ORIGINAL ARTICLES DERIVED FROM RESEARCH

EFFECT OF PEROXIDASE ON COMPOSITE RESIN ADHESION TO DENTAL ENAMEL AFTER WHITENING¹

Paula Alejandra Baldión Elorza², Laura Nathalia Viteri Lucero³, Edilberto Lozano Torres³

¹ Article derived from a research project to opt to the title of Dentist, as part of a project of the Research Group on Dental Materials (GRIMAD). It obtained financial aid from Universidad Nacional de Colombia's School of Dentistry, as part of its internal call for research proposals in 2009
² Dentist, specialist in Oral Rehabilitation, School of Dentistry, Universidad Nacional de Colombia. Professor at the School of Dentistry, Universidad Nacional de Colombia
³ Dentists, School of Dentistry, Universidad Nacional de Colombia

Baldión PA, Viteri LN, Lozano E. Effect of peroxidase on composite resin adhesion to dental enamel after whitening. Rev Fac Odontol Univ Antioq 2012; 24(1): 8-21.

ABSTRACT

INTRODUCTION: some reports suggest that residual oxygen released by whitening agents interfere with composite resin adhesion to dental structure. The objective of this study is therefore to compare composite resins' shear bond strength to dental enamel after whitening using 38% hydrogen peroxide pre- and post- treatment with peroxidase enzyme before adhesion.
METHODS: a total of 45 healthy human premolars were selected and divided into three groups of 15 teeth each. Group 1: control group (only adhesion); group 2: whitening and adhesion; group 3: whitening, application of peroxidase, and adhesion. After treatment, shear bond strength was measured with a Shimadzu testing machine in order to determine significant statistical differences among the three groups, with a confidence level of 95% and p < 0.05.
RESULTS: the control group obtained a shear bond strength of 12.8Mpa (± 3.2), the whitening group showed an average 3.5 Mpa (± 1.43), and the group treated with whitening and peroxidase presented an average 12.2 Mpa (± 3.12).
CONCLUSIONS: shear bond strength values significantly decreased with application of 38% hydrogen peroxide; however, a significant increase was also obtained by applying peroxidase before adhesion.

Key words: horseradish peroxidase, dental adhesion, dental whitening, composite resins, hydrogen peroxide.

INTRODUCTION

There exists some controversy on the effects of whitening agents on both soft and hard oral tissues. A rational and responsible use of such agents has been promoted, as well as selecting products that have been thoroughly studied, in order to provide safe and successful treatments while minimizing side unwanted effects. Enamel alterations reported by some studies include increased porosity, fissure formation, surface erosion, and demineralization zones at interprismatic areas,^1-6 as well as alterations in terms of chemical composition and mechanical properties.^{1, 7-13} Authors such as Dishman et al¹⁴ and Villarreal et al¹⁵ maintain that whitening agents interfere with composite resin adhesion to dental structure due to interaction with residual oxygen in form of free radicals that remain on tissues two to four weeks after completing the dental whitening procedure,^{7, 8, 10} thus blocking their adhesive properties. Nevertheless, some methods for neutralizing residual oxygen free radicals have been tested in order to reverse the unwanted physicochemical effects on both dental structure and enamel substrate adhesion.^{15, 16} Some strategies, such as waiting for a reasonable time after dental whitening¹⁷ or using antioxidant substances^{15, 18, 19} have been suggested as a way to removing residual peroxide from dental structures in order to perform adhesive restorations right after the whitening procedure while at the same time protecting the enamel from possible unwanted side effects of the whitening agents' residual peroxide, such as microstructural changes,^{2, 4, 5} surface weakening,²⁰ or alteration of its chemical composition.^{7, 9}

Studies such as the ones by Türkün et al1^{9, 21} and Gökçe et al²² have suggested the use of sodium ascorbate as an effective method to revert the oxidant effect of whitening substances; similarly, Rotstein et al¹⁸ found out that catalase locally used for three minutes was effective for totally eliminating the residual hydrogen peroxide remaining on dental structures after application of hydrogen peroxide at the intracoronal area.

The purpose of this study was to comparatively evaluate the effect of antioxidant treatments with peroxidase on composite resins' immediate bond strength to dental enamels that have been exposed to whitening agents with high concentrations (38%) of hydrogen peroxide.

MATERIALS AND METHODS

This was an in vitro experimental quantitative study on 45 healthy human premolars with complete root formation, recently extracted for orthodontic reasons and with no caries, fractures, dental wear, enamel faults, or previous restorations. The samples were stored in proper conditions for a period no greater than three months. Ethical considerations of sample collection, handling and disposal were respected, thus complying with Ministry of Health Resolution 008430 of 1993,²³ which considers this study to be of minimum risk; patients provided a signed consent for donating their teeth specifically for this study, which was approved by the Ethics Committee of Universidad Nacional de Colombia's School of Dentistry.

The samples were fixed on 0.5% chloramine for 24 h, and later stored on distilled water until the tests began. The teeth were introduced in acrylic resin casts in order to shape cube-like supports that later facilitated their insertion in the Shimadzu AG-SI Series testing machine. The adhesive area was previously delimited by means of a Contact^® paper circle with a central hole of 2 mm in diameter. 37% phosphoric acid was later added to adequately prepare the enamel surfaces; they were dried with paper towels, and two layers on Tetric N-Bond^® adhesive (Ivocar/Vivident, Germany, Liechtenstein) were later applied for the surfaces to get a bright shade. The samples were kindly ventilated and each of them (MV- D) was photopolymerized during 20 seconds with a Bluephase G2^® lamp (Ivocar/Vivadent, Gemany, Liechtenstein) on low mode, following the manufacturer's instructions.

The samples were sorted out in three groups: group 1: adhesion, surface cleansing with sodium bicarbonate and water, application of 37% phosphoric acid, rinsing, drying, adhesive, resin; group 2: whiteningadhesion, application of 38% hydrogen peroxide (Opalescence X-tra Boost^®/Ultradent Products, South Jordan, USA)—following the manufacturer's instructions, the whitening agent was mixed in an activator syringe, and two consecutive applications of 15 min each were performed until obtaining a layer no greater than 1 mm in thickness—, surface cleansing with sodium bicarbonate and water, application of 37% phosphoric acid, rinsing, drying, adhesive, and resin; group 3: whitening-enzyme; the enzymatic antioxidant used was horseradish peroxidase (Peroxidase from horseradish type I/ SIGMA-ALDRICH, St. Louis, USA) containing 50-150 units/mg solid, which was dissolved in a phosphate buffer, producing a final concentration of 472 mg/ml weight/volume at a pH of 6.9 to 7. This solution was stored at a temperature of -20 °C until it was used; at that moment, a thin layer of it was applied to the dental surface, letting it work for 15 min. Distilled water was used to rinse the samples; their surfaces were dried with paper towels, and a conventional adhesion process was performed.

After surface treatment and composite resin adhesion, each sample was tested at the Shimadzu AG-IS Series universal testing machine, setting the section sheet at 0,5 mm from the adhesive interface. A load of 50 N was applied at a constant speed of 1 mm/min until fracturing the sample; the values at which this fissure occurred were recorded. Considering adhesion area (A = p r2), given in mm, as well as the load magnitude (in newtons), the corresponding strength was calculated in Mpa (N/mm2); the obtained data were recorded in a table displaying the two variables of this study: Surface treatment per group and adhesion to dental enamel (shear bond strength in Mpa).

Data analysis was performed by means of a nonparametric test based on ANOVA Kruskal-Wallis one-way ranks in order to contrast the hypothesis of equality of treatment medians, and later with a non-parametric Mann-Whitney test to contrast the hypothesis of equality of the groups under study, by using the Social Sciences Statistical Package SPSS, 15.0 (SPSS Inc., Chicago, IL, USA). Central tendency measures (mean and standard deviation) were used to condense data presentation per group. Values of p < 0.05 were considered as statistically significant differences among the control group and the groups treated with whitener and peroxidase enzyme.

RESULTS

With a 5% significance level (95% confidence level), the null hypothesis of equality of means among treatments is rejected. This studied yielded statistically significant evidences of decreased composite resin shear bond strength to enamel after applying 38% hydrogen peroxide in comparison to the control group (p = 0.003). There were also signs of increased shear bond strength in the group treated with whitening and subsequent application of peroxidase in comparison to the shear bond strength of the group treated with whitening followed by immediate adhesion (p = 0.0025); and recovery of the initial shear bond strength value was observed as no statistical difference occurred between the control group (with adhesion only) and the group exposed to peroxidase (p = 0.069) (table 1).

These results show that group 1 (adhesion) presented a greater shear bond strength, with a value of 12.8 Mpa (± 3.2) in comparison to group 2 (whitening-adhesion), which shear bond strength values decreased 72.65% in comparison to group 1, with an average of 3.5 Mpa (± 1.43).

Application of peroxidase after whitening in group 3 produced a recovery of 95.3% in comparison to group 1 with an average of 12.2 Mpa (± 3.12) (figures 1 and 2).

DISCUSSION

Several reports have suggested that peroxide-based whitening agents may produce morphological and structural changes at the resin-enamel interface, thus interfering with composite resins adhesion to dental structure.^{8, 10, 14, 15}

The purpose of this study was to determine the effect of peroxidase enzyme on composite resins' adhesion to dental enamels previously exposed to 38% hydrogen peroxide; to this end, several adhesion tests were performed by applying a tangential load on three groups: group 1: control group, treated with adhesion only; group 2: whitening with subsequent adhesion, and group 3: whitening, treatment with peroxidase, and immediate adhesion.

The tests demonstrated a shear bond strength decrease of 8.93 Mpa of the resin immediately adhered to the whitened enamel in comparison to the control group, which was treated with adhesion only. This proves that using whitening agents with high concentrations of hydrogen peroxide during the times indicated by manufacturers reduces the composite resin's bond strength to dental enamel. This phenomenon has been analyzed by authors such as Bulut et al,²⁴ Miles et al,²⁵ and McGuckin et al,²⁶ who have explained this effect by the presence of residual peroxide or oxygen released by whitening agents—which inhibit the polymerization process of adhesion systems and composite resins—as well as by alterations of the enamel surface structure associated to erosion of the aprismatic layer and to lack of calcium and phosphorous.^{15, 27} This adhesion strength decrease has led some authors, such as Miranda et al²⁸ and Shinohara et al,²⁹ suggest that restorative procedures should be postponed two to four weeks after whitening, since reduction of composite resins bond strength to enamels that have been recently whitened has proven to be temporary.³⁰ This is explained because the enamel suffers a process of reminarlization³¹ that allows replacing ions of phosphate, calcium, and other lost minerals to the same elements or to other ions present in saliva. And this includes fluoride ions which favor the formation of crystals, thus providing a more stable base for adhesion. Supersaturation of calcium and phosphate ions in saliva plays an important role in the process of remineralization; however, authors such as Larsen et al32 suggest that this process requires time because, even though saliva and remineralizing solutions are supersaturated in comparison to the apatite present in the enamel, the total dissolved amount of calcium and phosphate is small, and therefore after precipitation of dissolved minerals only 1/20.000-1/30.000 of the mineralization solution volume is occupied by mineral. Moreover, the concentration gradients of the enamel mineralization solution are small, which explains the slow diffusion inside the structure;³² this is why it seems reasonable to wait the time recommended by authors such as Shinohara et al,²⁹ Basting et al,33 and Cavalli et al³⁴ in order to recover an enamel surface with less apparent changes.

Clinical situations such as old restorations in the anterior sector—that affect aesthetic appearance—, the need of starting orthodontic treatment or simply patient's availability for a cosmetic treatment that requires adhesive post-whitening precedures,^{25, 35} mean a difficult situation to the clinician at the moment of deciding whether to perform restorative treatment instead of waiting for some reasonable time to proceed with postwhitening adhesion. In consequence, dentists need to find a way of performing adhesive restorations right after the whitening procedure without risk of failing.

Villarreal et al¹⁵ maintain that the concomitant use of antioxidant substances reduces the adverse effect on resins' bond strength to enamel as well as morphological and chemical changes. Enzymatic antioxidants, such as catalase and superoxide dismutase,^{15, 18, 36} and non-enzymatic antioxidants,³⁷ such as 10% sodium ascorbate, have been tested as inhibitors of residual oxygen, and have proven to be able to reverse the negative effects of whitening agents on bond strength to enamel, with satisfactory results so far.^{19, 21, 24, 38} Rotstein et al¹² demonstrated that applying catalase for three minutes may totally remove residual hydrogen peroxide, and they recommend this as an intracameral post-whitening protocol in nonvital teeth to eliminate oxygen from both pulpal chamber and periodontal neighboring tissues in order to reduce potential damage.

Lai et al,³⁹ Türkün et al,¹⁹ and Feiz et al38 demonstrated that submerging samples in water for 24 hours³⁹ and using an antioxidant agent as sodium ascorbate inhibits the decrease of composite resins bond strength after whitening.

Peroxidase has also been tested in studies by Bowles et al⁴⁰ and Gimeno et al,⁴¹ who demonstrated that its use during or immediately after whitening with hydrogen peroxide may prevent harmful effects on soft tissues of the oral cavity and the pulp canal. The studies by Masakiet al,⁴² Spector et al,⁴³ and Björkman et al⁴⁴ on fibroblasts, epithelial and thyroid cell cultures demonstrated a protective effect on tissues thanks to the use of catalase and peroxidase in presence of oxygen free radicals.

In this study, an increase of shear bond strength was observed when applying peroxidase enzyme on the dental surface right after whitening with 38% hydrogen peroxide, as it demonstrated a significant recovery of composite resin adhesive properties by elimination of radicals released by the whitening agent, thanks to its enzymatic action in reduction-oxidation processes.^{15, 16, 18} This shows that the peroxidase enzymatic activity enables elimination of residual peroxide. Being a molecule of 40.000 Da in size, it has the capacity of penetrating the enamel matrix through its rods, and probably even reaching the dentinal tubules, thus allowing interaction with oxygen molecules, causing their oxidation and degrading into subproducts such as water, therefore avoiding inhibition of the polymerization process of composite resins and adhesive systems by conforming a stable tridimensional net of carbon-carbon bonds of methacrylate groups.^{38, 39}

Rocha et al⁴⁵ reported that applying catalase and glutathione peroxidase may increase bond strength to the enamel in comparison to a positive control group with application of hydrogen peroxide; nevertheless, catalase proved to have better activity than peroxidase. They explain this finding by the catalase's mode of action, which requires a reduced number of molecules to produce its enzymatic effect. However, the authors point out that none of the two enzymes has the capacity of entirely neutralize harmful effects, as bond strength was significantly lower than in the negative control group, thus concluding that using a more concentrated solution might yield better results. This would explain the difference with the results of the present study. Rocha et al⁴⁵ used peroxidase of bovine erythrocytes of 680 units/mg of proteins at a concentration of 10 mg/ml, while for the present study we applied horseradish peroxidase of 150units/mg of proteins at a concentration of 472 mg/ml, increasing the final quantity of weight/volume of the enzyme, with better results in terms of recovery of bond strength to dental enamel following whitening.

To meet the inherent conditions in enzymatic reactions, enzymes must be handled under conditions that favor their denaturation, as a way to producing their inactivation. Using enzymatic antioxidant agents, such as catalase, peroxidase, and superoxide dismutase, offers greater technical sensitivity for clinical application in comparison to non-enzymatic antioxidants. Enzymatic activity is conditioned by the following factors: effect of enzymatic concentration, temperature, pH of the solution, and its shaking. The effect of enzymatic concentration determines the reaction speed due to the proportion enzyme quantity/substrate. Reaction speed decreases as substrate concentration increases. In terms of temperature control, most enzymatic reactions occur at temperatures ranging from 0 to 60 °C. Optimum temperature is usually related to the temperature of the cellular environment from which the enzyme derives; for peroxidase activation, this temperature is 37 °C. It keeps its protein tertiary structure thanks to non-covalent bonds of a physical nature, which are highly sensitive to changes in temperature. Another important factor to consider is pH effect. Peroxidase enzyme has a characteristic optimum pH of 6,9 to 7,0; changes in the concentrations of H+ and OH- modify the solution's pH altering the interaction among the molecules' proteins, and therefore also their tridimensional structure and their activity. Finally, let's consider the shaking effect. When strongly shaken, they grow foam due to the low surface tension of protein solutions, which favors their denaturation.^{15, 46-48}

It is necessary to consider the protocol suggested for processing both enzymatic solution and peroxidase conservation, which allows catalytic activity to be performed on the substrate of residual hydrogen peroxide and free radicals in the dental surface. This protocol includes explicit guidelines for preparation, conservation, and application of the enzyme, which increase sensitivity and make the clinical technique difficult as its catalytic activity may be easily altered due to its biological nature. It is therefore important to assess applicability of this protocol for clinical handling of horseradish peroxidase (Horseradish Type I/SIGMA-ALDRICH, St. Louis, USA) in our context, as it is very costly and hard to get, just as happens with other types of antioxidant enzymes with similar characteristics, such as catalase or other kinds of peroxidase derived from blood, yeast, or green beans.

The presence of lyophilized powder peroxidase demanded preparation of a phosphate shock-absorbing solution, at a pH of 7, as a stable suspension for the enzyme to perform its catalytic action without denaturizing, and thus obtaining an easy-to-apply liquid or gel. Also, it was necessary to rigorously store the enzyme at -20° C and to carefully transport it, and to wait a very long time to have the product shipped to our country. These limitations suggest the need of finding new alternatives for enzyme production at lower costs and easier ways to acquire and distribute the products, by directly isolating the enzyme from products easily obtained and with simple separation lab procedures.

The findings of this study are considered important as they provide significant evidence to examine in greater detail the clinical procedures that allow reducing the harmful effects of whitening agents, since despite the abundant studies that prove the physiological,^49-53 chemical^{7, 9, 54} mechanical,^{1, 8-11, 13, 20, 55} and structura^{1-3, 6, 12} alterations produced by peroxides, it is still one of the most requested procedures by patients and one of the most practiced by dentists currently.

RECOMMENDATIONS

Further studies are recommended in order to find an alternative to peroxidase application, qualitatively assessing the catalytic activity in terms of speed of reaction and time, as well as microscopic evidence of its action on the enamel's microstructural changes.

It is necessary to determine the catalytic constants and the mean life of peroxidase in relation to 38% hydrogen peroxide applied on the enamel surface for dentistry purposes, in order to obtain a parameter and to determine the amount of the closest catalytic units that guarantee entire removal of residual hydrogen peroxide and its free radicals.

CONCLUSIONS

Limitations of the present study include, among others:

There is a significant statistical reduction of the shear bond strength of composite resins adhered to enamels that have been subjected to whitening with 38% hydrogen peroxide.

Recovery of shear bond strength values of composite resins occurs when applying the enzyme peroxidase on the dental surface right after application of 38% hydrogen peroxide.

ACKNOWLEDGEMENTS

To Universidad Nacional de Colombia's School of Dentistry for its financial aid to this study as part of the School's internal call for proposals.

CORRESPONDING AUTHOR

Judith Patricia Barrera Chaparro
Carrera 18 N.° 80-94. Bogotá D. C.
Fundación Universitaria San Martín
Facultad de Posgrados de Odontología
Posgrado de Ortodoncia
Correo electrónico: barrerajudith@gmail.com

REFERENCES