP, Brazil), and then evaluated with stereoscopic magnifying glass (Olympus 5ZX&, Olympus, São Paulo, SP, Brazil) to eliminate those samples with enamel defects, such as the presence of superficial cracks or hypoplasia.Dentin surfaces liver enzyme (powder, 2,000-5,000 units/mg protein, Sigma-Aldrich).26]29 Then, three drops of the H 2 O 2 liquid phase (100 mL) were added, and the product was mixed for 15 s.This procedure was performed immediately before each application of the product.In the HP group, the same procedure was performed but no chemical substance was incorporated.The manipulation of the bleaching gel (1 drop of thickening per 3 drops of H 2 O 2 ) was based on the manufacturer's instructions (FGM).In total, three 15-minute applications were performed in all bleached groups, and a 40-mL volume of bleaching gels was applied to enamel at each The bleaching gels were manipulated in a 1.5 mL tube, and the probe was submersed into the material.The pH was checked after 0.5, 5, 10, and 15 min.A total of 4 samples per group was manipulated for this analysis.

Trans-enamel and trans-dentinal cytotoxicity
Enamel/dentin discs were adapted to artificial pulp chambers (APC), as previously described. 29e APC/disc sets (sterilized in ethylene oxide) were placed in 24-well plates with dentin in contact with to experimental tubes to react with leuco crystal violet (0.5 mg/mL; Sigma-Aldrich) and horseradish peroxidase enzyme (1 mg/mL; Sigma-Aldrich).The final volume of reaction was adjusted to 3 mL with distilled water, and the optical density of the solutions was measured at 600 nm wavelength (Synergy H1, BioTek).To estimate the effects of chemical activators on H 2 O 2 diffusion, the HP group was considered 100% of H 2 O 2 diffusion, and diffusion percentages for chemically activated groups were calculated based on this parameter.

Color alteration measurement
For this analysis, the discs were subjected to staining with black tea to standardize the baseline color (n=8). 29This procedure was performed to Specimens were kept in 100% humidity to standardize the hydration pattern.For that, the discs were placed at the bottom of wells of 24-well plates in a way that dentin was maintained in contact with a cotton pellet embedded with deionized water, and the enamel was covered with a cotton pellet embedded in artificial saliva solution. 10,23The plates were kept in incubator for 72 h, at 37°C (Orion 515, Fanen, São Paulo, SP, Brazil).A color readout was then used to obtain baseline (BS) values for each disc.To be read, the discs were positioned in a white silicone matrix, leaving only the enamel surface exposed.for all bleached groups compared with NC; however, cell viability higher than that of the HP group was determined for the HP+MC, HP+PR, and HP+CT groups, with HP+PR featuring the greatest value (Figure 2a).Increased oxidative stress was detected for all groups related to NC; but all bleached groups associated with chemical activators featured oxidative stress substantially lower than that found in the HP group (Figure 2b). ) radicals. 30llowing the same catalytic mechanism, the aim was to reduce the amount of residual H 2 O 2 on tooth- to induce H 2 O 2 catalysis with Fenton and Fenton-like reactions, respectively, in which the decomposition of H 2 O 2 generates hydroxyl radicals (HO • ), [31][32][33] as shown in Figure 4 (Eq.1 and 2).In the presence of high concentrations of H 2 O 2 , HO • interacts with excess H 2 O 2 to promote a series of propagation reactions (Figure 4, Eq.3-5), resulting in the release of other free radicals, such as HO with no release of free radicals, minimizing the effectiveness of the reaction. 3,30Reactions related to this process are demonstrated in Figure 4 (Eq.6 and 7).Therefore, formation of these sub-products may be considered a disadvantage of the iron-based Fenton reaction.

Residual H2O2 quantification
We also observed a slight deposition of brown sub-products during manganese chloride reaction with the bleaching gel, which disappeared at the end of the reaction (Figure 1).According to the literature, soluble manganese is a stoichiometrically efficient catalyst for the generation of hydroxyl radicals; however, depending on pH regime, an amorphous manganese oxide precipitate occurs. 33  7). 32[26] Additionally, it was shown that the concentration of H 2 O 2 and the time for diffusion through enamel and dentin are significantly reduced in the presence of manganese-containing activators, 24,28  after one single 3x10-minute tooth-whitening session when a peroxidase-rich extract was incorporated into the dental product.The authors speculated that this positive effect was related to the enhanced free radical formation mediated by this enzyme.
Overall, the indication is that the release of free radicals mediated by the interaction of H 2 O 2 with chemical activators increase bleaching effectiveness, minimizing the amount of residual H 2 O 2 that diffuse through enamel/dentin to cause toxic effects to the cultured pulp cells.However, one important limitation of this study is that free radicals released from the bleaching gels and their trans-enamel and trans-dentinal diffusion were ignored.Therefore, the cytotoxicity observed here for all chemically activated groups may also be related to the release of such reactive molecules capable of diffusing through enamel/dentin to reach the cells.This effect may be related to the contradictory results found for H 2 O 2 diffusion on HP+MC group.Instead of achieving the lowest H 2 O 2 diffusion, this group had the same behavior as HP+FS and HP+CT groups for all the other parameters tested.We believe that manganese chloride increases the formation of free radicals in comparison with ferrous sulfate and catalase, but these free radicals may have diffused through enamel and dentin to play a role in the cytotoxicity to the pulp cells.
On the other hand, peroxidase also had the lowest amount of residual H 2 O 2 , which was statistically similar to manganese chloride.Nevertheless, the better cell parameters and whitening outcome found for HP+PR group compared with HP+MC may result from the oxidase cycle reaction, since the reactive species described as compound I and III may have played a role in the reaction of H 2 O 2 with tooth structure, resulting in a lower amount of free radical diffusion through enamel/dentin discs.Therefore, more studies are needed to demonstrate the pathway related to the minimization of the toxicity mediated by the chemical activators tested here.Non-stained discs were used in the biological assays to avoid the release of toxic components of black tea to the cells, as performed before. 7,10,23,29Since free radicals released by the bleaching gel were expected to react with the chromophores, we may speculate that a high amount of non-reacted molecules may have diffused to cause the toxic effects observed in this investigation.Also, several other relevant biological factors found in vivo, such as the presence of pulp pressure, extracellular matrix, and host immune cells, have been rejected in laboratorial tests to assess the biological and esthetic behavior of dental products. 18Hence, the results found in this in vitro study can be considered overestimated.
Torres, et al. 28 (2013) reported that the use of chemical activators to minimize the residual H 2 O 2 diffusion to the pulp chamber seems to be more evident when low-concentration bleaching gels are used.According to those authors, 35% H 2 O 2 formulations provide greater availability of residual H 2 O 2 , so, likely, the chemical activator is unable to reduce the rate of peroxide diffusion at secure levels.The literature has already shown that highconcentrated bleaching gels have intense potential to bleach teeth from the first 45-minute session. 16,17vertheless, the increase in bleaching effectiveness demonstrated in this study may be useful to fasten the whitening outcome on dark-colored teeth, such as tetracycline stained teeth; however, more studies are needed to prove the effectiveness of the formulations tested in these specific situations.The focus of the current studies is to reduce the concentration of in-office bleaching gels to minimize the negative effects on pulp cells. 38,39It has been previously demonstrated that a 50% reduction in the concentration of peroxide in bleaching gels with 35% H 2 O 2 results in a significant positive effect on cell viability; but the bleaching effectiveness is harmed. 7,23erefore, since the manganese chloride, catalase, and peroxidase were capable of minimizing the cytotoxicity of a highly concentrated bleaching gel by increasing its reactivity with tooth structure, it seems reasonable that they may be considered a promising alternative to improve the biocompatibility and esthetic outcomes of low-concentrated whitening products.However, considering the limitations of this in vitro study, further investigations are needed to clarify the benefits of chemical activations to commercial bleaching agents and their safety for clinical applications.Also, a costbenefit analysis should be performed, since the purified horseradish peroxidase enzyme used here has a high cost.Evaluating other sources of this enzyme and performing dose-response studies may clarify the benefits of this substance as a chemical activator of tooth bleaching products.

15 -
minute application, via a pipette coupled with a capillary piston tip (Microman E, Gilson, Middelton, WI, USA), which allows standardized pipetting of viscous liquid.Digital images were taken at a 45 cm distance from the specimens, with a DSLR camera (Nikon D3300; F 22, ISO 200, speed 180; Melville, NY, USA) coupled with macro lenses (90 mm; F2.8.Tamron, Colonia, Italy) and a circular flash (EM-140DG Macro flash, speed 1/16; Sigma Corporation of America, Ronkonkoma, NY, USA) to observe the reaction of the bleaching gel with the chemical activators during the 15-minute application time.The pH was measured with a bench top pH meter (HI2221 Calibration Check pH/ ORP Meter, Hanna Instruments, Woonsocket, Rhode Island, NE, USA), coupled with a microprobe (HI1131P General Purpose pH Electrode, Hanna Instruments).

1
mL of DMEM (Dulbecco's Modified Eagle Medium; supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L glutamine; Gibco, Grand Island, NY, USA).Enamel remained exposed to receive the treatments according to each group.Immediately after the bleaching procedure, the culture medium (extract) was collected and distributed into 100-mL aliquots, which were applied for 1 h to odontoblast-like MDPC-23 cells previously seeded in an 80% confluence pattern [1x10 4 cells/96-well plates in DMEM plus 10% fetal bovine serum (FBS), for 24 h at 37°C and 5% CO 2 ].Cell viability was assessed by incubation of the cells for 4 h in DMEM without FBS (Gibco) supplemented (10:1) with 5 mg/mL MTT solution (Sigma-Aldrich) at 37°C and 5% CO 2. The absorbance of formazan crystals in the viable cells was read (570 nm; Synergy H1, BioTek, Winooski, VT, USA), and the mean absorbance of the NC group was considered 100% of cell viability (n=8).Oxidative stress was evaluated in cells pre-treated with fluorescence probe carboxy-H 2 DCFDA (5 mM; Invitrogen, Carlsbad, CA, USA) (n=8), and then exposed for 1 h to the extracts.After this period, the fluorescence intensity was monitored at 59 nm excitation and 517 nm emission (Synergy H1, BioTek), and the fold increases were calculated after normalization with the NC group.Quantification of residual H2O2A 100-µL aliquot of the extract collected from the same samples of trans-enamel and trans-dentinal cytotoxicity assay (n=8) was transferred to tubes containing 900 mL of acetate buffer solution (2 mol/L, pH 4.5), to avoid H 2 O 2 degradation.Then, 500-µL of buffer solution plus extract was transferred SOARES DG, MARCOMINI N, DUQUE CC, BORDINI EA, ZUTA UO, BASSO FG, HEBLING J, COSTA CA randomly distribute specimens with similar L* and b* values (CIE L*a*b* system) among the control and experimental groups, obtaining standardized samples.Darkened samples were used to verify the potential of the different formulations to fasten the whitening outcome compared with the gel with no chemical supplementation.
A portable UV-VIS spectrophotometer with 4 mm aperture (Color Guide 45/0; BYK-Gardner GmbH, Geretsried, BAV, Germany) was positioned over the center of the 5.6 mm diameter disc, so that only the surface of the sample was read with no interference of the background color.The samples were read in sequence by the same operator.A total of three sequential readings was performed to eliminate bias from spectrophotometer positioning onto the sample.Bleaching protocol was performed on enamel surfaces, after each enamel was washed with deionized water and dried with filter paper.The specimens were incubated in 100% humidity for 72 h to allow for rehydration, and a post-bleaching (PS) color readout was obtained.Values of L* a* b* were recorded to obtain ΔL, Δa, and Δb values for each sample by the following equation: Δ(L, a or b) = PS value (L*, a*, or b*) -BS value (L*, a*, or b*).Overall color change of each specimen, expressed as ΔE, was calculated according to the equation ΔE=[(ΔL)²+(Δa)²+(Δb)²]½. Statistical analysis Sample size was calculated with DDS Research (Sample Size Calculator, average, two samples, a=5%; b=95%), and eight samples per group were stablished for each assay.Percentage of cell viability (MTT assay) (NC versus HP) and DE (NC versus HP) were used as parameters.Power calculation analyses were also performed by DDS Research (Statistical Power Calculator, average, two sample, two-tail test, a=5%) at the end of the experiment, showing 100% statistical power for each evaluation.Two independent experiments were performed for each assay.Data were compiled and analyzed by Kolmogorov-Smirnov and Levene tests.Since normal data were obtained, one-way ANOVA and Tukey's test were used for cell viability, as well as H 2 O 2 diffusion, oxidative stress, DE, DL, Da, and Db analysis.Data of pH measurement were analyzed with repeated measure two-way ANOVA and Dunnet's test to compare pH values at each timepoint with those from HP group.All statistical analyses were carried out at a significance level of 5%.Results Chemical activation characteristics Reaction immediately before manipulation and after 5, 10, and 15 minutes on tooth surfaces can be observed in Figure 1a.Bubbles were observed in all chemically activated groups and deposition of brown sub-products was detected only in groups HP+MC and HP+FS.pH values of the bleaching gels at each timepoint and the statistical analysis through time can be observed in Figure 1b.The statistical analysis through time can be observed in Figure 1b.Significant increase in the pH compared with HP group was detected for HP+FS at 5 min and for HP+MC at 0.5 min.The enzymes peroxidase and catalase had insignificant influence on the pH of the 35% H 2 O 2 gel.Biologic assaysConsiderable cell viability reduction was observedIncreased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel

Reductions in H 2 O 2
diffusion occurred for those extracts obtained from bleached groups associated with chemical activators compared with plain H 2 O 2 gel (HP group).The HP+MC and HP+PR groups featured significantly lower H 2 O 2 amounts on extracts compared with the other groups (Figure 2c).Bleaching effectiveness All bleached groups presented notably higher DE values than the NC group.HP+PR featured the highest DE value, which was significantly different from that of the other groups (Figure 3a).All the bleached groups promoted significant increase in DL and decrease in Db compared with NC group; nevertheless, only HP+PR featured significant differences with HP group for both parameters.Considerable reduction in Da related to NC group was detected for HP+PR and HP+MC groups.Discussion Catalyzed H 2 O 2 propagation has been used to increase the oxidative potential of H 2 O 2 , that, in turn, eliminates organic contaminants from aquifers and soil 3 .Depending on environmental conditions, H 2 O 2 may be dissociated into different molecules, releasing oxygen (O 2 ), water (H 2 O), and highly reactive free radicals, such as hydroxyl (HO • ), perihydroxyl (HO 2 • ), and superoxide (O 2 •

Figure 1 -Figure 2 -Figure 3 -Figure 4 -
Figure 1-(a) Representative digital images of bleaching gel reaction in the presence or not of chemical activators, immediately after manipulation (0 min) and after 5, 10, and 15 min on enamel/dentin discs.Note the bubbles in HP+FS, HP+MC, and HP+CT groups.Deposition of brown sub-products can be observed in groups HP+FS and HP+MC.(b) Graph representative of mean (standard deviation) values of pH for the bleaching gels at 0.5, 5, 10, and 15 min.Asterisks indicates significant difference with the HP group (repeated measures Two-way ANOVA/Dunnet's test, p<0.05) Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel 35% H 2 O 2 bleaching gel with manganese chloride, peroxidase, or catalase enhances tooth-whitening and reduces the amount of residual H 2 O 2 capable of diffusing through enamel and dentin, minimizing the toxic effects of this product to pulp cells.Enzymatic activation with peroxidase featured the best biological and esthetic results compared with those of the other chemical activators.
Increased whitening efficacy and reduced cytotoxicity are achieved by the chemical activation of a highly concentrated hydrogen peroxide bleaching gel