Effect of 10% Sodium Ascorbate and 10% α-tocopherol in Different Formulations on the Shear Bond Strength of Enamel and Dentin Submitted to a Home-use Bleaching Treatment
This in vitro study assessed the shear bond strength of human enamel and dentin submitted to a bleaching treatment with 10% carbamide peroxide and treatment with antioxidant agents containing 10% α-tocopherol and 10% sodium ascorbate formulated in solution and gel. Sixty human dental enamel slabs (E) and 60 human dental dentin slabs (D) were randomly divided into six groups (n=10). Groups E1 and D1 were negative control groups and the bleaching agent was not applied. The bleaching agent was applied daily for two-hours on the dental slabs of all the other groups and, during the remaining 22 hours, the specimens were stored in an artificial saliva solution for a total of 14 days. Groups E2 and D2 were positive control groups and they only received application of the bleaching agents. Antioxidant agents were applied in Groups E3 and D3 (10% sodium ascorbate solution), E4 and D4 (10% α-tocopherol solution), E5 and D5 (10% sodium ascorbate gel) and E6 and D6 (10% α-tocopherol gel) for two hours. Cylinders were made with microhybrid resin composite and a total-etch adhesive system for shear bond strength tests. These tests were performed in a universal testing machine at a speed of 0.5 mm/minute to obtain the values in MPa. ANOVA (p>0.05) showed no significant differences among groups E4, E5, E6 and E1. However, groups E3, E5 and E6 presented statistically similar values to group E2. The Kruskal-Wallis test showed no significant differences among D1 and all the other experimental groups; the same values occurred with D2, which did not differ from the experimental groups. Antioxidant treatment with 10% α-tocopherol solution was the only effective agent to revert the oxidizing effects of the bleaching treatment on enamel.SUMMARY
INTRODUCTION
The home-use dental bleaching treatment with 10% carbamide peroxide has become widely used and has been the choice for achieving better dental esthetic appearance in cases of darkened and/or stained teeth. Moreover, other esthetic procedures can also be performed, such as esthetic veneers on anterior teeth, replacement of old restorations with an unsatisfactory aspect or the closure of diastemas and other esthetic procedures. However, bleaching is frequently associated with restorative procedures in which esthetic restorations with resin composite or veneer may be necessary to replace old restorations, with the intention of obtaining a more acceptable color to conclude the case.1–2
However, many studies have reported a significant reduction in bond strength of restorative materials to dentin and enamel after home-use and in-office bleaching treatment.3–12 In order to avoid adhesive failures at the restorative material/dental surface interface, a waiting period that varies from 24 hours to three weeks is recommended,461113–14 since the reduction in bond strength of resin composite to post-bleaching treatment enamel or dentin has been shown to be temporary.1115–16
There are some studies that used sodium ascorbate solutions as an antioxidant agent.8–91217–21 Lai and others8 reported that hydrogen peroxide or sodium hypochlorite induced a reduction in the bond strength of resin composite to dentin, a situation that was reverted to with the use of antioxidant sodium ascorbate.9 Kimyai and Valizadeh20–21 evaluated the use of sodium ascorbate formulated as a hydrogel or solution and found no differences between the preparations, although bond strengths were significantly increased following the use of sodium ascorbate treatments.
However, there are no studies that assessed Vitamin E or α-tocopherol22 as an anti-oxidizing agent. Alpha-tocopherol is the most active component of the Vitamin E complex, and this organic substance is the most powerful antioxidant in the lipid phase of the human body.23–24 The extremely critical role of alpha-tocopherol in protecting against free-radical reactions becomes apparent when considering the vast number of diseases and conditions, such as aging, many types of cancer, atherosclerosis and other circulatory diseases, arthritis, cataract formation, senile dementia (Alzheimer's type) and respiratory disease induced by pollution–thought to be caused by these reactions.25 Thus, the current study evaluated the shear bond strength of human dentin and enamel submitted to bleaching treatment with 10% carbamide peroxide, then treated with antioxidant agents containing 10% sodium ascorbate and 10% α-tocopherol formulated in solution and gel.
METHODS AND MATERIALS
1. Experimental Design
The factor under study in this experiment was treatment protocols at six levels:
Artificial saliva (negative control group)—saliva
10% carbamide peroxide (positive control group)—10%CP
10% carbamide peroxide + 10% sodium ascorbate solution—10%CP + 10%SAS
10% carbamide peroxide + 10% α-tocopherol solution—10%CP + 10%ATS
10% carbamide peroxide + 10% sodium ascorbate gel—10%CP + 10%SAG
10% carbamide peroxide + 10% α-tocopherol gel—10%CP + 10%ATG
The experimental units consisted of 60 slabs of human enamel (E) and 60 slabs of human dentin (D). Ten slabs of each substrate (n=10) were randomly distributed among the treatment agents. Shear bond strength was assessed quantitatively and expressed in MPa.
2. Preparation of Tooth Slabs
Thirty non-erupted human third molars extracted for reasons not related to that of the research and kept stored in thymol solution (0.1%; pH 7.0) after extraction were used in this experiment. The teeth were debrided with scalpel blades and periodontal curettes. A cross-section was made, dividing the root and coronary portions. Longitudinal sections allowed enamel slabs measuring 3 mm x 3 mm and 2 mm thick to be obtained. Those sections with cracks or stains were excluded.
The slabs were embedded in polystyrene resin by using PVC molds 2.0 cm in diameter, leaving the external enamel surfaces uncovered by the resin. After 24 hours, the slabs were removed from the molds and flattened to obtain the smooth surfaces required for shear bond strength tests.
The enamel specimens were flattened with aluminum oxide discs (Norton, São Paulo, SP, Brazil) of sequentially decreasing granulation (400, 600, 1200) cooled under running water and polished with diamond pastes (pastes of 6, 3, ½ and ¼ μm) and felt discs (Top, Ram and Gold, Arotec, Osasco, SP, Brazil) under mineral oil cooling (Red mineral oil, Arotec, Osasco, SP, Brazil). Dentin specimens were flattened with aluminum oxide discs (Norton, São Paulo, SP, Brazil) of sequentially decreasing granulation (600 and 1200) under running water-cooling. No polishing with felt discs was done to avoid residual diamond paste accumulating inside the dentinal tubules. Between disc granulations and pastes, the specimens were ultrasound washed (Ultrasound washer, Unique, Brazil) by placing the specimens in distilled and deionized water for a 10-minute period to eliminate residue.
One hundred and twenty slabs of enamel and dentin were randomly divided into the treatment agent groups (n=10) and kept in a humid environment for one day until the beginning of the treatment applications.
3. Treatment Agent Specification
The treatment agents used in this experiment are specified in Table 1, according to the lot number, composition, mean pH value and manufacturer.
4. Application of Treatment Agents
Before starting to apply the treatment agents, an individual mold was made for each specimen, using 0.4 mm thick flexible polymer in a vacuum plasticizer (Vacuum plasticizer P7, BioArt Equipamentos Odontológicos, São Paulo, SP, Brazil).
For groups E1 and D1, slabs were kept in an artificial saliva solution that was changed every two days throughout the experiment.
The bleaching agent applications were made in groups E2, E3, E4, E5, E6, D2, D3, D4, D5 and D6 using a calibrated syringe that placed 0.02 ml of each treatment agent on the dental slabs. The individual molds were placed over the specimens, immersing them in 13.5 ml of artificial saliva solution (pH=7.0) in individual containers. These containers were closed and kept daily for two-hours 37°C (± 1°C).
The individual molds were removed and washed with distilled and deionized water. The treatment agents were removed from the dental slabs by washing with distilled and deionized water using a soft toothbrush (Colgate, São Paulo, SP, Brazil), making five back-and-forth movements over the slabs.
During the remaining 22 hours a day, the slabs were kept in 13.5 ml of artificial saliva solution in individual closed containers at 37°C (± 1°C). The artificial saliva solution used in the current study, which was changed every two days, was the remineralizing solution described by Featherstone and others26 and modified by Serra and Cury.27
At the end of the 14-day treatment phase, the enamel and dentin slabs were kept in their individual containers in 13.5 ml of artificial saliva solution at 37°C (± 1°C) for one day.
5. Application of Antioxidant Agents
Before application of the antioxidant agents formulated in solution, an individual mold was also made for each specimen using a 0.4 mm-thick flexible polymer in a vacuum plasticizer.
After concluding the treatment phase with bleaching agents as described above, the specimens from groups E3, E4, D3 and D4 received solution applications using a calibrated syringe to place 0.02 ml of each antioxidant agent onto the dental slabs. The individual molds were placed over the specimens with the intention of preventing the antioxidant agent solution from being diluted in the artificial saliva, immersing the specimens in 13.5 ml of artificial saliva solution (pH=7.0) in individual containers that were closed and kept at 37°C (± 1°C) for a two-hour period. The individual molds were then removed, the treatment agents washed from the dental slabs with distilled and deionized water for 30 seconds and immersed in 13.5 ml of artificial saliva solution (pH=7.0) in individual containers that were closed and kept at 37°C (± 1°C) for one day.
Specimens from groups E5, E6, D5 and D6 received gel applications using a calibrated syringe to place 0.02 ml of each antioxidant agent on the dental slabs. The individual molds were placed over the specimens in order to prevent the antioxidant agent gels from being diluted in the artificial saliva. They were then immersed in 13.5 ml of artificial saliva solution (pH=7.0) in individual containers that were closed and kept at 37°C (± 1°C) for two-hours. The individual molds were then removed, along with the treatment agents, using a soft toothbrush, making five back-and-forth movements over the slabs and washing with distilled and deionized water. They were then immersed in 13.5 ml of artificial saliva solution (pH=7.0) in individual containers that were closed and kept at 37°C (± 1°C) for one day.
6. Shear Bond Strength Tests
Microhybrid resin composite (Z250, 3M ESPE, St Paul, MN, USA) cylinders were made using an adhesive system (Single Bond, 3M ESPE) according to the manufacturer's recommendations after storage for one day. The location of the shear bond strength tests was delimited by placing a piece of adhesive paper with a 3 mm in diameter hole punched in it over the enamel or dentin surface. A 5 mm high and 3 mm in diameter Teflon ring mold was placed against the specimen to receive two layers of filling material—a microhybrid resin composite—and each layer was light polymerized for 20 seconds. The ring mold was removed, the filling material was stored for one day in individual containers in a humid environment at 37°C and the shear bond strength tests were performed in a testing machine (EMIC Equipamentos e Sistemas de Ensaio Ltda, São José dos Pinhais, PR, Brazil) at a speed of 0.5 mm/minute with a 250 kgf load cell on the enamel or dentin surface parallel to the machine line of travel. A steel knife-edge was placed over the specimen so that the shear force was directed at the bond surface. The shear bond strength values obtained were expressed in MPa.
7. Fracture Mode Analyses
To analyze the fracture modes after the shear bond strength tests, the enamel or dentin surfaces were examined visually under a stereoscopic loupe at 30× magnification. The types of fractures were considered and classified as adhesive (lack of adhesion), cohesive in enamel/dentin (failure of the tooth substrate), cohesive in resin (failure of resin composite) or mixed (adhesive and cohesive failures).
8. Statistical Analysis
An exploratory analysis showed that the data did not meet the pre-suppositions for a parametric analysis. After data transformation (square root), the normality required for a parametric analysis was obtained for enamel but not for dentin. Therefore, a non-parametric test for dentin was indicated. Analysis of Variance (ANOVA) was used to compare the experimental groups of enamel using the Multiple Comparison test for individual verifications (Tukey Test). For dentin, the data was evaluated by the non-parametric Kruskal-Wallis test to compare the treatment agents using the Multiple Comparison test for individual verifications (Dunn Method). The statistical analysis was assessed by the statistical program Bioestat 4.0 with a 5% significance level.
RESULTS
Tables 2 and 3 show the means and standard deviations of shear bond strength tests for enamel and dentin, respectively.
For enamel, ANOVA (p>0.05) showed no significant differences among groups E4, E5, E6 and E1. However, groups E3, E5 and E6 presented statistically similar values to group E2. Thus, an antioxidant effect on Group E4 can be inferred.
For dentin, the non-parametric Kruskal-Wallis test showed no significant differences among negative control group D1 and all the experimental groups. The same occurred with positive control D2, which did not differ from the experimental groups.
In the fracture mode analysis, there was a higher percentage of cohesive in resin failures for enamel and dentin slabs (Figure 1).
Citation: Operative Dentistry 34, 6; 10.2341/09-029-L
DISCUSSION
The action mechanism of bleaching agents is based on a complex oxidation reaction, with the release of oxygen-free radicals that penetrate through the pores of enamel prisms reaching dentin and, via a chemical process, causing the “breakup of organic molecules that pigment the dentin, converting the molecules in carbon dioxide and water, which was released together with singlet oxygen.28 Oxygen and hydroxyl or perhydroxyl ions that are released when a bleaching agent is applied to dental structure are responsible for inhibiting resin material polymerization and interfering in resin penetration into enamel.3–4911131529–30 Perdigão and others31 reported that the reduction in shear bond strength after bleaching treatment may not be related to the presence of residual oxygen in the enamel structure but, instead, by the accumulation of oxygen in dentin, which may be an oxygen reservoir site, or by changes in the enamel and dentin mineral/protein content.
To avoid these and other severe effects caused by oxidant agents, there are three known enzymatic systems in the body that are found in cells, such as cytosol, mitochondria and perox-ysomes.32 The first is composed of two types of superoxide reductase enzymes; the second is composed of glutathione, together with two enzymes (se-glutathione peroxidase and glutathione reductase), and the last is formed by the catalase enzyme,33 which acts in the dismutation of hydrogen peroxide in water and oxygen.32–34 Among the biological antioxidants of low-molecular-weight acquired by the human being, carotenoids, bilirubin, ubiquinone and uric acid can also be pointed out. Nevertheless, the most important micro-molecules are tocopherols and ascorbic acid.35–37 Oxidative damage may be inhibited by the antioxidant action of Vitamin E, glutathione, Vitamin C and carotenoids, constituting one of the main endogenous defense mechanisms of the body.38 The treatment of oxidative stress with Vitamins E and C, alone or in combination, resulted in an increase in the activities of the scavenging enzymes catalase, superoxide dismutase, gluthatione-S-trans-ferase and levels of reduced glutathione with a decrease in lipid peroxidation.39
Some studies reported the effects of sodium ascorbate solution as an effective method to neutralize the accumulation of oxygen and its byproducts on the surface of enamel or dentin, improving bond strength and avoiding failures in adhesive resin polymerization.8–917–21 However, Torres and others29 evaluated the effects of six antioxidant agents, including sodium ascorbate, on the bond strength of enamel submitted to bleaching treatment, with the result of a significant increase in bond strength in the group treated with catalase. However, none of the antioxidant agents was capable of completely neutralizing the deleterious effects of bleaching on bond strength. The same inefficient effect of sodium ascorbate was observed in the current study, both in the form of solution and gel.
In the present study, it was also observed that the group that received application of the antioxidant agent α-tocopherol in solution was statistically similar to the negative control and differed from the positive control group for enamel, being the only efficient antioxidant treatment for the application protocol adopted.
According to Lai and others,9 the sodium ascorbate solution needs to be applied for at least one-third of the bleaching treatment period, which could be clinically impractical. The protocol tested in the current study compared the use of gel or solution preparations, since the use of gel preparation should be more practical and clinically more acceptable for application, since the patient could apply the antioxidant gel in molds previously made by the dentist soon after the end of the bleaching treatment for the same two-hour period as that of the bleaching treatment, in an endeavor to restore the changed redox potential of the oxidized bonding substrate.
Some studies reported that alcohol application on bleached enamel increased bond strength, although the values did not return to the levels of the non-bleached group.640 The presence of alcohol in the composition of the 10% α-tocopherol solution formulated for this study may have contributed to the good response in reversing the compromised bond strength of bleached enamel since 10% α-tocopherol was not miscible in water solutions. Thus, the phenomenon observed may be system-specific, not only due to the antioxidant agent α-tocopherol but also to the presence of alcohol.
The fact that none of the antioxidant treatments differed statistically for the dentin groups after the bleaching treatment may be related to the substrate characteristics. Titley and others41 reported that dentin and dentinal fluid can act as a peroxide and oxygen reservoir, which may be present at the bonding interface, inhibit the polymerization reaction and reduce bond strength.1529 Thus, an increase in the period of time of the antioxidant application, which may vary according to the antioxidant agent, and/or an increase in the concentration used for dentin, may be indicated in an endeavor to neutralize the free oxygen present in a higher amount in dentin than in enamel. It may also be necessary to examine the stability of the activity of these antioxidant agents in the solution and gel formulations.
Alpha-tocopherol formulated in solution resulted in a significant increase in bond strength of bleached enamel compared with the positive group. However, a period of at least one week after the bleaching treatment is still recommended before proceeding with bonding procedures.4613–14 Further studies should be conducted to evaluate the antioxidant potential of sodium ascorbate and α-tocopherol in solutions and gels for the purpose of determining the stability of their activity, increasing the concentration for a more effective action, especially in dentin and reducing the application period.
CONCLUSIONS
It was concluded that only the alpha-tocopherol solution was able to minimize the accumulation of oxygen on the enamel surface after the bleaching treatment.
Fracture mode analysis for groups after shear bond strength test
Contributor Notes
Robson Tetsuo Sasaki, undergraduate student, School of Dentistry, São Leopoldo Mandic, Campinas, SP, Brazil
Flávia Martão Flório, DDS, MS, ScD, professor, Department of Preventive Dentistry, São Leopoldo Mandic Research Center, Campinas, SP, Brazil
Roberta Tarkany Basting, DDS, MS, ScD, PhD, professor, Department of Restorative Dentistry, São Leopoldo Mandic Research Center, Campinas, SP, Brazil