Abstract
Objective
This study evaluated in vitro, the effects of carbamide peroxide 10% (CP) associated with Carbopol® (CP-ct) and Aristoflex® (CP-at) thickeners on human gingival fibroblasts (HGF) cytotoxicity and assessed in situ their effects on dental enamel.
Material and methods
The cytotoxicity was analyzed using MTT - Vybrant® proliferation test. For in situ stage, 144 bovine enamel/dentin blocks were randomized into seven groups (n=12). Samples were stained, fixed in intraoral palatal devices and bleached for 4 h, during 14 days, with: Carbopol thickener (ct), Aristoflex thickener (at), CP-ct, CP-at, CP without thickener (CP-wot), Commercial CP (CP-com). The samples had their microhardness (SMH), roughness (Ra) and color analyzed using a microdurometer, a rugosimeter and a spectrophotometer, respectively. The analyses were performed at baseline and 24-h after completion of tooth bleaching.
Results
Different thickeners were similar regarding their cytotoxicity. The experimental gels with Carbopol exhibited lower SMH values, while the groups treated with CP exhibited higher Ra values. For the color change results, the groups treated with CP had values above the acceptability and perceptibility limits.
Conclusion
CP-at was able to promote an effective bleaching with less alterations of the tooth surface compared to the CP-ct. Hence, Aristoflex stands as a promising thickener in conjunction with CP in order to preserve the physical properties of dental enamel after home bleaching.
Key words: Tooth Bleaching, Dental Esthetics, Biocompatible Materials, Dental Enamel, Fibroblasts
Keywords: MeSH terms: Tooth Bleaching, Bleaching Agents, Biocompatible Materials, Dental Enamel, Fibroblasts, Dental Esthetics
Introduction
Among bleaching gels, the gold standard treatment is carbamide peroxide 10 wt% (1). The bleaching treatment can be regarded as safe and minimally invasive (2). However, there are concerns remaining about its unfavorable effects on dental tissues such as alterations of the surface roughness or the tissue microhardness (3, 4), apart from its possible cytotoxic effects on the gingiva (5).
The mechanism by which the tooth bleaching occurs is associated with hydrogen peroxide and its precursor, carbamide peroxide (CP), oxidizing capacity. These radicals are oxidizing agents which are able to break chromogenic molecules. Besides, they are able to remove these pigments by diffusion out of the enamel and dentin structure. Consequently, this reduces the light absorption, thus making the tooth lighter (6–8).
CP-based bleaching gels formulations usually comprise Carbopol (carboxypolymethylene) as thickener which is an acidic and ionic polymer derived from carboxylic acid. Carbopol increases the bleaching gels viscosity (9), hence is able to promote better retention in tray and maintenance on the dental surface. In other studies, it has been proven that Carbopol could reduce the enamel microhardness due its capacity to bind to calcium (10, 11), thus preventing the saliva minerals from being incorporated into the dental structure.
Considering the Carbopol limitations, this study intended to evaluate a bleaching gel that contained a thickener based on sulfonic acryloyldimethyltaurate acid copolymer and vinylpyrrolidone (Aristoflex), which is a pre-neutralized synthetic polymer that has been already assessed in an in vitro study (12). Aristoflex aids in the formulation of crystalline gels with an adequate consistency. This thickener has some important properties: It is stable in an acid pH has a cationic behavior, and it acts as an inert thickener within the formulations. In the pharmaceutical production it is already used as a thickener in personal care formulations. Moreover, it is also incorporated in oral hygiene products such as whitening toothpastes, mouth rinses, and gels (13), without posing long-term risks to human health.
As previously mentioned, this study evaluated the in vitro cytotoxicity and in situ physical alterations of dental enamel, such as color, roughness, and microhardness, after treatment with an experimental bleaching gel that contains carbamide peroxide 10 wt% and sulfonic acryloyldimethyltaurate acid copolymer and vinylpyrrolidone (Aristoflex). The study null hypotheses were: 1) Aristoflex would not be toxic to human gingival fibroblasts (HGF) cells; 2) 10 wt% carbamide peroxide gel containing Aristoflex would not interfere with the bleaching efficacy, enamel roughness, and microhardness.
Material and methods
Cytotoxicity Analysis: Based on initial cytotoxicity tests, dental bleaching gel solutions with different concentrations were formulated. First, the pH values of gels were measured in triplicate with a digital pH meter (Phs-3b, Phtek, Sao Paulo, SP, Brazil). The pH meter had on a pH electrode with a temperature sensor connected to an ion analyzer. The electrode was put inside 3 g of each gel and the results were recorded (Table 1).
Table 1. Manufacturers, basic composition and pH of products.
| Product | Manufacturer | Composition* | pH |
|---|---|---|---|
| Carbopol thickener (ct) | Drogal Manipulations (Piracicaba, Brazil) |
Carbopol 940, nipagin, glycerin, amp-95 and deionized water | 3.72 |
| Aristoflex thickener (at) | Aristoflex AVC, nipagin, glycerin, amp-95 and deionized water. | 4.95 | |
| Carbamide peroxide 10 wt% + carbopol thickener (CP-ct) | Carbamide Peroxide 10 wt%, sodium fluoride, carbopol 940, nipagin, glycerin, amp-95 and deionized water | 4.56 | |
| Carbamide peroxide 10 wt% + Aristoflex thickener (CP-at) | Carbamide Peroxide 10 wt%, sodium fluoride, Aristoflex AVC, nipagin, glycerin, amp-95 and deionized water | 6.97 | |
| Carbamide peroxide 10 wt% (CP-wot) | Carbamide Peroxide 10 wt% and deionized water | 7.74 | |
| Whiteness Perfect or Commercial CP 10 wt% (CP-com) | FGM (Sta Catarina, Brazil) |
Carbamide Peroxide 10 wt%, potassium nitrate, carbopol neutralized, sodium fluoride and deionized water | 5.45 |
*MSDS data sheet.
For the cytotoxicity analysis based on a previous study (14), human gingival fibroblasts (HGF) were cultivated in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with antibiotics (10000 U penicillin and 10 mg/mL streptomycin - Vitrocell Embriolife, Campinas, SP, Brazil) and 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and humidified atmosphere containing 5% CO2. Once an approximate 80% confluence was reached, in order to detach them, the cells were washed with 0.25% trypsin/EDTA (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After detachment, the cells were centrifuged at 3000 rpm for 5 min at 4 °C. The supernatant was discarded and a new was added and mixed with the cells which were further seeded in 96-well culture plates (Corning Costar Corp., Cambridge, MA, USA).
Considering the final concentration in the culture medium, the cells were exposed to various gels concentrations as follows: 10 mg/mL; 5 mg/mL; 2.5 mg/mL; 1.25 mg/mL; 0.63 mg/mL; 0.31 mg/mL.
1) Carbamide Peroxide 10 wt%+ Carbopol (CP-ct); 2) Carbamide Peroxide 10 wt%+ Aristoflex (CP-at); 3) Carbamide Peroxide 10 wt% + Carbopol / commercial (CP-com); 4) Aristoflex gel (at); 5) Carbopol gel (ct); 6) Triton X-100 0.1% (Sigma-Aldrich): negative control; 7) DMEM: positive control.
Culture plates (n = 7) were incubated at 37 °C for 4 h (bleaching treatment time). After the incubation had been completed, the cytotoxicity was determined by Vybrant® MTT cell proliferation assay (Thermo Fisher Scientific, Waltham, MA, USA). The MTT is an indirect method to determine the cell viability and proliferation according to the mitochondrial succinate dehydrogenase activity.
Within living cells, MTT [3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide], a yellow tetrazole salt, is reduced by succinate enzyme complex to Formazan [(E, Z)-5-(4, 5-dimethylthiazol-2-yl)-1, 3-diphenylformazan], that later precipitates as insoluble purple-gray crystals. After the crystal dissolution, the final solution color intensity (optical density), measured with the spectrophotometer, is the measurement of the cell viability.
For the cell counts, a Neubauer counting chamber was used. For this, the cells were seeded 24 h before the incubation with the gels extracts. For cytotoxicity assessment, 100 µL of solution (5 x 104 cells/mL) was added to each well of the culture plates. Then, the gel extracts were added to the wells. After incubation had been finished, the liquids were aspirated and the wells were washed gently twice with phosphate buffered saline (PBS pH 7.4; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 200 µL of 0.3 mg/mL of MTT solution in DMEM was placed into the wells. The plates were further incubated for 3 h with 5% CO2 at 37 °C. Finally, after having been emptied, the wells were filled with 200 μL of ethanol. The absorbance was read using a micro-spectrophotometer (Asys UVM340, Biochrom, Cambridge, England) at 570 nm to determine the optical density. The assays were made at two different times and all of them in quadruplicate. Based on da Silva et al. (15), the products cytotoxicity was scored.
In situ stage: This was a randomized double-bind in situ study. Freshly extracted bovine incisors, without fractures or enamel stains, were chosen and stored in 0.1 wt% thymol at 4 °C. Enamel/dentin blocks were prepared for color and roughness analysis (4 x 4 x 2.5 mm), and for microhardness analysis (4 x 4 x 2 mm). Decreasing-grit silicon carbide abrasive papers (#1200, #2500, and #p4000 - Isomet, Buehler, Lake Bluff, IL, USA) were employed to regularize the enamel surface and achieve an adequate height. To polish the samples, felts (Top-, Ram- and Supra-, Arotec, Cotia, São Paulo, SP, Brazil) with metallographic diamond pastes (6 μm - Top, 3 μm - Ram, 1 μm – Supra, Arotec) and lubricant (Arotec, Cotia, São Paulo, SP, Brazil) were used. In the course of each polishing step, in order to remove the polishing debris residuals, the samples were water-sonicated for 10 min.
After preparation, the samples were stained by putting them into a black tea solution (Leão Junior SA, Curitiba, PR, Brazil). The tea solution was renewed daily. After completing 6 days of staining, the samples were stored in artificial saliva, daily renewed, during 14 days in order to achieve the color stabilization (16). Subsequently, the samples were sterilized with ethylene oxide and were stored in water at 4 °C until the treatment.
After signing an informed consent form, twelve volunteers (six males and six females), aged 18-35 years, agreed to participate in this study (Ethics Committee register: 79720117.1.0000.5418). The inclusion criteria met by the volunteers were: normal salivary flow (checked by sialometry) and the absence of caries and/or periodontal disease- Patients who do not absorb any fluoride (by either fluoridated water intake, or use of fluoride toothpaste), wear orthodontic appliances, use medication that altered their salivary flow, smoke, or had fixed or removable prostheses were excluded.
A maxillary impression of each volunteer was obtained with alginate (Hydrogum, Zhermack, Badia Polesine, RO, Italy). Using the cast models, palatine acrylic-resin-based devices were created containing 6 cavities of 35 mm2 wide and 3 mm deep. Inside the cavities, the teeth samples were inserted and stabilized in the palatal device employing sticky wax (ASFER - Chemical Industry Ltda, SP, Brazil). In each cavity, color/roughness and microhardness samples were placed from each group and exposed to saliva. The groups were in diverse places in each patient, considering the salivary ducts of the parotid gland location.
Before the beginning of all treatments, the palatal devices stayed in a volunteer's mouth for 24 h to allow the salivary pellicle development. Volunteers were taught to remove the device only in order to consume food and drink. Meanwhile, the samples had to remain inside a water-filled device to avoid dehydration. One day after the device had been taken out of from the mouth and dried with absorbent paper and, the teeth samples were bleached. For each sample placed in the device, a single bleaching gel treatment was applied daily during 14 days. For this scope, the volunteers were trained to do the gel application over the samples’ enamel surfaces, and let them undisturbed during 4 h at room temperature. Throughout the bleaching treatments, the device had to remain in a water-filled device. It is relevant to point out that the water did not get in contact with the area covered by the bleaching gel. After the treatment time was over, gels were rubbed out using cotton swabs, washed with running water, and dried with absorbent paper. Subsequently, the device was placed back to the patient's palate.
Microhardness Analysis (SMH): An enamel surface microhardness (SMH) analysis was performed using a Knoop microhardness tester (HMV 2000, Shimadzu, Kyoto, Japan). At the samples central region, a Knoop diamond indenter applied a static load (50 gf/5 s). Analyses were taken initially - after staining (initial), and 24 h after the bleaching treatments finished (final) (Figure 1). For each sample, the average of five 100 μm equidistant indentations was used for statistical analyses.
Figure 1.
_363-375-f1.jpg)
In situ phase experimental design summaries
Roughness Analysis (Ra): For the purpose of surface roughness (Ra) assessment, a roughness tester (Surftest 211, Mitutoyo, Suzano, SP Brazil) was used. The Ra was also assessed at initial and final stages (Figure 1). On the sample surface, three equidistant measurements were made. The instrument was programed to have a cutoff point of 0.25 mm, reading extent of 1.25 mm, load of 5 N and speed of 0.1 mm/s. The average of three measurements of the same sample was considered in the statistical analysis.
Color Analysis (ΔE*ab, ΔE00, WID): To analyze the color, a spectrophotometer was used (CM 700D, Minolta, Osaka, Japan) which was previously calibrated following the manufacturer's specifications. Samples were put in a polytetrafluoroethylene-based device in ambient light condition (MiniMatcher MM-1, GTI Graphic Technology, Newburgh, NY, USA) as to systematize the environment. The measurements were made after staining (initial) and 24 h after the treatment end (final). The ΔE*ab, ΔE00 and Whiteness Index for Dentistry (WID) values were obtained through the following formulas:
_363-375-e1.jpg) |
where ΔL*, Δa* and Δb* are the differences between the assessment times (initial- final) (Figure 1) for the L*, a* and b*values, correspondingly. ΔL', ΔC' and ΔH' stand for the differences in light value (Light), chroma (Chroma) and hue (Hue), respectively, employing CIEDE2000 metric. SL, SC and SH are factors to regulate the coordinate values as a function of variation in color difference. While KL, KC and KH enable to do a correction concerning the experiment settings, and RT is a parameter that takes in account the interaction between chroma and hue changes in blue region.
The following perceptibility and acceptability limits were taken as reference: (17-19)
- Delta Whiteness Index for Dentistry (ΔWID): 50%:50% perceptibility (0.72 ΔWID); 50%:50% acceptability (2.62 ΔWID)
- CIELAB and CIEDE2000: 50%:50% perceptibility (1.2 ΔE*ab and 0.8 ΔE00) and 50%:50% acceptability (2.7 ΔE*ab and 1.8 ΔE00).
Statistical Analyses: Statistical analyses were performed using GraphPad® Prism 6.0 software (GraphPad Software, San Diego, CA, USA). The normality and equality of variances were checked with the Shapiro-Wilk and Levene's tests. Cytotoxicity, ΔE*ab and ΔE00 data were analyzed by one-way analysis of variance (ANOVA) and Tukey test. For the microhardness and roughness data, mixed models for repeated measures and Tukey-Kramer test were used. ΔWID was analyzed by non-parametric Kruskal Wallis and Dunn tests. A 5% significance level was considered for all the analyses.
Results
Cytotoxicity: Cytotoxicity results are shown in Figure 2. Without CP, the Carbopol and Aristoflex gels had cytotoxicity values above the IC50 independently of their concentration. Still, above the 5 mg/mL concentration all groups that had CP in their formulation exhibited cytotoxicity values below IC50.
Figure 2.
_363-375-f2.jpg)
Cell viability (%) based on the employed gel concentration. #p < 0.05 compared to carbopol (ct) and *p < 0.05 compared CP 10% + carbopol (CP-com). IC50: concentration that after exposure causes 50% cell death or cell growth inhibition or other utilized cytotoxicity metric.
Microhardness (SMH): For the SMH, the interaction among groups and times was significant (p < 0.05). At the initial time amid groups no statistical difference was found (p > 0.05). The groups treated with Carbopol: Carbopol thickener (ct) and Carbamide Peroxide 10 wt% + carbopol thickener (CP-ct) SMH decreased significantly (p < 0.05). At the final assessment (24 h after end of treatment) the ct group SMH values were lower than the rest of the groups, and the CP-ct group had significantly lower SMH compared to other groups, however the difference was not significant when compared to Carbamide Peroxide 10% with Aristoflex (CP-at) (p < 0.05), Table 2.
Table 2. Mean (standard deviation) microhardness (KHN) of enamel before and after being submitted to different bleaching treatments.
| Group | Time | |
|---|---|---|
| Initial | Final | |
| Aristoflex thickener – at | 293.87 (15.75) Aa | 280.27 (25.53) Aa |
| Carbopol thickener – ct | 293.71 (16.43) Aa | 151.00 (36.27) Bc |
| Carbamide peroxide 10 wt% with carbopol (experimental) – CP-ct | 292.52 (16.56) Aa | 250.28 (24.13) Bb |
| Carbamide peroxide 10 wt% with aristoflex (experimental) – CP-at | 292.97 (15.41) Aa | 275.09 (36.28) Aab |
| Carbamide peroxide 10 wt% (without thickener) – CP-wot | 292.66 (14.94) Aa | 285.85 (9.32) Aa |
| Carbamide peroxide 10 wt% with carbopol (commercial product - control group) – CP-com | 292.72 (15.21) Aa | 280.60 (8.62) Aa |
Means followed by distinct letter (uppercase in horizontal and lowercase in vertical comparison) differ from each other (p≤0.05); p (group) <0.0001; p (time) <0.0001; p(interaction)<0.0001.
Roughness (Ra): Regarding Ra (Table 3), the factors groups and times had a significant interaction (p < 0.05). At the initial time, the Ra between the groups was no significantly different (p > 0.05) and all of them showed significant Ra increase over time (p < 0.05). At the final assessment, the higher Ra was for the group treated with the Carbopol thickener (ct) (p < 0.05), but the difference was not significant when compared to Aristoflex thickener (at) (p > 0.05).
Table 3. Mean (standard deviation) roughness (Ra) of enamel before and after being submitted to different bleaching treatments.
| Group | Time | |||
|---|---|---|---|---|
| Initial | Final | |||
| Aristoflex thickener – at | 0.12 (0.01) Ba | 0.15 (0.02) Aab | ||
| Carbopol thickener – ct | 0.11 (0.01) Ba | 0.16 (0.02) Aa | ||
| Carbamide peroxide 10 wt% with carbopol (experimental) – CP-ct | 0.11 (0.01) Ba | 0.14 (0.02) Ab | ||
| Carbamide peroxide 10 wt% with aristoflex (experimental) – CP-at | 0.11 (0.01) Ba | 0.14 (0.01) Ab | ||
| Carbamide peroxide 10 wt% (without thickener) – CP-wot | 0.12 (0.01) Ba | 0.14 (0.01) Ab | ||
| Carbamide peroxide 10 wt% with carbopol (commercial product - control group) – CP-com | 0.12 (0.01) Ba | 0.14 (0.01) Ab | ||
Means followed by distinct letter (uppercase in horizontal and lowercase in vertical comparison) differ from each other (p≤0.05); p (group) <0.0001; p(time)<0.0001; p(interaction)<0.0001.
Color: The groups treated with gels that only contained thickeners (ct and at) displayed significantly lower ΔE*ab (p < 0.05) (Figure 3). Boxplot graphs also display the perceptibility and acceptability limits.
Figure 3.
_363-375-f3.jpg)
Boxplot of the ΔE * ab according to the group and time. Different letters denote significant differences (p≤0.05).
Similarly, the gels that contained only the thickeners (ct and at) ΔWID values were significantly lower than the rest of the groups (p < 0.05) (Figure 4). Color variations in the carbamide peroxide 10 wt% with Carbopol (CP-ct) carbamide peroxide 10 wt% with Aristoflex (CP-at), and carbamide peroxide 10 wt% with Carbopol commercial product (CP-com) groups were beyond the perceptibility and acceptability limits.
Figure 4.
_363-375-f4.jpg)
Boxplot of the delta whiteness index for dentistry (ΔWID) according to the group and time. Different letters denote significant differences (p≤0.05).
As for ΔE00 (Figure 5), the groups that contained only the thickeners (ct and at) had also lower values when compared to the rest (p < 0.05). All groups with 10% carbamide peroxide in their formulation displayed ΔE00 values above the perceptibility and acceptability limits.
Figure 5.
_363-375-f5.jpg)
Boxplot of ΔE00 according to the group and time. Different letters denote significant differences (p≤0.05).
Discussion
According to the obtained results, the first hypothesis, that the association of Aristoflex with carbamide peroxide 10 wt% (CP) would not be cytotoxic for human gingival fibroblasts (HGF) cells, was accepted. The usage of CP for the bleaching of vital teeth has been well-established and can be broadly implemented (20). Nonetheless, CP is placed into custom-made trays. Since these trays touch the gingival tissue, there exist the possibility of irritations and ulceration wounds caused by the CP and all its by-products as they have contact with oral cells. Besides, they have toxic effects on HGF (5, 21).
Previous in vitro studies employed HGF to assess potential cytotoxic components within the bleaching gels (5, 22). The current study demonstrated that the usage of CP-ct and CP-at for 4 h generated a comparable cytotoxicity curve. Nevertheless, the bleaching gel concentration augmentation induced the reduction of the fibroblast viability, particularly over 2 mg/mL. It is possible to assume that this effect is directly associated with the CP concentration, making this component liable for the product cytotoxic effect. Nevertheless, it is relevant to emphasize, that even at their highest concentrations, none of the thickeners (Carbopol and Aristoflex) without CP were cytotoxic.
Preceding studies have stated that mineral dissolution might occur throughout tooth bleaching procedure resulting in changes of the enamel surface (8, 23, 24). Based on the results of Basting et al., 2001, the bleaching gels acidic properties, extended application times, and the addition of Carbopol are considered to be likely the main responsible factors for dental structure surface alterations (25). Even if bovine enamel was evaluated in the current study, considering its physical-chemical similarity to human enamel (8), it is reasonable to deduce that this study obtained results that could be comparable to the results obtained with human tooth. Based on previous research (12, 13), thickeners are polymers that, due to their bioadhesive capabilities, interrelate with dental structure. Bioadhesive capacity is associated with potential ionic bonds between polymers and the dental structure, generating a sort of "film" (meaning, a polymer coating that is located on the dental structure). This "film" has the capacity to generate a barrier that precludes the saliva-induced remineralization which is essential to reduce or hamper the hydrogen-peroxide-induced mineral loss generated during the bleaching treatment (11, 24).
Of these thickeners, Carbopol is an anionic polymer with a high capacity to bind to the dental surface due to its high affinity with it. This closeness creates a “film” in a properly-adhered and thick way that would finally preclude the saliva-induced remineralization (12, 13). Conversely, Aristoflex exhibits some properties that are different from Carbopol, starting by its cationic nature. Besides, it has low affinity for the dental structures, which results in the establishment of a not so closely-attached “film”. Therefore, it may favor the saliva-induced remineralization (11, 12).
Our results are in accordance with previous research (12, 13). In the comparison of the initial and final times of our study, a reduction in the SMH of the experimental groups that had Carbopol (ct and CP-ct) could be noticed. For the final assessment, the experimental groups that had Carbopol exhibited lower SMH values, while the experimental group that had carbamide peroxide 10 wt% with Aristoflex (CP-at) displayed more intermediary values than the rest of the groups.
Even if the commercial gel (CP-com) could have neutralized Carbopol thickener incorporated, which is able to decrease the occurrence of potential alterations on the enamel surface, the precise amount of this compound has remained unknown. Since the formulation details are undisclosed, it is challenging to determine how each particular component could have effects on the ultimate properties of the material. Nevertheless, it is possible to speculate that the Carbopol levels within the FGM gel are not sufficiently high to modify the enamel SMH, which happened with experimental gels that contained Carbopol. The salivary fluid in the oral environment is overloaded with minerals such as calcium and phosphate that have the capacity to assist in the process of remineralization (24, 26) or in the demineralization decrease (8). Along with the process of remineralization, the minerals from saliva could be deposited in an irregular manner on the enamel surface and this could increase the surface roughness (10) similarly as it occurs during the dissolution of the minerals. In the present research, all the groups presented an increase in Ra levels which could be related to the mineral restructuration that occurs over the surface of the enamel after the alternation between demineralization and remineralization processes. After completion of a 24-hour treatment, Carbopol gel (ct) displayed higher levels of Ra compared to the rest of the groups. It can be assumed that these results might be associated with acidic pH value (3.72) of this gel resulting in higher mineral loss. As a matter of fact, mineral loss in deeper levels (50, 75 and 100 µm), could not be restored by the saliva-induced remineralization methods (8, 27). Therefore, it can be assumed that the measured microhardness values may tend to decrease as well as roughness increment.
To assess color alterations after bleaching in dental research, the ∆E and ∆E2000 formulas have been routinely employed, and are created according to the CIE L*, a*, b* system. Still, it is relevant to mention that while ∆E adopts the same value for all CIEL*, a*, b* coordinates, ∆E2000 takes into account parametric elements to evaluate CIEL*, a*, b* color alterations. Specifically, by using the CIEL*, a*, b* spectral dimension it is conceivable to assess bleaching alterations by means of changes in one or more of its coordinates (L*: luminosity; a*: red-green axis and b*: yellow-blue axis) (28, 29). The whitening index (white level), that is WID, has been used more recently in the dental research although it is also based on the CIELab system. When distinguished from the other indices, WID intends to achieve a higher correlation to visual perception (17, 28). The effectiveness of the bleaching gels assessment is relevant, and the visual perception thresholds and color alteration acceptability act to check the quality control.
The samples used in the present study had their color standardized through a staining process with a black tea solution (7, 11). In the present study, all the samples presented similar behavior: the groups that had carbamide peroxide (CP-ct, CP-at, and CP-com) presented values over the perceptibility (PL) and acceptability (AL) limits with all the formulas - ∆E (PL: 1.2 / AL: 2.7), ∆WID (PL: 0.72 / AL: 2.62), and ∆E2000 (PL: 0.8 / AL: 1.8). This shows that the bleaching effectiveness of the CP-at is being comparable to the groups CP-ct and CP-com in terms of how noticeable and acceptable were the results of color changes. It means that the use of Aristoflex as a thickener did not hinder the bleaching effectiveness.
Nonetheless, Aristoflex (at) and Carbopol (ct) groups displayed color alterations above the PL with all the formulas. However, this color alteration was lower than the one observed in the carbamide-peroxide-based groups. Even if the thickeners cannot by themselves bleach the tooth, along with the bleaching treatment, these polymers have an important role to modulate the chemical reaction by liberating reactive oxygen species from hydrogen peroxide, which is actually responsible for bleaching (11). Additionally, the alteration of color could be associated with the acidic properties of the thickeners that are related to the demineralization of the surface and the enamel morphology alteration. This alteration could affect the samples surface reflectance patterns by inducing the color alteration.
Therefore, the second null hypothesis was partially accepted. Carbamide peroxide 10 wt% gel containing Aristoflex altered neither the effectiveness of bleaching gel nor the microhardness of the enamel between the initial and final times. Yet the roughness of the enamel had an increment. The current study design is restricted since it has not assessed some relevant aspects of the safety of experimental bleaching gels (e.g. trans-enamel-dentin cytotoxicity) or the bleaching effectiveness maintenance (e.g. after 14-days of bleaching). Further research is needed to consider the aforementioned features. In a similar manner, in vivo and clinical studies that can verify the current results are still required. Although the present study is corroborated by previous research, it points to the fact that Aristoflex is a favorable alternate thickener for at-home bleaching treatment.
Conclusion
In conclusion, the association of carbamide peroxide 10 wt% with Aristoflex is a promising alternative to the already commercially available Carbopol-containing bleaching products. It has exhibited significant improvements on whitening efficiency, microhardness and roughness of dental enamel without generating cytotoxicity to human gingival fibroblastic cells.
Acknowledgments
The authors thank to Drogal manipulations products for the donation of their products. Thanks to the Coordination for the improvement of Higher Education Personnel (CAPES – Brazil) Finance Code 001, and to the São Paulo Research Foundation (FAPESP) grants #2018/24446-1 and #2019/202721 for supporting and stimulating the current research. The founding agencies did not were involved within the study design, data collection, and analysis, decision to publish or the manuscript preparation.
Footnotes
Conflict of interest
The authors declare they have no conflict of interest.
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