Results

The ANOVA test showed significant differences in the tested groups (p=0.00; F=19.32 to 7 degree of freedom). The means and standard deviations of the bond strength values for the groups and the results of the Tukey test are listed in Table 2.

The PC group showed significant lower bond strength mean than the NC group. All of the experimental groups (SA, CA, GP, AC, ET and SB) showed bond strength means significantly lower than the NC group. The CA group showed significantly higher bond strength mean than the bleached PC group and the groups SA, GP, AC, ET and SB showed no significant differences compared to PC group (Table 2).

The Figure 1 compares the mean bond strength values of the different groups. A significant reduction of the bond strength mean for the bleached groups compared to the unbleached group is evident. At the same time, a trend of increase of the bond strength values for the groups that received the neutralizer agents is noted. Nevertheless, there was no reversal of the compromised bonding for the bleached groups compared to the NC group.

Discussion

In the present study, it was observed that the bleaching procedure resulted in a significant decrease of bond strength values compared to the unbleached group (Table 2 , Figure 1). The bleaching agents release free radicals as the nascent oxygen and hydroxyl or peri-hydroxyl ions when they are applied to the dental structure. Free radical is any molecule that has one unpaired electron, providing it high reactivity. These molecules are able to react with the electron-rich regions of the pigments inside the dental structure, breaking down large pigmented molecules into smaller, less pigmented ones². On the other hand, this property could be deleterious to the bonding of resinous materials. One theory proposed to explain the influence of bleaching agents on bonding suggests that peroxides and their byproducts present inside the dental structure are capable to interfere with the polymerization process of the adhesive material^4-5,12-13.

Some eletronic and optical microscopic studies showed that the resin tags in the bleached enamel were sparse, short, poorly defined, structurally incomplete and, in some areas, completely absent^5,12-14. It is supposed that the bonding agent applied on the etched enamel is not completely polymerized, impairing the mechanical retention and decreasing the bond strength⁵. Bubble-like structures and voids were observed inside the adhesive layer, suggesting that the oxygen released by the hydrogen peroxide is trapped within the adhesive during light-activation^4,11-12. According to McGuckin et al.⁴, the void areas present at the interface decrease in bonds formed at longer time intervals after bleaching cessation, indicating a gradual elimination of HP and its byproducts with consequently increase of the bond strength.

According to Titley et al.¹⁵, the dentin and dentinal fluid can act as a peroxide and oxygen reservoir. The reservoirs of gaseous or dissolved oxygen products could persist until removed by pulpal microcirculation and diffusion from the external surface. A greater surface diffusion would be expected based on a reduced pulpal microcirculation¹⁶. Thus, levels of peroxide or oxygen higher than normal may be present in bonding interface, inhibiting the polymerization reaction and reducing bond strengths⁴.

Various anti-oxidant agents were tested in this study. It is possible that, reestablishing the altered redox potential of the oxidized bonding substrate, the anti-oxidants allow free radicals polymerization of the adhesive resin without its premature termination¹⁰.

Free radicals are deficient electrons and the neutralization process results in oxidation of the neutralizer agent⁷. They are classified in three categories: the full-time prevention, the active detoxication against an oxidative stress and the passive detoxication. The role of the full-time prevention is to prevent the overproduction of free radicals by inactivating molecules, which are responsible for their generation. The active detoxication is based on three enzymes that compose the basic system of anti-oxidant defense system. They are superoxyde dismutase, catalase and glutathione peroxidase and can be found in cells where the oxidative stress is greater, as cytosol, mitochondria and peroxysomes¹⁷.

To control the HP, the human body uses two enzymes. The catalase accelerates the transformation of HP into O₂ and water^17-18. The amount of peroxide decomposed is directly proportional to the concentration of enzyme. The rate of this reaction is rapid and little energy is involved as catalase accelerates the decomposition of HP by more than 100.000.000 fold. Rotstein⁸ investigated the efficacy of catalase applied following intracoronal bleaching and observed the total elimination of HP after 3 minutes.

The role of glutathione peroxidase is also to reduce HP. When two molecules of glutathione react between each other, they produce two hydrogen ions (H⁺) which react with HP and result in two water molecules¹⁹.

In the present study, it was observed that the application of both catalase and glutathione peroxidase resulted in greater bond strength means than the positive control group (Table 2, Figure 1). However, there was significant difference only when catalase was applied, showing its better activity in comparison to glutathione peroxidase. This result is probably related to the catalase mechanism of action, which needs a reduced number of molecules. Nevertheless, both enzymes were unable to completely neutralize the pH, because the bond strengths were significant lower than the NC group. It can be supposed that if more concentrated solutions were used, the results would probably be more expressive.

Another line of defense of the human body, the passive detoxication, allows the reduction of free radicals which could have survived the two other lines of defense. As examples, we have vitamin E, vitamin C or ascorbic acid, flavonoid and beta-carotene^18,20-21. The ascorbic acid is a reducing agent capable to donate two high-energy electrons to scavenge the free radicals²². Previous studies showed that the application of 10% sodium ascorbate was effective to reverse the compromised bonding to oxidized dentin and enamel^3,10-11,23. The use of the sodium ascorbate, instead of the ascorbic acid, is recommended to avoid the potential double-etching effect of this mild acid on etched teeth¹⁰. The pH of the solution used in the present study was around 7.76, with no demineralizing effect. Since vitamin C and its salts are nontoxic and are commonly used in the food industry as antioxidants, it is unlikely that their intra-oral use will result in adverse biological effects or clinical hazard^10-11. In this study, the sodium ascorbate application increased the bond strength means compared to the positive control group, but the difference was not significant. According to Lai et al.¹¹, 10% sodium ascorbate needs to be applied for at least onethird of the bleaching time and sometimes, this may not be clinically acceptable. In this study, it is probably that more consistent effects could be observed with the increase of the application time.

Some previous studies showed that the use of adhesive systems containing organic solvents like ethanol²⁴ or acetone⁹ can reverse the negative effects of bleaching on bonding. It was also observed that the alcohol application on bleached enamel increased the bond strengths, although the values did not return to the levels of the non-bleached group^9,13,25. These effects may be due to two factors. The first one is related to the fact that the bleached enamel is more porous and therefore, has more water containing oxygen. The high pressure solvents associated to resin monomers are able to displace the water and to improve the impregnation of the dental substrate^13,26. By the other hand, it was supposed that the solvent could interact with the free radicals present in enamel and inactivate them^{7, 13}. In the present study, we aimed to test the anti-oxidant ability of ethanol and acetone, so they were applied on enamel and then the surface was rinsed to remove residues. Thus, all of the groups were similarly hydrated and the anti-oxidant effects of the different treatments could be individualized.

It is known that enamel, on the contrary of dentine, can be completely air-dried and the dehydrate action of solvents is unnecessary. The adhesive used is essentially composed by resinous monomers without the addition of solvents, enabling the independent study of the effects of the tested treatments. It can be observed in Table 2 that the application of solvents had no significant effect on bond strength, showing its inability to neutralize free radicals.

The aqueous solution of 7% sodium bicarbonate has been indicated by the manufacturer to neutralize the damaging action of the bleaching gel on soft tissues. According to them, its action is based on the fact that the high pH of the sodium bicarbonate solution (around 8.67) can destabilize the peroxide molecule and result in its decomposition and inactivation. The treatment of bleached enamel with sodium bicarbonate resulted in a mild increase, non-significant effect on bond strength means compared to the positive control group (Table 1, Figure 1).

Since a great number of human teeth are difficult to be obtained, bovine enamel was used as substitute to bond strength tests. Although there are differences in density and porosity of human and bovine enamel²⁷, the mechanism of acid etching is similar²⁸. According to Nakamichi et al.²⁸, bovine teeth can be used as substitute to human teeth in bond strength tests. Nevertheless, if the presence of peroxide in the interprismatic spaces is the explanation for the adverse influence on adhesion, the effect in bovine enamel may be greater compared to human enamel, due to their inherent differences of structure and size of interprismatic areas²⁷. While using bovine teeth does not lead to results that are identical with those obtained when human teeth are used, they produce results that are comparable and certainly useful in evaluating the influence of various treatments on enamel bond strengths¹.

In this study, it was observed that the catalase resulted in the most efficient treatment, although within the tested parameters, it was not able to completely reverse the deleterious effects of bleaching on bonding. Nevertheless, this substance is very unstable, loosing quickly its activity, and is high costly. Thus, the preparation of a commercially disposable product would be hardly viable.

Therefore, it is still recommendable to wait a period of around two weeks after the bleaching treatment to proceed the bonding process. Additional studies are needed to test higher concentrations of anti-oxidant agents, reducing the time needed to an efficient neutralizing action.

It was concluded that only the catalase application resulted in significant increase of bond strengths, in relation to the positive control group and none of the treatments was able to completely neutralize the deleterious effects of bleaching on bond strength.

Acknowledgements

The authors thank FUNDUNESP for the financial help and the above mentioned manufacturer for supplying the bleaching agent. The authors have no financial interest in the companies whose products are mentioned in this article.

References

1. Titley KC, Torneck CD, Smith DC, Abdifar A. Adhesion of composite resin to bleached and unbleached bovine enamel. J Dent Res. 1988; 67: 1523-8.
2. Garcia-Godoy F, Dodge WW, Donohue M, O’Quinn JA. Composite resin bond strength after enamel bleaching. Oper Dent. 1993; 18: 144-7.
3. Türkün M, Kaya A.D. Effect of 10% sodium ascorbate on the shear bond strength of composite resin to bleached bovine enamel. J Oral Rehabil. 2004; 31: 1184-91.
4. Mcguckin RB, Thurmond BA, Osovitz S. Enamel shear bond strengths after vital bleaching. Am J Dent. 1992; 5: 216-22.
5. Dishman MV, Covey DA, Baughan LW. The effects of peroxide bleaching on composite to enamel bond strength. Dent Mater. 1994; 9: 33-6.
6. Cavalli V, Reis AF, Giannini M, Ambrosano GM. The effect of elapsed time following bleaching on enamel bond strength of resin composite. Oper Dent. 2001; 26: 597-602.
7. Rose RC, Bode AM. Biology of free radical scavengers: an evaluation of ascorbate. FASEB J. 1993; 7: 1135-42.
8. Rotstein I. Role of catalase in the elimination of residual hydrogen peroxide following tooth bleaching. J Endod. 1993; 19: 567-9.
9. Barghi N, Godwin JM. Reducing the adverse effect of bleaching on composite-enamel bond. J Esthet Dent. 1994; 6: 157-61.
10. Lai SC, Mak VF, Cheung GS, Osorio R, Toledano M, Carvalho RM et al. Reversal of Compromised Bonding to Oxidized Etched Dentin. J Dent Res. 2001; 80: 1919-24.
11. Lai SC, Tay FR, Cheung GS, Mak VF, Carvalho RM, Wei SH et al. Reversal of Compromised Bonding in Bleached Enamel. J Dent Res. 2002; 81: 477-81.
12. Titley KC, Torneck CD, Smith DC, Chernecky R, Abdifar A. Scanning electron microscopy observations on the penetration and structure of resin tags in bleached and unbleached bovine enamel. J Endod. 1991; 17: 72-5
13. Kalili T, Caputo AA, Mito R, Sperbeck G, Matyas J. In vitro toothbrush abrasion and bond strength of bleached enamel. Pract Period Aesthet Dent. 1991; 3: 22-4.
14. Sundfeld RH, Briso AL, De Sa PM, Sundfeld ML, Bedran-Russo AK. Effect of time interval between bleaching and bonding on tag formation. Bull Tokyo Dent Coll. 2005; 46: 1-6.
15. Titley KC, Torneck CD, Ruse ND, Krmec D. Adhesion of a resin composite to bleached and unbleached human enamel. J Endod. 1993; 19: 112-5.
16. Edwall L, Olgart LG. Influence of cavity washing agents on the pulpal microcirculation in the cat. Acta Odontol Scand. 1972; 30: 39-47.
17. Scriver CR, Beaudet AL, Sly WS, Valle D. The metabolic basis of inherited disease. 6th ed. New York: McGraw-Hill; 1989. p.1551-61, 2779-2801.
18. Gutteridge JM. Biological origin of free radicals, and mechanisms of anti-oxidant protection. Chem Biol Interact. 1994; 91: 133-40.
19. Wendel A. Glutathione peroxidase. In: Jacoby WB. Enzymatic basis of detoxification. New York: American Press; 1980, v.1, p.333-53.
20. Carr AC, Tijerina T, Frei B. Vitamin C protects against and reverse specific hypochlorous acid- and chloramines-dependent modifications of low-density lipoprotein. Biochem J. 2000; 2: 491-99.
21. Smith MJ, Anderson R. Biochemical mechanisms of hydrogen peroxide and hydpochlorous acid mediated inhibition of human mononuclear leukocyte functions in vitro: protection and reversal by anti-oxidants. Agents Actions. 1992; 36: 58-65.
22. VanDuijn MM, Tijssen K, VanSteveninck J, Van Der Brack PJ, Van Der Zee, J. Erythrocytes reduce extracellular ascorbate free radicals using intracellular ascorbate as an electron donor. J Biol Chem. 2000; 275: 27720-5.
23. Kaya AD, Turkun M. Reversal of dentin bonding to bleached teeth. Oper Dent. 2003; 28: 825-9.
24. Sung EC, Chan SM, Mito R, Caputo AA. Effect of carbamide peroxide bleaching on shear bond strength of composite to dental bonding agent enhanced enamel. J Prosthet Dent. 1999; 82: 595-9.
25. Kum KY, Lim KR, Lee CY, Park KH, Safavi KE, Fouad AF et al. Effects of removing residual peroxide and other oxygen radicals on the shear bond strengrh and failure modes at resintooth interface after tooth bleaching. Am J Dent. 2004; 17: 267-70.
26. Perdigão J, Francci C, Swift EJJr, Ambrose WW, Lopes M. Ultra-morfological study of the interaction of dental adhesives with carbamide peroxide-bleached enamel. Am J Dent. 1998, 11: 291-301.
27. Travis DF, Glimcher MJ. The structure and organization of, and the relationship between the organic matrix and the inorganic crystals of embryonic bovine enamel. J Cell Biol. 1964; 23: 447-97.
28. Nakamichi I, Iwaku M, Fusayama T. Bovine teeth as possible substitutes in the adhesion tests. J Dent Res. 1983; 62: 1072-81.

Copyright 2006 - Piracicaba Dental School - UNICAMP São Paulo - Brazil