Assessment of a novel bleaching agent formula containing 35% hydrogen peroxide and titanium tetrafluoride: an in vitro study

This study developed experimental gels containing titanium tetrafluoride (TiF4) combined with commercial 35% hydrogen peroxide (HP), and evaluated bleaching efficacy and pH of the gels, and mineral content and morphology of enamel submitted to these treatments. In phase-1, different stock gels mixed with TiF4 were combined with HP. In phase-2, the selected gels were tested on enamel/ dentin specimens (n=8): HP; HP and Natrosol+TiF4 (HPnT); HP and Natrosol+Chemygel+TiF4 (HPncT); HP and Aristoflex+TiF4 (HPaT). Bleaching was performed in four sessions (3x15min-application/ session). Color (CIEL*a*b*) and whiteness index (WID) were measured after each session, whereas whiteness index differences (ΔWID), color alteration (CIELab–ΔE, CIEDE2000-ΔE00), enamel morphology and pH, at end of bleaching therapy. The change in Knoop microhardness (ΔKHN) was compared before and after bleaching. Data were analyzed by two-way repeated measures ANOVA and Bonferroni (CIEL*, a*, b*), one-way ANOVA and Tukey (ΔWID, ΔE, ΔE00), and LSD (ΔKHN) tests (α=5%). SEM and pH measurements were submitted to descriptive analysis. No differences were observed in lightness (L*) or WID among the groups (p > 0.05), but HP exhibited lower b* values (p<0.05), higher ΔWID than HPnT, and the highest ΔE among the groups (p < 0.05). No differences in ΔE00 were observed between HP and HPncT (p > 0.05), and HPncT showed higher ΔKHN than HP (p < 0.05). HP presented pH values closer to neutral (6.9), whereas experimental agents showed acidic pH values (2.3–3.9). No morphological changes were observed in HP or HPncT groups. HPncT was able to bleach the enamel and maintain enamel microhardness and surface integrity, even at low pH.


Introduction
Minimally invasive operative dentistry focused on high esthetic criteria requires safe and effective techniques, procedures, and products. In this respect, dental bleaching is a common clinical procedure, because it is undoubtedly an effective technique, 1 regardless of the hydrogen peroxide (HP) concentration used. 2,3 Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript.
However, safety has always been a concern, since literature reports damage to the dental structure promoted by HP agents, including a decrease in microhardness, 3 an increase in roughness, tooth sensitivity, morphological changes and enamel mineral loss. 4 These adverse effects may be transitory, considering the remineralization action played by saliva or topical fluoride application. 5 Therefore, using remineralizing agents like sodium fluoride (NaF), 6 or incorporating NaF into bleaching agents could be feasible alternatives for controlling or reversing enamel mineral loss. 7,8 Although the ability of sodium fluoride to reduce the solubility of components of the mineralized structure has already been proven, 9 other fluoride agents, such as titanium tetrafluoride (TiF 4 ), have drawn the interest of researchers to their therapeutic properties, such as reversing mineral loss, and triggering remineralization of mineral structures. 10,11,12 According to previous studies, TiF 4 can prevent demineralization, because it forms a titanium-rich layer on top of the enamel. 11,13,14 S olut io n s o r va r n i s h e s w it h d i f f e r e nt concentrations of TiF 4 (mostly 4%) have been designed and indicated for application on erosion lesions, owing to their ability to inhibit mineral loss. 14 The effectiveness of 4% TiF 4 in remineralizing artificial white spot lesions has been found to be similar to 2% NaF. 11 TiF 4 has an extremely low pH (≈ 1.2) 11 that might favor remineralization, 15 and that is believed to form a new compound (hydrated hydrogen titanium phosphate) by a process of dissolutionprecipitation. 16 During application, titanium ions interact with phosphate compounds, replacing the calcium ions on the hydroxyapatite dental surface. 15,17 Based on the findings for the remineralizing ability of TiF 4 , 18 its incorporation into bleaching agents could benefit esthetic procedures by preventing structural/morphological changes in enamel, and the damage promoted by HP. So far, only one in vitro study has performed topical application of a TiF 4 solution after in-office bleaching treatment. According to the results, 4% TiF 4 was as effective as NaF in preventing bleached enamel mineral loss. 19 Based on the aforementioned, this in vitro study aimed to test an experimental gel containing TiF 4 , with different stock gels, and evaluate the bleaching efficiency and pH of the gels, and the mineral content and morphology of the enamel submitted to a commercial 35% HP bleaching agent associated with the experimental gel formulations. TiF 4 powder was homogenized by producing experimental agents with different stock gels (Natrosol, Aristoflex and Chemygel), before combining them with a 35% HP commercial agent (HP).
Therefore, the null hypotheses were that the experimental TiF 4 gels, compared with a commercial HP agent: a) would not interfere in the bleaching effectiveness; b) would not change the enamel mineral content of the bleached enamel; and c) would not affect the surface morphology of the bleached enamel surface.

Methodology Experimental design
This in vitro study evaluated the color alteration, the enamel mineral content and the surface morphology of enamel blocks submitted to a 35% HP agent (HP, Whiteness HP 35% FGM, Joinville, SC, Brazil) with or without different TiF 4 -based formulations. The experimental TiF 4 -based formulations were obtained and tested using a study divided in two phases: 1-preliminary manipulation of different stock gels combined with TiF 4 , and 2-testing of the selected TiF 4 formulations combined with 35% HP.
In phase 1, different reagents and stock gels were combined with TiF 4 to obtain a gel-based TiF 4 that had a homogeneous appearance, and that could be applied to enamel, after which the gels obtained were analyzed visually. The most suitable combinations obtained in phase 1 were selected and tested in phase 2, in which enamel blocks were prepared, selected according to surface microhardness, and randomly divided into the following groups (n = 8) (independent variable at four levels): HP (control); HP combined with Natrosol + 4% TiF 4 (HPnT); HP combined with Natrosol + Chemygel + TiF 4 (HPncT); HP combined with Aristoflex + TiF 4 (HPaT). The response variables (dependent variables) were color evaluation (according to CIEL*, a*, b* parameters) and whiteness index for dentistry (WI D ), measured at baseline and 24 h after each bleaching session. Following the bleaching process, whiteness index differences (ΔWI D ), color alteration (CIELab -ΔE and CIEDE2000 -ΔE 00 ), changes in Knoop microhardness (ΔKHN), and enamel morphology (scanning electron microscopy -SEM) were evaluated. Phase 1. Preliminary manipulation of gel-based TiF 4 Different stock gels and diluents were tested (Tables 1 and 2) to produce the experimental gel able to dissolve TiF 4 , and having adequate viscosity and homogeneity. In this phase, no analytical method was used, except for the visual analysis of the gel combination tested. The experimental gels were manipulated under controlled temperature (20°C). Based on scientific literature, 4% TiF 4 in powder form was defined as the standard concentration for all manipulations, 20 and the weight equivalence (g) of the others compounds was calculated and weighed on an analytical precision balance (Chyo JEX-200, YMC Co Ltda, Tokyo, Japan). First, the TiF 4 powder was crushed with a plastic stick into the plastic beaker, and mixed with the amount of stock gel required to  According to the data reported in Tables 1 and  2, and based on visual analysis, the combinations selected for phase 2 were those capable of homogenizing TiF 4 without forming clusters or precipitation, and of maintaining gel viscosity (as opposed to liquid), compared with the commercial bleaching gel combinations. The experimental groups selected were: a. 96% Natrosol and 4% TiF 4 ; b. 48% Natrosol, 48% Chemygel and 4% TiF 4 ; c. 96% Aristoflex and 4% TiF 4 .

Phase 2. Testing of the selected gel-based TiF 4 Specimen preparation
Thirty-two enamel/dentin bovine blocks free of caries and defects were obtained from the buccal surface of sound bovine incisor crowns. The blocks with standard dimensions (5x5 mm) were polished with silicon carbide paper (#600, #1500 and #4000-grit) (Buehler ® , Lake Bluff, USA), and cleaned ultrasonically for 10 min (Marconi ® , Piracicaba, Brazil) at the intervals of the polishing steps.
The dentin surface was isolated with transparent acid resistance varnish (L'Apogée Alfaparf, Campo Grande, Brazil), and the blocks with the exposed enamel surface were immersed in black tea solution (Dr. Oetker, São Paulo, Brazil) for 24 h at room temperature (25°C). After staining, the specimens were stored in distilled water for one week, which was changed daily. 21,22 The surface microhardness (Knoop Hardness Number -KHN, Shimadzu ® Corporation, Kyoto, Japan) was determined, and enamel blocks with mean values of 292.9Kg/mm 2 +/-29.01 were selected and randomly allocated into four treatment groups (n = 8), as described in Table 3.

Bleaching treatment protocol
The control group (HP) was submitted to the bleaching protocol, according to the manufacture's instruction: the solution was manipulated in the plastic container -it consisted of one drop of thickener and three drops of 35% hydrogen peroxide, both from the commercial product. Manipulation of the experimental agents was performed with 4% TiF 4 combined with the stock gels (Natrosol, Natrosol + Chemygel or Aristoflex), as described in phase 1. One drop (or 0.05g) of these experimental gel-based TiF 4 alternatives was incorporated into the commercial bleaching agent (HP), as descripted for the control group, and mixed manually with a plastic spatula immediately before applying the solution to the enamel surface. Bleaching treatment consisted of three applications of 15 min each, in four sessions at 72-h intervals. 23 Following the bleaching protocol, the specimens were washed with distilled water and stored in artificial saliva (1.5 mM CaCl, 0.9 mM NaH 2 PO 4 , 150 mmol/l KCl, pH 7.0) at 37°C, 24 changed every 24 h.

Color measurement
The color parameters were evaluated before bleaching (baseline), and 24 h after each of the four bleaching sessions. The evaluation was performed with the Vita Easyshade spectrophotometer (Vita-Zahnfabrik, Bad Säckingen, Germany), attached to a three-fingered laboratory clamp (Jack lift -Q219, Quimis), with standard height, and with the tip pointing downwards to the dental block. The specimen was placed over an opaque white ceramic background, and the color was measured under a controlled light environment. Three color measurements were taken on each specimen in different directions, by rotating the specimen and the ceramic underneath, without displacing the spectrophotometer, considering that variations may occur if the tip is placed at a distinct angle. 25 Mean values of the CIELab parameters (L*, a*, and b*) were obtained. Later, the whiteness index for dentistry (WI D ), whiteness index differences (ΔWI D ), color change (ΔE) and CIEDE2000 color difference (ΔE 00 ) were determined according to the following equations: 26,27,28 Color changes were evaluated between the bleaching sessions: ΔE 0 (1 st session -baseline), ΔE 1 (2 nd session -baseline), ΔE 2 (3 rd session -baseline) and ΔE 3 (4 th session -baseline).
CIEDE2000 was performed according to the L*, a* and b* parameters, and to the c* and h* parameters calculated by a software program validated by Sharma et al. 29

Microhardness analysis
Knoop microhardness measurements (KHN, Shimadzu, Kyoto, Japan) were performed on the enamel surface before and after 24 h of the last bleaching application (4 th ) to calculate the change in Knoop microhardness (ΔKHN). Five indentations were made 500 µm from the margin and 100 µm apart, under a 490.3mN load for 10 sec. Mean values were obtained for each specimen. 12

pH measurement
Bleaching gels (control and experimental agents) were submitted to pH measurement (MS Tecnopon, Piracicaba, Brazil) using a small ion-electrode device (Sensoglass, São Paulo, Brazil). 3,30,31,32 The pH meter was calibrated with three standard solutions (pH 1.0, 4.0 and 7.0) (Dinâmica ® , Diadema, Brazil) and 0.01 accuracy. The pH of each group was evaluated at four different times (baseline, 5, 10 and 15 min, corresponding to a single bleaching application of 15 min) in triplicate (three pH readings).

Scanning electron microscopy
Three specimens from each group were selected and observed under scanning electron microscopy (SEM) (JEOL-JSM, 6460LV, Tokyo, Japan). The specimens were left to dry in an incubator for 24 h, sputter-coated with gold (MED 010, Balzers, Liechtenstein), and submitted to evaluation using SEM (1,500x magnification), operating at 15 kV in vacuum mode (45 Pa). 12

Statistical analysis
The normal distribution and homoscedasticity of the data were confirmed by the Shapiro-Wilk and Levene tests (p > 0.05), and parametric analyses were then performed. CIELab parameters (L*, a*, b*), and the whiteness index for dentistry (WI D ) were analyzed by two-way repeated-measures ANOVA and Bonferroni's post-hoc test. Whiteness index differences (ΔWI D ), color alteration (ΔE), and CIEDE2000 color difference (ΔE 00 ) were tested by one-way ANOVA and Tukey's post-hoc test. Changes in Knoop microhardness (ΔKHN) were analyzed by one-way ANOVA and LSD post-hoc test. The analyses were performed using SPSS 21.0 (SPSS, Chicago, IL, USA), with a significance level set at 5%. SEM evaluation and pH measurement were submitted to descriptive analysis. The test power was determined by GPower 3.1 (GPower, ASA Group, Autenzell, Bayern, Germany), considering the main variables of the study (surface microhardness and the color parameters of CIELab, whiteness index for dentistry -WI D , whiteness index differences -ΔWI D , color change difference -ΔE and CIEDE2000 -ΔE oo ) (β > 0.9, α = 0.05).

Color measurement
The test power showed values higher than 0.9578 (95%) for all the methodologies performed, thus ensuring high sample value confidence.
The CIELab color parameters (L*, a* and b*) values for HP, and the experimental agents at baseline and after bleaching applications are shown in Table 4. The L* coordinate did not differ among the bleaching sessions or the groups (p > 0.05). At the end of bleaching, HP and HPncT groups showed no differences in a* mean values (p > 0.05), but were higher than groups HPaT and HPnT (p < 0.028). Furthermore, only HPaT showed a decrease in the a* value at the end of bleaching (p < 0.01). The yellow appearance of the teeth (b* mean values) decreased after HP (p < 0.014) and HPncT applications (p < 0.03), unlike the other groups that maintained similar b* values (p > 0.05). HP displayed lower b* values (p = 0.038) than HPnT after the bleaching treatment, denoting that values were closer to blue than yellow.
The whiteness index for dentistry (WI D ) and the color change (ΔE) values for all the treatment groups throughout the bleaching application period are shown in Table 5. No differences in WI D were observed among the groups at any bleaching application, or throughout the bleaching sessions (p > 0.05), except for HP between baseline and the 2 nd , 3 rd and 4 th applications. HP had higher ΔE values, based on the 2 nd bleaching application, compared with HPnT at ΔE 1 , and compared with all the experimental agents at ΔE 2 and ΔE 3 (p < 0.004). Moreover, no differences were found among the experimental groups at any color change evaluated (ΔE 0 -ΔE 3 ) (p > 0.05).  The whiteness index difference (ΔWI D ) and color change (CIELab -ΔE and CIEDE2000 -ΔE 00 ) values for all the treatment groups are shown in Table 6. HP had a higher whitening effect (ΔWI D ) than HPnT (p = 0.002), but it was no different from HPncT and HPaT (p > 0.05). HP exhibited higher color change values (ΔE and ΔE 00 ) than the experimental groups (p < 0.047), but no differences in color change (ΔE 00 ) were found between HP and HPncT (p > 0.05). Figure 1 shows the results for the change in Knoop microhardness (ΔKHN, before and after bleaching treatment) among the bleaching formulations. HPncT increased the enamel microhardness, and had a higher ΔKHN than any other group (p < 0.022). HP and HPaT decreased enamel microhardness with no statistical difference between them (p = 0.716). HPnT caused the highest microhardness loss among the groups, after bleaching (p < 0.006).

pH-measurement
The control group (HP) presented the highest pH values (6.9) among the groups (Figure 2

Scanning electron microscopy
The representative enamel surface areas of the control and experimental groups are shown in Figure 3. The HP group showed a flat and relatively smooth surface, whereas the HPnT and HPaT groups presented similar surface topography, revealing rough enamel areas, and the presence of enamel cracks. In contrast, HPncT displayed a regular, flat surface, with no topographical enamel alterations.

Discussion
The primary goal of this research was to determine if the experimental gels containing TiF 4 (a potential remineralizing agent) combined with 35% hydrogen peroxide would influence the bleaching efficacy of the oxidizing agent (HP). The bleaching ability was determined by measuring color according to three distinct parameters (L*, a*, and b*), which individually indicate variations of lightness (L* -black; +white) and chrome (a*-green; +red and b* -blue; +yellow). 33 For bleaching purposes, changes in the L* and b* parameters denote bleaching, since lightness increases (L* values), and the yellow appearance of the teeth (b* values) decreases. 33 According to the results, no increase in lightness (L* values) was observed    after dental bleaching regardless of the bleaching gel tested, but the yellow appearance of the HP and HPncT treated groups decreased (b*). This result was expected for the HP group, but was a positive outcome for the HPncT group. Variations in the a* parameter indicate whether color is changing to red (a* > 0) or green (a* < 0), and although this parameter could hardly apply to bleaching, specimens in this study were stained with black tea solution; 21 therefore, variations in the a* parameter could denote removal of the black tea stains. After bleaching, all the groups showed negative values (a* < 0), indicating the ability of all the agents to decompose the red stains promoted by the tea, but HPaT and HPnT showed the lowest a* values.
The whiteness index for dentistry (WI D ) indicates that higher values correspond to whiter teeth, and lower WI D (including negative values) denotes darker teeth. The positive WI D results for all the groups indicated the whitening ability of the agents, 27 and no differences were observed between the experimental groups and the control (HP) for any of the bleaching applications. Moreover, the ΔWI D values for HPncT and HPaT were similar to HP. However, the commercial HP gel showed greater color changes (ΔE) than the experimental agents. Although ΔE is an important parameter for observing color changes based on the L*, -a*, -b* parameters, we should bear in mind that it should not be interpreted individually, but together with other variables, like WI D and ΔWI D , which correlate color with proximity to white. 27 In addition, all the groups showed ΔE higher than 2.7, which is the limit previously established for observing color changes clinically. 34 Another formula used in dentistry to evaluate color change is CIEDE2000, which uses the CIEL*a*b* parameters, but provides adjustments in the way color difference is evaluated to make it acceptable for human accuracy (ΔE 00 ≥1.8). 28,35 According to the results, no differences in ΔE, ΔWI D , or ΔE 00 were found among the HPaT, HPncT, and HPnT experimental groups; however, only HPncT exhibited two of these parameters (ΔWI D and ΔE 00 ), similar to the control group (HP). Overall, the color results seem to suggest that hydrogen peroxide was able to diffuse through the enamel and dentin, and react with organic and bioorganic matter, or biological residual products of dental tissues, and achieve bleaching by breaking down the stain. The stain removal occurred even when HP was combined with the experimental TiF 4 -based gels. 4,33,36 On the other hand, the different stock gels tested (Aristoflex, Natrosol or Natrosol with Chemygel) in combination with an acidic agent (TiF 4 ) may have interfered with the decomposition rate of HP. 37 Therefore, although we expected the experimental agents to perform comparably to HP, or preferably better in every color parameter, the first hypothesis was not accepted, since the TiF 4based gels interfered with the bleaching efficacy of the commercial HP agent.
In the present study, the reason for using different stock gels was based on having to dilute TiF 4 before it could be incorporated into the bleaching agent (phase 1). TiF 4 is a highly unstable agent in water 17 , and the first trials used Chemygel, a water-free petroleum polymer-based gel. Unexpectedly, Chemygel alone had unsatisfactory results, because it was unable to homogenize TiF 4 , even after sonication and centrifugation. Subsequently, other stock gels were tested (Aristoflex, Natrosol and Natrosol combined with Chemygel), and these combinations were able to homogenize TiF 4 .
Stock gels are used as vehicles for bleaching formulations, because they turn solid or liquid compounds into gel consistency, and thus prolong the decomposition rate of oxygen ions. 37, 38 Aristoflex is a versatile polymer, and is frequently used as a vehicle in cosmetic products. 39 Natrosol is a cellulose-based polymer found in some existing bleaching products. It remains stable in acidic media (such as TiF 4 solution or gel) without damaging the enamel structure. 37,40 Previous studies have observed that Natrosol promotes acceptable color change when combined with HP, and minor enamel surface alterations after bleaching, thus qualifying this agent as a promising substitute for Carbopol. 38 However, although TiF 4 is unstable, the results of this study indicate that the combination of Natrosol with Chemygel performs better in regard to color change, compared with the other experimental formulations. The fact that HPncT was able to change the enamel color to one that was closer to that obtained for the HP group may indicate that the addition of TiF 4 with Natrosol and Chemygel did not completely hamper the ability of HP to release free radicals, and oxidize the stained molecules. Nevertheless, adjustments in the formulation made by adding catalyst substances could be of interest.
The control group (HP) -a stable commercial reference -decreased enamel microhardness, a result confirmed by a previous study. 3 In fact, this was expected, and was why TiF 4 -based gels were combined with HP. However, not all the TiF 4 -based gels performed appropriately. HPnT, for instance, had the lowest microhardness difference among the groups, and the smallest surface morphological alterations, as observed under SEM. In addition, although HPaT succeeded in bleaching, it also decreased the enamel microhardness and promoted morphological alterations in different parts of the enamel, ultimately compromising the performance of this gel.
As opposed to t hese results, t he HPncT experimental agent increased microhardness after bleaching, and no morphological changes or surface defects were detected on the enamel, representing a very important finding. Thus, since experimental formulations decreased (HPnT) or increased (HPncT) the enamel microhardness difference, and promoted enamel surface morphology alterations (HPnT and HPaT) compared with the control group (HP), the 2 nd and 3 rd null hypotheses were rejected, respectively.
The pH value plays a major role in enamel mineral content balance. 41 It can influence the kinetics and stability of the stock gel, 38 and how TiF 4 interacts with enamel. For example, TiF 4 combined with Natrosol + Chemygel (HPncT) was able to maintain enamel microhardness and surface characteristics even in acidic pH (final pH = 3.91), unlike HPnT (final pH = 2.30), which included only Natrosol as a stock gel, and which had lower microhardness values. Yu et al. 15 observed that the TiF 4 solution performed better (remineralizing potential) in acidic media. This could explain the remineralizing action of HPncT, compared with that of the HP group. It should be pointed out that higher pH values interfere in the storage stability of HP, and increase the bleaching efficacy of peroxide-based gels, 41 which could explain why the commercial HP had superior color results for most color parameters, compared with the experimental acidic TiF 4 gels.
The TiF 4 mechanism is based on its ability to deposit titanium ions on the enamel surface, thus ensuring acid resistance and producing a layer with hydrated titanium phosphate, titanium oxide and calcium fluoride, able to prevent acid penetration. 10,17,42 This layer is formed on enamel treated with solutions or varnishes containing 4% TiF 4 , 13,14,15,42 and is not homogeneous because of differences in the concentration of inorganic content (phosphate crystals and hydroxyapatite) on the enamel surface. 11 One limitation of the current study was that microhardness assessment was limited to the surface, and no cross-sectional microhardness evaluation was performed to determine the integrity of the enamel subsurface. Another limitation was that no chemical analysis was performed on the enamel to confirm whether a layer with hydrated titanium phosphate or titanium oxide actually was formed. In addition, although the experimental gel containing 4% TiF 4 , Natrosol and Chemygel combined with 35% HP showed promising results (since it increased enamel microhardness and showed no morphology changes), no post-bleaching microhardness evaluation was performed, and the ability of artificial saliva to control mineral loss over time 43 was not assessed. Possibly, the reduction in microhardness caused by the commercial HP gel and the experimental HPaT could be reversed even in in vitro conditions, and the microhardness results observed would not represent a major concern over time.
Since no reports in the literature describe the combination of stock gels/TiF 4 /HP, further laboratory investigations are essential to evaluate the enamel color change of TiF 4 associated with different bleaching products that have distinct pH values. Moreover, the post-bleaching enamel mineral content, viscosity and cytotoxicity of this experimental gel must still be determined.

Conclusions
The different stock gels (Aristoflex, Natrosol or Natrosol with Chemygel ® ) evaluated were able to homogenize 4% titanium tetrafluoride (TiF 4 ). Furthermore, 35% hydrogen peroxide combined with an experimental gel containing TiF 4 , Natrosol and Chemygel (HPncT) was able to control enamel mineral loss and enamel morphological alterations, even at low pH values.