Effect of bromelain enzyme on the microleakage of composite resin restorations after external tooth bleaching: An in vitro study.

AIMS: The aim of this study is to evaluate in vitro the effect of application of bromelain enzyme on the microleakage of composite resin restorations after external tooth bleaching using spectrophotometric evaluation. SUBJECTS AND METHODS: Buccal Class V cavities were prepared on the surface of fifty intact premolars, which were randomly divided into five groups. All cavities were filled with composite resin. Group I: Teeth were not bleached but restored (n = 10). External bleaching with 35% hydrogen peroxide was carried out for the rest of the specimensGroup II: Cavities were restored immediately after bleaching (n = 10)Group III: Cavities were restored after a delay of 3 weeks (n = 10)Group IV: Cavities were treated with sodium ascorbate after bleaching and then restored (n = 10)Group V: Cavities were treated with bromelain enzyme solution after bleaching and then restored (n = 10). Microleakage was assessed by the dye extraction method using a spectrophotometer. STATISTICAL ANALYSIS USED: The data were analyzed statistically by comparison of mean microleakage and post hoc test using SPSS 2.0 software.
RESULTS: Group I displayed the least amount of microleakage, whereas Group II showed the greatest amount of microleakage (P < 0.05). Groups III, IV, and V showed a significantly lower amount of microleakage compared to Group II (P < 0.05). There was no statistically significant difference between Groups IV and V.
CONCLUSIONS: Microleakage increased significantly after external bleaching with 35% hydrogen peroxide, and decreased when the bleached teeth were treated with antioxidants. Ten percent bromelain enzyme was effective in decreasing microleakage; however, its efficacy was similar to 10% sodium ascorbate. Copyright:

INTRODUCTION

With the mounting demand for esthetics, vital tooth bleaching procedure has gained popularity over the last couple of decades.[12] External tooth bleaching offers a fast, economical, and a conservative option for the treatment of discolored teeth.[3] Different bleaching agents such as hydrogen peroxide and carbamide peroxide have been commonly used in different concentrations to attain a desirable result.[145]

The bleaching procedure may give rise to a number of complications, which may include postoperative sensitivity, pulpal irritation, tooth structure alterations, to decreased bond strength of composite resin restorations to the enamel.[6] Another important complication following bleaching is an increase in microleakage of composite restorations, which was extensively reviewed and reported in a systematic review by Attin et al.[7]

Bleaching agents mainly act through a complex oxidation reaction, which results in the release of oxygen and other free radicals.[8] An increase in microleakage of composite resin restorations associated with bleaching occurs mainly because of the presence of residual peroxide and residual oxygen. This tends to interfere with the adhesion of restorative materials by inhibiting resin polymerization.[9]

Different methods have been suggested to avoid this complication postbleaching, which may vary, including removal of the superficial enamel layer, use of adhesives containing organic solvents, pretreatment of enamel with alcohol, cleansing the cavity with antioxidants and a postbleaching period varying from 24 h to 3 weeks.[7]

The most commonly recommended method is to delay the bonding procedure until 2–3 weeks after bleaching.[1011] Studies have shown that residual peroxide and oxygen dissipate over a period of time, eliminating any interference with bonding. However, this is a time-consuming method for both the practitioner as well the patient.[12]

Hence, the need for a quicker and convenient method lead to the utilization of several antioxidants such as sodium ascorbate, ascorbic acid, butylhydroxyanisole, catalase, ethanol, acetone, glutathione, peroxide, α-tocopherol, and sodium bicarbonate. According to research, only a few of these antioxidants were able to decrease the microleakage of composite resin restorations.[12] The interest in natural antioxidants of plant origin has greatly increased in recent years. Numerous natural antioxidants such as proanthocyanidins, lycopene have successfully shown to have a positive effect on bleached enamel.[13]

One such natural antioxidant is bromelain enzyme. It is a complex mixture of proteolytic enzymes derived from pineapple. It has a wide range of therapeutic benefits and has found applications in medicine, health, food, and cosmetics.[14]

To our knowledge, there is insufficient literature on microleakage of composite restorations following bleaching. Till date, no study has investigated the effect of bromelain enzyme on bleached enamel.

The aim of this study was to evaluate and compare the effect of bromelain enzyme on the microleakage of composite resin restoration after external tooth bleaching with 35% hydrogen peroxide.

SUBJECTS AND METHODS

Preparation of antioxidant solution

To prepare 10% sodium ascorbate solution 10 g of sodium ascorbate (Kemphasol, India) in the form of powder was dissolved in 100 ml of distilled water. Furthermore, 10 g of bromelain enzyme (Source naturals, USA) in the form of powder was collected from the capsule and dissolved in 100 ml of distilled water to make 10% bromelain enzyme solution.

Sample preparation

Fifty intact premolars that had been freshly extracted for orthodontic purposes were used. Teeth with caries, hypoplastic areas, or cracks and teeth that had a history of restorative/endodontic treatment were excluded. After extraction, the soft tissue, dental calculus, and stains were immediately removed from the surfaces of the teeth with the help of an ultrasonic scaler. Teeth were then stored in normal saline at 37°C until further use.

Experimental groups

The stored teeth were randomly divided into five experimental groups (n = 10 per group) after bleaching with 35% hydrogen peroxide (Pola office, SDI Limited, Bayswater, Victoria 3153, Australia) [Table 1]. Before bleaching, tooth surfaces were polished with oil- and fluoride-free fine pumice and water using a brush and a slow-speed hand-piece. All tooth surfaces were then rinsed and dried with an air syringe.

Table 1

Distribution of specimens and study group

Groups	Bleaching agent	Antioxidants used	Composite restoration
Group I (n=10)	None	None	Done immediately
Group II (n=10)	35% hydrogen peroxide	None	Done immediately
Group III (n=10)	35% hydrogen peroxide	None	Done 3 weeks after bleaching
Group IV (n=10)	35% hydrogen peroxide	10% sodium ascorbate solution for 10 min	Done immediately
Group V (n=10)	35% hydrogen peroxide	10% bromelain enzyme solution for 10 min	Done immediately

Group 1 (unbleached/control group)

Group 1 teeth underwent cavity preparation and restoration but were not bleached. Box-shaped Class V cavities were made on the buccal surface, around the cementoenamel junction, using a round diamond bur in high-speed hand-piece under air and water-cooling. Occlusal margins and gingival margins of the prepared cavities were located in the enamel and the root, respectively. The prepared cavities were approximately 2 mm in height, 3 mm in the mesiodistal direction, and 2 mm in depth. The samples were etched with 37% phosphoric acid for 15 s, rinsed with water for 20 s, and bonded, followed by composite buildup. Following this, composite resin (Tetric N-Ceram, shade A3, Ivoclar Vivadent) was placed into cavities and polymerized in 1.5–2 mm-thick layers for 20 s. The composite resin was then finished with fine-grit finishing bur, and the teeth were preserved at 37°C in artificial saliva (E saliva, Entod Pharma Ltd. India) that was changed daily for 10 days. The same operator carried out the standardized procedure.

Group 2 (immediate restoration group)

In Group 2, the teeth were first bleached by the following method and then immediately restored with composite resin. The teeth were bleached with 35% hydrogen peroxide, Pola Office (SDI Limited, Bayswater, Victoria 3153, Australia) with three applications of 8 min each. Following bleaching, the specimens were thoroughly rinsed with water for 1 min. The bleached teeth were stored in fresh artificial saliva at 37°C between treatments. After the final bleaching, the cavities were immediately prepared and restored as described above for Group 1.

Group 3 (delayed restoration group)

In Group 3, the teeth were bleached as described above and then, after the final rinse, immersed in artificial saliva at 37°C. After 3 weeks, cavities were prepared, and composite resin restorations were placed as in Group 1.

Group 4 (sodium ascorbate treated group)

In Group 4, the teeth were bleached, and the cavity prepared immediately as described above for Group 2. The specimens were exposed to 10% sodium ascorbate for 10 min using a cotton pellet before filling with composite resin. After removal of the cotton pellet, the cavity was rinsed with distilled water for 2 min and dried gently with air to prepare it for composite resin restoration. Composite resin restorations were placed as in Group 1.

Group 5 (bromelain enzyme-treated group)

After bleaching, a novel antioxidant agent, 10% bromelain enzyme was applied to cavities in the bleached teeth, as described above for Group 4. The samples were restored, as described in Group 1.

Microleakage test (using dye extraction method)

All surfaces of the teeth, except for a 1-mm zone surrounding the restoration margins, were covered with three coats of nail varnish, the apices of the roots were sealed with sticky wax and immersed in 2% methylene blue solution for 24 h. After 24 h, the samples were rinsed under tap water for 30 min, and nail varnish was removed using an ultrasonic scaler. Then, the samples were placed in vials containing 65 wt% nitric acid for 3 days in centrifugal tubes. The vials were centrifuged at 14,000 rpm for 5 min, and following that, the supernatant solutions thus collected were used to determine absorbency in an ultraviolet (UV) visible spectrophotometer at 550 nm using concentrated nitric acid as the blank. The results were recorded as a measure of transmission of light. The data were analyzed statistically by comparison of mean microleakage and post hoc test using SPSS 2.0 software (Spss Inc., Chicago, IL, USA).

RESULTS

The results are presented in Table 2:

Table 2

Descriptive statistics showing mean, standard deviation of microleakage for all the groups

	n	Mean	SD
Group 1	10	0.02620	0.001033
Group 2	10	0.06670	0.005851
Group 3	10	0.03180	0.003676
Group 4	10	0.03860	0.006293
Group 5	10	0.03880	0.003706

SD: Standard deviation

Mean average microleakage of the control Group I (0.026 ± 0.001) was significantly lower than all other groups except Group III (delayed restoration group) (0.031 ± 0.003) (P < 0.05)

Group II (immediate bonding) had the highest value (0.066 ± 0.005) among all the groups

Among the experimental groups, Group IV (0.038 ± 0.006) and Group V (0.038 ± 0.003) showed significantly lower microleakage values as compared to Groups II and III. However, there was no statistically significant difference between the two.

DISCUSSION

Different variables affect the longevity of direct restorations, which could be clinician related, patient related or restoration related.[15] The ability of restoration to bond to tooth structure helps achieve a stronger and durable restoration. Failure of composite restorations may be attributed to numerous factors such as polymerization shrinkage, secondary caries, fracture of the restoration, and microleakage at the tooth-restoration interface.[16] Various research articles have reported diminished bond strength of the composite restorations to enamel following bleaching.[3121718192021] However, to date, relatively few studies have researched on the increase in the microleakage of composite resin restorations following tooth bleaching, specifically external tooth bleaching.[12]

The present study demonstrated that external bleaching with 35% hydrogen peroxide (Groups II–V) significantly increased the microleakage values of restorations compared to the unbleached group (Group I) [Table 3], which was in accordance with a study done by Han et al.[12] The presence of residual peroxide and oxygen postbleaching is the main reason for this increase in microleakage.[789] Titley et al.[22] suggested that the dentin and dentinal fluid acts as a free radical reservoir, which interferes with resin attachment and inhibits resin polymerization.

Table 3

One-way analysis of variance for intragroup and intergroup comparison

	Sum of squares	df	Mean square	Significant
Between groups	0.010	4	0.002	0.000
Within groups	0.001	45	0.000
Total	0.011	49

Dishman et al.[23] noted that resin tags in bleached enamel were short, sparse, poorly defined, structurally incomplete, or completely absent when restored. This explains the results displayed by Group II (immediate restoration) wherein the microleakage was the highest.

The peroxide ions decompose or are released through surface diffusion upon storage for 2–3 weeks, thereby reversing the structural changes caused by bleaching.[24] This particular finding was supported by the result obtained in Group III (delayed restoration) where the microleakage value had decreased and was similar to Group I (unbleached group). Thus, concluding that composite restorations done after a period of 2–3 weeks eventually eliminates any reduction in microleakage associated with bleaching. However, this is time-consuming for both the practitioner and the patient.[12]

Hence a quick and effective method to reverse the effect of residual free radicals is the application of antioxidants to the cavity. Therefore, this study is aimed to investigate the effect of a new antioxidant (Bromelain enzyme) on the microleakage of composite resin restorations after external tooth bleaching with 35% hydrogen peroxide.

In the present research, the authors noted significant differences (P < 0.05) when comparing microleakage in the unbleached control group to that of groups that had antioxidant treatment (Groups IV and V) [Table 4]. As reported by Han et al.[12] this suggests that antioxidants play an important role in decreasing microleakage when restorations are done immediately after bleaching.

Table 4

Post hoc multiple comparison test for comparing the different groups using Tukey’s test

Groups	Mean difference (I-J)	SE	Significant
Group 1
Group 2	−00.040500*	00.002021	00.000
Group 3	−00.005600	00.002021	00.059
Group 4	−00.012400*	00.002021	00.000
Group 5	−00.012600*	00.002021	00.000
Group 2
Group 1	0.040500*	0.002021	0.000
Group 3	0.034900*	0.002021	0.000
Group 4	0.028100*	0.002021	0.000
Group 5	0.027900*	0.002021	0.000
Group 3
Group 1	0.005600	0.002021	0.059
Group 2	−0.034900*	0.002021	0.000
Group 4	−0.006800*	0.002021	0.013
Group 5	−0.007000*	0.002021	0.010
Group 4
Group 1	0.012400*	0.002021	0.000
Group 2	−0.028100*	0.002021	0.000
Group 3	0.006800*	0.002021	0.013
Group 5	−0.000200	0.002021	1.000
Group 5
Group 1	0.012600*	0.002021	0.000
Group 2	−0.027900*	0.002021	0.000
Group 3	0.007000*	0.002021	0.010
Group 4	0.000200	0.002021	1.000

*P<0.05 significant. SE: Standard error

Moreover, in the present study, except for the unbleached group, the lowest microleakage values were observed in samples treated with antioxidants, such as sodium ascorbate (Group IV), or with bromelain enzyme (Group V).

However, there was no statistically significant difference between Group IV (sodium ascorbate) and Group V (bromelain enzyme).

Sodium ascorbate is a potent antioxidant capable of quenching the reactive free radicals. It neutralizes the effect of the residual oxygen layer, allows free radical polymerization of resin base materials by restoring the altered redox potential of the oxidized bonding substrate, thus reversing the compromised bonding.

The utilization of plant extracts as a viable alternative to chemical and synthetic antioxidant have been encouraging.[13] Bromelain enzyme is still a recent entity in restorative dentistry, and as yet, there has been only a single study that has been conducted to test its deproteinizing properties.[25] In the current study, the authors researched on its ability to function as an antioxidant. The antioxidant property of bromelain enzyme may be from the amino acid content, which can scavenge the radical by donation of the hydrogen atom.[14]

35% hydrogen peroxide (Pola office) was used for three applications of 8 min each. The reason for the gel to be refreshed is due to the fast degradation of peroxide. Studies have shown that hydrogen peroxide used for tooth whitening has a pH around 7 immediately after application, but when placed for a longer period, the pH of the gel decreases to 5, which may increase tooth sensitivity.[26]

In the present study, the results were recorded as a measure of absorbance of light. According to Beer–Lambert's law, the absorbancy of the solution is directly proportional to the concentration of absorbing species in the solution and the path length. Hence, it can be interpreted that the absorbance of the solution is directly related to the amount of microleakage. Thus, a UV spectrophotometer was used to calibrate the amount of microleakage.[27]

The study utilizes a dye extraction method to quantify the amount of microleakage instead of dye penetration study, as dye extraction provides reliable results because it measures all the dye taken up and gives a quantitative reading.[2829]

The future scope for in vitro studies utilizing an experimental antioxidant formulation such as the one described in this article includes the assessment of microstructure and bond strength characteristics of treated teeth. Further, it should be determined whether the higher microleakage rates that were observed in vitro subsequent to bleaching translate to significant deterioration of restorations in actual clinical situations and whether this deterioration can be reduced by using an effective antioxidant formulation. In addition, determining the minimum time required for antioxidant treatment after external tooth bleaching is necessary.

CONCLUSIONS

Microleakage increased significantly after external bleaching with 35% hydrogen peroxide and decreased when teeth were treated with antioxidants

Bromelain enzyme had similar efficacy in reducing microleakage as that of sodium ascorbate.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.