Efficacy and Safety of Bleaching Gels According to Application Protocol
This study evaluated bleaching efficacy, enamel microhardness, and roughness of highly concentrated hydrogen peroxide (HP) gels (35%–40%) using different application protocols. Gel decomposition and pH alteration were also analyzed. Bovine enamel/dentin specimens were divided into groups according to the bleaching gel—Pola Office Plus (POP–SDI, 37.5% HP), Opalescence Boost (OPB–Ultradent, 40% HP), Whiteness HP (WHP–FGM, 35% HP)—and application protocol—single application (SA) and multiple application (MA) during the in-office session. Deionized water was used in control group (no bleaching). Thus, seven final groups were obtained (n=15/group). Color (CIE L*a*b*), surface microhardness (SMH), and roughness (Ra) were assessed before/after treatments. The pH of gels was measured, and HP concentration was determined with potassium permanganate titration method in different times. Data were submitted to analysis of variance and Tukey tests (5%). All gels presented similar and clinically acceptable bleaching efficacy (ΔE>2.7) for both SA and MA, as well as no significant differences for SMH and Ra comparing the two protocols in the same gel. Peroxide decomposition significantly increased with time, but final gel concentrations were still high after 45 minutes (32.29% POP; 38.45% OPB; and 32.74% WHP). The pH decreased over time (initial - after 45 min) for WHP (6.83±0.07 - 5.81±0.06), but minimal alterations were observed for POP (8.09±0.09 - 7.88±0.07) and OPB (7.82±0.11 - 7.87±0.07). Peroxide decomposition was very low for all gels tested, and pH remained stable for POP and OPB gels. Bleaching protocol did not influence whitening efficacy and hazardous effects over enamel, thus potentially there was no clinical significance. Therefore, for the products tested, there is no evidence for recommending the gel change during the bleaching session.SUMMARY
Objectives:
Methods and Materials:
Results:
Conclusions:
INTRODUCTION
Dissatisfaction with tooth color is a matter of concern among several populations around the globe, with indexes varying from 20% to 66% in adult populations.1 Studies with quality-of-life questionnaires also show tooth color alteration as an important issue2 and is often associated with the desire for aesthetical treatments, such as tooth whitening.1,3,4 This is considered a noninvasive treatment for patients who desire to change their tooth color and involves the use of gels containing hydrogen or carbamide peroxide in different concentrations3 that are directly applied over the enamel surface. The mode of action of these gels is not quite fully understood, but it is based on the peroxide diffusion through tooth structures, releasing free radicals able to oxidize chromophores in less complex structures, changing the light reflection pattern and resulting in a whiter appearance.1,3,5
The in-office technique uses high concentrated hydrogen peroxide gels (20% to 40%) applied for short periods, usually from 30 to 60 minutes. Although the bleaching efficacy of different in-office products has been shown, different concentrations and application protocols are available, varying according to the manufacturer.6
Side effects such as tooth sensitivity is commonly reported in clinical practice, whereas deleterious effects over enamel surface, such as reduction of microhardness and increase of roughness, remains controversial.7–10 As the peroxide solution is more stable in an acidic environment, its decomposition is higher when pH increases.11 Therefore, the bleaching gels usually become available in two bottles or dual-barrel syringe, one with the peroxide solution (acid) and the other with the thickener (alkaline), resulting in a final pH closer to neutral when both substances are mixed, probably minimizing the deleterious effects over enamel. Over time, peroxide decomposition generates free radicals, which tend to reduce the pH,12 with potentially hazard consequences to dental tissues and reduction of whitening efficacy. The release of free radicals by the bleaching gels is directly proportional to their peroxide concentration,13 but over time, the peroxide decomposition might reduce the amount of free radicals available inside the gel layer. The pH reduction and the peroxide decomposition are the reasons some manufacturers indicate changes of the gel during the application protocol. Nevertheless, the level of peroxide decomposition during the bleaching procedure, as well as the final peroxide concentration and pH after the recommended application time, has not been properly reported in the literature. Some authors show that after a certain concentration, a higher peroxide content of the bleaching gel does not necessarily increase the bleaching effect,14,15 whereas others reported a direct relation between concentration and color change.7 Also, a previous in vitro study showed low correlation between color alteration and multiple applications of a highly concentrated peroxide gel (35%) during the bleaching session,12 whereas a clinical report showed better results when the gel was replaced at the same session.16 Therefore, the real need for multiple applications during the bleaching session remains uncertain, regarding both whitening efficacy and potential deleterious effect on hard tooth structures. The peculiarities of each bleaching gel formulation, based on the presence of different ingredients, may influence its chemical behavior during the clinical application as well as the recommended protocol. Some gels present higher peroxide concentration, besides different thickening agents and viscosities. However, all of them claim to produce an adequate bleaching outcome and achieve the desired esthetic improvement. Thus, the aim of this study was to evaluate different application protocols for in-office dental bleaching regarding color, enamel surface microhardness, and roughness, as well as the peroxide decomposition and pH alteration of the gel during its application. The null hypotheses tested were that a) there is no difference of bleaching efficacy, enamel microhardness, and roughness between single and multiple application of the gels, as well as peroxide concentration and pH over time, and 2) the different bleaching gels, despite the application protocol, do not present significant differences on the evaluated parameters.
METHODS AND MATERIALS
Experimental Design
This study followed a factorial 3×2 design, considering bleaching systems (at three levels: Pola Office [POP], Opalescence Boost [OPB], and Whiteness HP [WHP]) and application protocol (at two levels: multiple and single) as experimental factors. Deionized water was the control group. The treatment sequence was randomized using an online software (www.random.org). The PICO question was stated: P, bovine teeth; I, in-office bleaching gels applied in single mode; C, in-office bleaching gels applied in multiple mode; and O, tooth color change, microhardness, roughness, peroxide concentration, and pH alteration. The research question based on the primary outcome (efficacy) was: Do in-office bleaching gels applied in single mode present similar efficacy compared with multiple application?
Sample Size Calculation
The sample size was determined based on data from a previous study.7 With the parameters of 0.05 as the significance level and 0.9 as the power test, 12 specimens per group were determined using G*Power 3.1 software (Heinrich-Heine-Universität, Düsseldorf, Germany). Due to possible loss, 15 specimens per group were prepared.
Specimens Preparation
Freshly extracted bovine incisors were stored in thymol solution (0.1%, pH 7.0) under refrigeration (5ºC). Enamel/dentin cylindrical specimens (n=105) were obtained from the buccal surface of the crowns using a custom-made diamond trephine mill (4 mm diameter) and standardized with 1.1 mm of enamel and 1 mm of dentin, as previously described.7
The specimens were embedded in white self-curing acrylic resin (Acritec, Panambra, SP, Brazil) using a cylindrical silicone mold (6 mm in diameter and 3 mm in depth).17 The specimens were ground flat and polished with water-cooled sequential silicon carbide abrasive papers (#P1200, #P2400, and #P4000 grit, Extec Corp, Enfield, CT, USA) in a polishing machine at 300 rpm for 30, 60, and 120 seconds, respectively. In between each paper change and after polishing, the specimens were sonicated to remove debris.
Specimens were evaluated in a stereomicroscope (×20; Stemi 2000, Carl Zeiss, Oberkochen, Germany) to certify the absence of cracks and imperfections and stored in artificial saliva during seven days for rehydration and stabilization of mineral content. The composition of the artificial saliva used was as follows: 0.33 g of KH2PO4, 0.40 g of NA2HPO4, 1.27 g of KCl, 0.16 g of NaSCN, 0.58 g of NaCl, 0.17 g CaCl2, 0.16 g of NH4Cl, 0.02 g urea, 0.03 g glucose, 0.002 g ascorbic acid in 1l deionized water, with pH of 6.8.18
Groups Division and Bleaching Protocol
Using the Excel software (Microsoft Corp, Redmond, WA, USA), specimens were randomly allocated into seven groups (n=15) according to the bleaching gel and application protocol, with deionized water as the negative control, as presented in Figure 1. Table 1 presents the gel composition and application protocols. For the multiple application technique, the exact manufacturer’s recommendation was followed. For the single application technique, the sum of application time was calculated for each product.
Citation: Operative Dentistry 46, 2; 10.2341/19-253-L
The gels were applied on the specimens surface using a positive displacement pipette (Pos-D, Mettler Toledo, Oakland, CA, USA) recommended for viscous materials to standardize the volume of 15 μL over each specimen. During the treatment, specimens were stored in 100% relative humidity at 30°C to simulate oral conditions. This temperature was observed in a previous in vivo evaluation in which the bleaching gel layer was applied on the patient’s teeth (CRG Torres and AB Borges, 2017, unpublished data). For the groups with multiple applications, the gel was removed with suction tip, and a new layer of the gel was immediately placed over the samples. After the final gel removal, samples were washed with deionized water. In the negative control group, specimens were treated exactly as for experimental groups, except that water was applied instead of bleaching gels. The same specimens were used for color, microhardness, and roughness experiments.
Color Measurement
Initial color was measured using a reflectance spectrophotometer (CM-2600d, Konica Minolta, Osaka, Japan), adapted to a small reading area. The device was adjusted to use the CIE Standard Illuminant D65, with 100% of ultraviolet spectral region and specular component included. Also, the observer angle was set at 2º, and the device was calibrated with a white and a black standard. Three consecutive measures were obtained for each specimen, which were averaged. The results of the color measurement were quantified in terms of the CIE L*a*b* coordinate system.
The specimens were placed in the equipment immediately after removal from the artificial saliva to avoid dehydration. Color reading was performed at baseline and seven days after the bleaching procedure period in which they were kept immersed in artificial saliva at 37ºC. Color alteration was measured calculating the changes of L* (ΔL), a* (Δa), and b* (Δb) color coordinates. Total color change was defined by calculating ΔE and ΔE00 (CIEDE2000), using the following formulas:
and
where luminosity is represented by L, saturation represented by C, and the hue represented by H. The rotation factor (RT) is used to correct the deflection in the blue region of the direction of the ellipse axis for visual perception, that is, the interaction between chroma and hue differences in the blue region.19
Surface Microhardness Measurements
Surface enamel microhardness was measured using a microhardness tester (FM-700, Future Tech, Tokyo, Japan), coupled with a Knoop indenter using a load of 50 g and 10-second dwell time. Three indentations were performed in each sample, distancing 100 μm between them, and the values were averaged. Samples presenting variation in 10% from initial microhardness values were replaced. Microhardness was measured in three different moments: baseline (surface microhardness [SMH]b) and immediately after bleaching (SMHa), to calculate the demineralization potential of the treatment (SMHde), and after seven days from the treatment (SMH7), to evaluate the saliva remineralization potential (SMHre). The percentage of microhardness variation was calculated using the formulas:
and
Surface Roughness Measurements
Surface roughness average (Ra) of all samples was measured using a contact profilometer (Mahr Surf GD 25, Mahr, Göttingen, Germany), with a standard cutoff of 0.25 mm, a transverse length of 2.5 mm, and a stylus speed of 0.5 mm/s, obtaining the arithmetic average of the roughness profile (Ra). The equipment was set for three surface readings, with a distance of 0.25 mm between them, and the mean value was considered the Ra for each sample. Roughness was also measured in three distinct moments: before the treatments (Rab), immediately after bleaching (Raa), and after seven days of the treatments (Ra7). The percentages of roughness alteration were calculated after treatment and after seven days using the formulas:
and
To guarantee that the specimens were always placed at the same position in the profilometer, a custom-made specimen holder was used.
pH and Hydrogen Peroxide Concentration Measurement
Thirty additional bovine crowns were used for this measurement, because of the requirement of a higher gel volume to allow pH and peroxide decomposition analysis. A surface area of 2 cm x 1 cm at the buccal surface was delimited using nail varnish. Crowns were randomly divided in three groups, according to the bleaching gel. The amount of 0.05 ± 0.01 g of the gel was placed over the delimited area.
The pH was measured using a pH meter (Seven Multi, Mettler Toledo, Schwerzenbach, Switzerland) coupled with a specific electrode for small sample sizes (HI1083D pH Electrode, Hanna Instruments, Woonsocket, RI, USA), calibrated with standard solutions (pH 4.00 and 6.86). Measurements were performed immediately after placing the gel over the tooth, and after 8, 15, 20, 35, 40, and 45 minutes. These times were chosen based on different partial and total application times recommended by the different manufacturers.
The peroxide concentration was assessed by titration with potassium permanganate (KMnO4) method, using an automatic potentiometric titrator (HI 902, Hanna Instruments). The hydrogen peroxide concentration was verified by oxidation-reduction reaction in which potassium permanganate reacts with hydrogen peroxide in an acidic environment: 2KMnO4 + 5H2O2 + 4H2SO4 = 2KHSO4 + 2MnSO4 + 5O2 + 8H2O.20
The bleaching gels samples were collected from the specimen’s surface and weighed. Then, the sample was diluted in deionized water (10 mL), with a dual asymmetric centrifugal mixer (Speed Mixer DAC 150.1 FVZ, Hauschild Engineering, Hamm, Germany), at 3500 rpm for 30 seconds. The solution was mixed with 20 mL of 1 M sulfuric acid solution over a magnetic stirrer. Aliquots of potassium permanganate solution were dispensed to the initial solution, and hydrogen peroxide concentration was calculated when reaction reached the turning point, by the following formula:
in which C = hydrogen peroxide concentration (w/w); V = volume of potassium permanganate solution added during titration; Cf = correction factor of 0.1 M potassium permanganate solution; and M = mass of bleaching gel sample collected in mg.20
Statistical Analysis
Normal distribution was tested by the Kolmogorov-Smirnov test. Color data was submitted to two-way analysis of variance (ANOVA), for application protocol and bleaching products, followed by Tukey test. The comparison between each group and the negative control was performed by Dunnett test. Microhardness, roughness, pH, and hydrogen peroxide concentration data were submitted to two-way repeated measures ANOVA, followed by Tukey test. The significance level adopted for all tests was 5%.
RESULTS
The results of two-way ANOVA for color analysis showed statistically significant differences for “bleaching gel factor” (ΔE, p=0.0034, and ΔE00, p=0.0006) but not for the “application protocol factor” (ΔE, p=0.0891, and ΔE00, p=0.0625). The WHP gel presented significantly lower color change compared with POP and OPB for both ΔE and ΔE00 analyses. Dunnett test showed that all bleaching gels, despite the application protocol, exhibited higher color change compared with the negative control group. Also, higher values of ΔE and ΔE00 compared with the acceptability threshold were observed for all products tested (Figures 2 and 3). The mean values of the L*, a*, and b* parameters are presented in Figure 4. In general, an increase of luminosity (L*) and a decrease of yellowing (b*) were detected for all tested groups in comparison to control.
Citation: Operative Dentistry 46, 2; 10.2341/19-253-L
Citation: Operative Dentistry 46, 2; 10.2341/19-253-L
Citation: Operative Dentistry 46, 2; 10.2341/19-253-L
Two-way repeated measures (RM)–ANOVA showed significant differences for the microhardness data (p<0.001 for interaction). Tukey test revealed that after bleaching, the groups POP/single application (SA) and WHP/multiple application (MA) presented a decrease of microhardness compared with the control, but neither gels nor protocols presented significant differences after seven days (Table 2). No differences between MA and SA for the same gel were observed.
Regarding roughness, two-way RM-ANOVA showed difference only for the time (p=0.0015), with %Rade presenting higher values than %Rare (Table 3) but not for the gels (p=0.85) or the interaction (p=0.30). Tukey test showed significant increase of roughness immediately after bleaching, which reduced after seven days.
Table 4 presents data of gel concentration and decomposition for the groups tested. RM-ANOVA showed significant difference for the interaction of factors (p<0.001). The gels concentration overall reduced over time. POP and WHP presented, in general, higher decomposition compared with OPB.
Table 5 shows data of gels pH for the groups tested. WHP presented the lower pH and the higher alteration with time. POP and OPB showed slight alkaline pH values, and OPB maintained stable pH values throughout the study. RM-ANOVA showed significant difference for interaction of factors (p<0.001), with significantly lower pH for WHP gel compared with POP and OPB.
DISCUSSION
Despite being widely used, the scientific evidence for guiding the bleaching gels mode of application is not well established, since there are several marketed products with different peroxide types, concentrations, and application protocols. Thus, this study aimed to promote a better understanding of the chemical behavior of bleaching gels and its influence on color change, microhardness, and roughness according to the application protocols, aiming to optimize bleaching efficacy with safety.
Bovine teeth were used as substrate since they are easier to find and to standardize in laboratory conditions. Although they are not identical to human teeth, they present similar calcium to phosphorous ratio, organic composition, and rehydration potential. Morphologically, the bovine substrate presents higher average diameter of enamel crystallites but no significant difference in number and diameter of dentinal tubules. It must be highlighted that the results obtained in laboratory tests with bovine teeth may not be directly extrapolated to clinical conditions, although they may provide valid comparison among tested treatments.21,22
The first null hypothesis tested was accepted since there were no significant differences of whitening efficacy, enamel surface microhardness, and roughness comparing the different protocols during the in-office session. Although some manufactures recommend that the whitening gel must be renewed during its contact with the tooth structure, the effect of the product changes is not fully proved. The results from this study support that multiple applications of the gel during the whitening procedure are not necessary, corroborating previous findings.12,23
Although some manufacturers suggest light activation of the bleaching gels as an option, this procedure was not adopted in our study, since its potential increase on bleaching efficacy is controversial.24 In addition, light sources may play a role in pulp temperature increase,25 potentially affecting the risk of sensitivity.26
All groups presented significant tooth color change when compared with the negative control, despite the protocol employed, suggesting that the success of bleaching is mainly related to the peroxide diffusion and not to its continuous reapplication.27 When different products were compared, lower color alteration was observed for WHP with both protocols, thus rejecting the second null hypothesis. Nevertheless, ΔE values were higher than 2.7 for all products tested. This value has been previously defined as the minimal color change value clinically accepted by observers.28 Corresponding value for the ΔE00 is 1.8.28
It might be speculated that the indication for multiple application of the gels during the bleaching session is due to the peroxide decomposition rate. However, the present results indicate that the gels decomposition rate was very low, varying from approximately 8% to 11% after 45 minutes (Figure 2), as also observed previously.16,27 This means that at the end of the bleaching session, the gels concentration was approximately 32% for POP and WHP and 38% for OPB, which is still very high. These data suggest that tooth color change is not directly proportional to the hydrogen peroxide decomposition rate, since all the groups presented satisfactory bleaching outcome. Furthermore, this indicates that the results may be dependent on the maintenance of a minimal peroxide concentration able to oxidize the organic components and produce tooth bleaching. Since there is no relevant reduction of the peroxide concentration, the gels efficacy does not change with the application protocol.
It must be highlighted that decomposition pattern is directly related to the gel chemical constituents and physical characteristics, mainly in relation to pH, polymer thickener type, and gel viscosity. Gels used in this study present different thickener agents, as polyvinylpyrrolidone and carbomers. These substances act on mechanical and rheological properties of gels and, consequently, on their bioavailability,29 which can increase bio-adhesion properties, influencing peroxide release. Additionally, the in-office bleaching gels are applied directly on isolated tooth surface, where saliva is not present, differently from at-home bleaching in which gels are applied inside trays. It has been shown that saliva contributes to higher gel decomposition.30,31
The diffusion of hydrogen peroxide through dental tissues is related to the contact time of the gel with the enamel surface.27,32 Thus, the longer contact will result in higher penetration of hydrogen peroxide.27 This reinforces the concept that multiple applications are not necessary, since the peroxide released from the gel is maintained for longer periods. Besides reduction of costs, the single application also saves time and decreases the risk of reaching soft tissues.27
Adverse effects such as tooth sensitivity and enamel damage must also be considered in clinical practice. Differening from our results, Reis and others (2011)16 reported in a clinical trial that multiple application with refreshing of gel (3x15 min) is preferable to single application (45 min) using 35% hydrogen peroxide, not only in terms of color change efficacy but also in patients’ reports of higher tooth sensitivity during prolonged application, which was attributed to the lower pH of the gel. In our study, the WHP gel presented a slightly acid pH (around 6.8 initially) and a variation of 15% at the end of the session (pH of 5.81 after 45 min), while POP and OPB gels presented a slight alkaline pH (8.09 and 7.82, respectively), with no relevant variation throughout the bleaching session. It must be pointed out that the higher pH favors the hydrogen peroxide decomposition, potentially maximizing bleaching efficacy, which might be related to the lower bleaching efficacy of the WHP product.11
Surface microhardness and roughness analyses showed that the bleaching procedure presented no relevant deleterious effect on enamel, regardless the application protocol used and the bleaching gel. Although a reduction of the microhardness data has been observed for some groups immediately after bleaching (Table 2), values were reverted after seven days, being similar to nonbleached teeth (control group). Chemical modifications in enamel after interaction with bleaching agents are expected and have been previously reported.33–35 These alterations have been attributed to the reduction of calcium and phosphate from the enamel and the slight erosive effect promoted by the peroxide gels, even with neutral pH gels.34 However, exposure to saliva and remineralizing agents may recover the dental structure composition.8,33 Also, according to ISO 28399:2011,36 the reduction of enamel microhardness after bleaching should be not higher than 10%, and all products tested presented values close to this range. Finally, significant roughness alterations were observed immediately after bleaching, which were also reverted after saliva exposure, and thus, with potentially no clinical relevance.37
Thus, it was observed that prolonged application of the bleaching gels is not more hazardous to enamel than the shorter ones, and both protocols present the same efficacy in terms of color alteration. The clinicians might benefit from single application, making the in-office bleaching easier and faster, since there is no need for gel removal and reapplication.
CONCLUSION
The hydrogen peroxide decomposition during the bleaching session was minimal for all gels tested. The pH remained stable for both POP and OBP gels. The bleaching protocol (single or multiple gel applications) did not interfere with bleaching efficacy, enamel surface microhardness, and roughness. Lower color change was observed for WHP compared with OPB and POP, but still clinically acceptable (ΔE higher than 2.7). Thus, the products tested can be considered efficient and safe, and no gel changes during the bleaching session are required.
Group division and bleaching application protocol.
Graph showing results for ΔE. The horizontal line represents the acceptability limit (2.7). Different letters show significant differences among groups (p<0.05).
Graph showing results for ΔE00. The horizontal line represents the acceptability limit (1.8). Different letters show significant differences among groups (p<0.05).
Mean and standard deviation of L*a*b* parameters for each gel with different applications protocol.
Contributor Notes
Clinical Relevance
Some bleaching protocols require change of the gel during the in-office whitening session. The results of this study indicate no need for this change, since there were similar bleaching efficacy and adverse effects on enamel comparing single and multiple applications of the hydrogen peroxide gels.
*Alessandra Bühler Borges, DDS, MSc, PhD, associate professor, Department of Restorative, São Paulo State University - UNESP, Institute of Science and Technology, São José dos Campos, São Paulo, Brazil
Fabrícia Stabile de Abreu, DDS, MSc student, Department of Restorative Dentistry, São Paulo State University - UNESP, Institute of Science and Technology, São José dos Campos, SP, Brazil
Mariane Cintra Mailart, DDS, MSc, PhD student, Department of Restorative Dentistry São Paulo State University - UNESP, Institute of Science and Technology, São José dos Campos, SP, Brazil
Rayssa Ferreira Zanatta, DDS, MSc, PhD, Department of Restorative Dentistry, São Paulo State University - UNESP, Institute of Science and Technology, São José dos Campos, SP, Brazil; Department of Restorative Dentistry, School of Dentistry, Taubaté University, UNITAU, Department of Dentistry, Taubaté, São Paulo, Brazil
Carlos Rocha Gomes Torres, DDS, PhD, associate professor, Department of Restorative Dentistry, São Paulo State University - UNESP, Institute of Science and Technology, São José dos Campos, São Paulo, Brazil.