Catalase and sodium fluoride mediated rehabilitation of enamel bleached with 37% hydrogen peroxide

Ruchi Thakur; Anand L Shigli; Divya Sharma; Gagan Thakur

doi:10.4103/0970-4388.165702



ORIGINAL ARTICLE

Year : 2015 \| Volume : 33 \| Issue : 4 \| Page : 324-330

Catalase and sodium fluoride mediated rehabilitation of enamel bleached with 37% hydrogen peroxide

Ruchi Thakur¹, Anand L Shigli², Divya Sharma³, Gagan Thakur⁴
¹ Department of Pedodontics and Preventive Dentistry, RKDF Dental College and Research Centre, Bhopal, Madhya Pradesh, India
² Department of Pedodontics and Preventive Dentistry, Bharati Vidyapeeth Dental College, Sangli, Maharashtra, India
³ Department of Pedodontics and Preventive Dentistry, Modern Dental College and Research Centre, Indore, Madhya Pradesh, India
⁴ Department of Oral and Maxillofacial Surgery, RKDF Dental College and Research Centre, Bhopal, Madhya Pradesh, India

Date of Web Publication

18-Sep-2015

Correspondence Address:
Dr. Ruchi Thakur
Dental Avenue, C 16, Vidya Nagar, Hoshangabad Road, Bhopal - 462 026, Madhya Pradesh
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/0970-4388.165702

Abstract

Background: Bleaching agents bring about a range of unwanted changes in the physical structure of enamel which needs to be restored qualitatively and timely. Catalase being an antioxidant ensures the effective removal of free radicals and improvement in fluoride mediated remineralization from the enamel microstructure which if retained may harm the integrity and affect the hardness of enamel. Materials and Methods: Thirty freshly extracted incisors were sectioned to 6 slabs which were divided into 5 groups: Group A, control; Group B, treatment with 37% hydrogen peroxide (HP); Group C, treatment with 37% HP and catalase, Group D, treatment with 37% HP and 5% sodium fluoride application, Group E, treatment with 37% HP followed by catalase and 5% sodium fluoride. Scanning electron microscope and microhardness analysis were done for all slabs. One-way ANOVA test was applied among different groups. Results: Vicker's microhardness number (VHN) of Group B and C was significantly lower. No significant difference between VHN of Group B and C. VHN of Group D was significantly higher than Group A, B, and C; but significantly lower than Group E. VHN of Group E was significantly higher than any other experimental group. One-way ANOVA revealed a highly significant P value (P = 0.0001) and so Tukey's post-hoc Test for the group comparisons was employed. Conclusion: Subsequent treatment of bleached enamel with catalase and fluoride varnish separately results in repairing and significantly increasing the microhardness.

Keywords: Antioxidants, catalase, dental bleaching, hydrogen peroxide, remineralization, sodium fluoride

How to cite this article:
Thakur R, Shigli AL, Sharma D, Thakur G. Catalase and sodium fluoride mediated rehabilitation of enamel bleached with 37% hydrogen peroxide. J Indian Soc Pedod Prev Dent 2015;33:324-30

How to cite this URL:
Thakur R, Shigli AL, Sharma D, Thakur G. Catalase and sodium fluoride mediated rehabilitation of enamel bleached with 37% hydrogen peroxide. J Indian Soc Pedod Prev Dent [serial online] 2015 [cited 2021 Feb 25];33:324-30. Available from: https://www.jisppd.com/text.asp?2015/33/4/324/165702

Introduction

A large number of dental bleaching treatments are available either in gel composition or increased peroxide concentrations. ^[1] These could be for professional use only containing high concentrations of peroxides (35-37%) and patient applied with low carbamide (10-20%) and hydrogen (3-7.5%) peroxide concentrations. ^[2] However, the use of high concentration peroxides may cause tooth sensitivity, alteration in surface morphology of enamel, reduction in bond strength of resin materials to possibly carcinogenicity. ^[3],[4]

Hydrogen peroxide (HP) is a relatively unstable molecule and in the presence of decomposition catalysts, enzymes and saliva, HP quickly disassociates into highly reactive oxidizing agents that is, three types of free radicals (hydroxyl radicals, per-hydroxyl radicals, and superoxide anions), reactive oxygen molecules and HP anions. ^[5] These free radicals are highly reactive as they have one unpaired electron. HP generally is maintained in a weakly acidic state to increase its shelf life by halting the ongoing dissociation. In the weakly acidic state, it produces a weak free radical - nascent oxygen (O). Perhydroxyl (HO ₂ ) on the other hand is a stronger free radical and is produced when the alkalinity of HP is maintained between pH 9.5 and 10.8. It is the highly unstable free oxygen component in each that reacts with, oxidizes and attacks the long-chained, dark-colored chromophore molecules (atoms or groups in a molecule that cause color) in enamel. This attack splits the chromophore molecules into smaller, less colored and more diffusible molecules, which results in whitening of the teeth. The end result depends on the concentration of HP, the time the radicals are in contact with teeth and how efficiently the radicals reach chromophore molecules. This process is known as oxidation which may go on till it results in the production of undesired combustion end products. So when desired whiteness is achieved, a scavenger against uncontrollable free radicals is required which can be catalase. Catalase comprises four polypeptide chains which are as long as more than 500 amino acids. Four porphyrin heme (iron) rings are present which react with HP at a pH of 7 (range being 6.8-7.5). End products of this reaction are oxygen and water which helps in maintaining a hydrated layer on the enamel surface postbleaching. Catalase accelerates the decomposition of HP by more than 100,000,000 fold. ^[6]

Jones and Wynne-Jones ^[7] proposed a mechanism for catalase action. It was shown that the mechanism could also comprehend the available information on the catalytic action of Fe ³⁺ ion, both in the decomposition of HP and as a mediator of coupled "peroxidatic" oxidations. The metal acts as a redox catalyst for the catalyst reaction through three reactions.

Human body constitutes its own antioxidant defense system in the presence of superoxide dismutase, catalase and glutathione peroxides usually in cells with increased oxidative stress such as cytosol, mitochondria, and peroxysomes. ^[8] Similarly, catalase in dental tissue reduces the oxidative stress by preventing overproduction and facilitating the removal of residual free radicals.

Topical fluoride (in the form of potassium nitrate, calcium phosphate, etc.) have been added to bleaching agents to reduce the mineral loss and dentin dehydration as the maintenance of physiological dentinal moisture can reduce the hypersensitivity. ^[9] However, an in vitro study reported that carbamide peroxide (CP) combined with ﬂuoride or calcium did not prevent the reduction in enamel microhardness. ^[10] Contrarily, an X-ray photoelectron spectroscopy study revealed that the additional sodium ﬂuoride in HP generates ﬂuoridated hydroxyapatite and calcium ﬂuoride crystals on enamel surfaces. ^[11] Dental bleaching agents penetrate into dentin at certain depths to decompose intrinsic chromatic pigments. ^[12],[13] Therefore, recent clinical evidence of associated calcium ﬂuoride formation raises new concerns about the permeability of enamel. The travel of reactive oxygen molecules and free radicals may be impeded by crystal deposition. The purpose of this study is to suggest that catalase if applied postbleaching helps in neutralizing the reactive oxygen molecules and free radicals and at the same time, it hydrates the enamel surface. This can be finally followed by topical fluoride application at the end of the bleaching cycle to remineralize and restore the surface hardness. Therefore, this present study suggests that remineralization of bleached enamel can be better brought about by topical fluorides provided the residual free radicals have been neutralized by catalase.

Materials and Methods

Specimen preparation

Thirty freshly extracted human central incisors stored in 0.1% buffered thymol solution (pH = 7) for no longer than 4 weeks after the extraction were cleaned of gross debris and examined under magnification (×20) for stains, microcracks, and/or surface defects. Teeth were sectioned at cemento-enamel junction and the crowns were further sectioned with diamond disks using a low-speed hand piece to obtain 6 enamel slabs (approximately 3.5 × 2.5 mm ² ) [Figure 1]. One hundred fifty-six enamel slabs were thus obtained (26 incisors ×6 sectioned enamel slabs = 156 enamel slabs). Five slabs (coinciding to following 5 groups - A, B, C, D, and E) [Figure 2] from each were randomly designated to each group while remaining 1 slab from each was discarded.

Figure 1: Extracted incisor crowns were sectioned as per markings shown in the figure to obtain 6 enamel slabs (approximately 3.5 × 2.5 mm 2 in dimension) from each central incisor

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Figure 2: Split plot design

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Experimental groups

Experimental design was as follows [Table 1]:

Table 1: Description of experiment groups

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Control Group: A.
Experimental Groups: B, C, D, and E.

All surfaces but labial were coated with dental varnish. The uncoated labial enamel surface was sequentially polished by 400, 600, 1200 grade sandpaper. These slabs were subjected to steam sterilization to avoid bacterial contamination and thereafter immersed and stored in deionized water at 37°C in the respective vials till the commencement of the study.

As per the study design stated in [Table 1], agents were applied on slabs as follows:

Regimen for 37% HP ([Gel form available in syringe] - Vishal Dentocare Pvt. Ltd.) application included 2 applications of 15 min each at a 7 day interval. After treatment the slabs were thoroughly washed with deionized water for 10 s and stored in artificial saliva (pH 7.0 - Pure Pharma Ltd.) at 37°C which shall be changed every 24 h, until the next treatment.
Regimen for sodium fluoride (sodium fluoride IP-50 mg [equivalent to 22600 ppm of fluoride] - Fluoritop-SR ^® Varnish: Slow Release Topical Dental Fluoride - ICPA Health Products Ltd.) - One drop was taken for each time and using an applicator brush, varnish was painted on the tooth surface.
Regimen for catalase (enzymatic activity units/mg protein 2000.00-5000.00 Hi-Media Labs Pvt. Ltd.) included an application for 3 min. Later was washed with deionized water and placed in a respective vial containing artificial saliva.

Microhardness testing

Slabs were positioned perpendicularly to the long axis of the indentor to record the Vicker's hardness number (VHN). The individual testing of the enamel slabs was blinded and a 1 kg (maximum) load Vicker's indentor was attached to a microhardness tester (Lecia, VMHT 30 A, M/S Lecia, Germany) which performed three measurements in 5 s to determine the VHN of each slab.

Scanning electron microscope

The individual testing of the enamel slabs was blinded and enamel surfaces were sputter coated with gold in a vacuum evaporator, and photo-micrographs of representative areas were taken at ×2000 magnifications.

Results

Vicker's microhardness

VHN of Group B and C was significantly lower than any other group. There was no significant difference between VHN of Group B and C. VHN of Group D was significantly higher than VHN of Group A, B, and C; but significantly lower than VHN of Group E. VHN of Group E was significantly higher than any other experimental group [Figure 3]. Statistical analysis by means of One-way ANOVA [Table 2] revealed a highly significant P value (P = 0.0001), and so a further Tukey's post-hoc Test [Table 2] for group comparisons was employed.

Figure 3: Mean values of Vicker's hardness number in control and experimental groups

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Table 2: Mean and SD of VHN in control and experimental groups

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Scanning electron microscope

Group A [Figure 4]a showed a layered deposition of minerals mostly from the artificial saliva (black arrows) and scratch lines due to mechanical wear (white arrows).
Group B [Figure 4]b showed crater like depressions (black arrows) with shiny margins (white arrows) displaying the "edge effect".
Group C [Figure 4]c showed different size rounded elevations (white arrows) suggesting protein deposits from catalase and tiny depressions (black arrows).
Group D [Figure 4]d showed homogenously even enamel surface suggesting remineralization with a multilayered appearance imparting the characteristic "edge effect" similar to [Figure 4]b. Layer containing minerals (described as an organicomineral membrane) can also be seen as isolated white spots (black arrows).
Group E [Figure 4]e showed smooth and regular enamel surface closely imitating the untreated enamel surface.

Figure 4: (a) Depositions from saliva (black arrows) and scratch lines (white arrows) (b) uneven surface (black arrow) and "edge effect" (white arrows) (c) depressions less than approximately 3 microns in diameter (black arrows). Elevations suggesting exposed enamel rods (white arrows) (d) homogenous enamel surface suggesting remineralization with multilayered appearance "edge effect" (white arrows). Organicomineral membrane seen as white spots (black arrows) (e) smooth enamel surface imitating untreated enamel surface with an overlying hydrated superficial layer (white arrows)

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Specimens in Group D [Figure 4]d and Group E [Figure 4]e appeared smooth and regular suggesting the effective postbleaching remineralization unlike Group B [Figure 4]b which appeared more rough and uneven highlighting the effects of high concentration bleaching agent. Specimens in Group C [Figure 4]c, however, showed a relatively smoother surface as compared to Group B [Figure 4]b.

Discussion

Changes in the chemical or morphological structure of enamel must be of prime concern when bleaching is used as a treatment for whitening teeth. Various scanning electron microscope (SEM) studies have demonstrated demineralization, surface defects, and degradation of sound enamel. Surface microhardness measurement is a simple method for determining the mechanical properties of enamel and it is related to a loss or gain of minerals. Lewinstein et al. ^[14] observed a slight reduction in enamel surface microhardness following 35% HP or 35% CP treatments on human enamel similar to our study [Figure 3] which he stated was completely reversed when treated with a 0.05% ﬂuoride solution. Rodrígues et al. ^[15] noted a slight reduction in surface microhardness following in ofﬁce 37% CP treatment.

McGuckin et al. ^[16] attributed that HP presented enamel pattern similar to type II acid etching pattern however our study shows rounded elevation in a concentrated area in [Figure 4]c suggestive of either protein deposits from catalase (which can be confirmed by further studies by noticing if they tend to disappear by the use of Sodium hypochlorite over the surface) or some mineral deposits. Titley et al. ^[17] reported the presence of a "white precipitate" as shown in [Figure 4]b giving the enamel a frosted appearance after application of 35% HP. However, Spalding et al. ^[18] with a different school of thought has described it as shinning areas characterized by light strips. They correspond to the "edge effect" - a contrast factor for secondary electrons in SEM - which is generated from the protrusions and circumferences of objects on the specimen surface, causing them to appear brighter than even surfaces similar to as shown in [Figure 4]b and d.

Lopes et al. ^[19] established that erosions in the enamel surface following bleaching did not present a uniform pattern and their intensity varied. There was also an apparent increase in superficial porosity characterized by a greater quantity of Tome's process pits that were observed on the fragments. It may be speculated that this aspect could explain tooth hypersensitivity, a side effect experienced by some people who undergo the tooth bleaching process. Indeed, most bleaching agents are acidic, which is not favorable to enamel, dentin, and cementum.

Artificial saliva was used for in vivo simulation; however, its remineralizing potential is not equivalent to natural saliva. Kuroiwa et al. ^[20] observed formation of a layer containing minerals (organicomineral membrane) on the enamel that can also be identified as granular and thin depositions similar to [Figure 4]d. Remineralization of bleached enamel is a conventional modality for reduction in the potential caries risk, remineralization and reduction in enamel solubility by increasing the fluoride content of the carbonate substituted hydroxyapatite enamel crystallite. Calcium fluoride and fluorapatite are the known major products deposited on the enamel and dentin surfaces after their exposure to high and low concentration topical fluoride vehicles, respectively.

Reduced bond strength have also been attributed to deeper penetration of HP ^[21] and residual oxygen that interferes with the resin infiltration into the etched enamel or inhibits polymerization. ^[22] Pretreatment with antioxidant agent allows free radical polymerization of the adhesive resin to proceed without premature termination by restoring the altered redox potential of the oxidized bonding substrate thus reversing the compromised bonding. ^[3] Also in presence of peroxide, it has been shown that hydroxyl radicals in the apatite lattice are substituted by peroxide ions and produce peroxide-apatite. When peroxide ions decompose, substituted hydroxyl radicals re-enter the apatite lattice, resulting in elimination of the structural changes caused by the incorporation of peroxide ions. ^[8] Catalase which is an antioxidant has been proved to be significantly effective in the removal of harmful residual HP from the dental tissues and help restore tooth integrity. Rotstein ^[7] investigated the efficacy of catalase following intracoronal bleaching and observed the total elimination of HP into the extraradicular medium after 3 min. This suggests that catalase also provides an appreciably short working time thereby reducing chairside time.

It is speculated that reducing the HP concentration (by the action of antioxidant agent - catalase) could reduce tooth sensitivity after first bleaching application. After final bleaching cycle, removal of HP would facilitate more fluoride uptake on the enamel surface as suggested in our study owing to the increase in the microhardness [Figure 3]. In a wet or humid environment, peroxide is hydrolytically cleaved and radicals interact with the fluoride of the gels or the enamel apatite reducing enamel fluoride acquisition or retention. ^[23] It is thereby observed that fluoridation of bleached enamel should be better performed by a fluoride gel applied after bleaching than by fluoridated CP bleaching agent as in the present study [[Table 1] - Group E]. Specimens in Group E showed a smooth surface on SEM and significantly increased microhardness as compared to Group D, signifying that treating bleached enamel with catalase and sodium fluoride can better restore the morphology and hardness. Therefore within the limitations of this in vitro study, we may conclude that although clinically effective, bleaching regimes adversely affect enamel surface, this damage can be repaired by subsequent use of catalase and fluoride remineralizing agents. Further in vivo studies however should be undertaken in order to clinically evaluate the effectiveness of the application of catalase and topical fluorides on bleached enamel.

Conclusion

The use of the 37% HP applied under the recommended treatment regimen causes distinct but variable morphological changes to the enamel surface. Subsequent treatment of damaged enamel with catalase and fluoride varnish (Fluoritop SR - NaF) in this study resulted in repair processes taking place and significant increase in the microhardness.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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