Comparison of Bromelain Enzyme, Sodium Hypochlorite, and Titanium Tetrafluoride on Shear Bond Strength of Restorative Composite to Dentin: An in vitro Study.

STATEMENT OF THE PROBLEM: Sufficient bond strength of composite restoration leads to its durability and survival; therefore, preparation of dentin surface for higher bond strength is essential.
PURPOSE: Our aim is to assess the deproteinizing effect of 3% bromelain enzyme and compare it to 4% titanium tetrafluoride (TiF4) and 5% sodium hypochlorite (NaOCl) regarding the shear bond strength (SBS) of composite resin to dentin. MATERIALS AND
METHOD: In this experimental study, 40 intact extracted human maxillary premolars were selected, and the occlusal surfaces of the teeth were sectioned at a depth of 2 mm from dentinoenamel junction. The teeth were divided into 4 groups (n=10). In Group 1, the teeth were etched with 37% phosphoric acid gel. In Group 2, the teeth were etched and deproteinized with 5% NaOCl. In Group 3, the teeth were etched and deproteinized with 4% TiF4. In Group 4, the teeth were etched and deproteinized with 3% bromelain enzyme. In each specimen, composites with 3 mm diameter and 2 mm height were prepared and cured. The test specimens were then stored in distilled water at room temperature for 7 days before conducting the SBS test (MPa). By universal testing machine at a crosshead speed of 1 mm/min, the results were analyzed using one-way ANOVA and Tukey test.
RESULTS: One-way ANOVA test demonstrated that pretreatment of dentin with a bromelain enzyme, TiF4 solution, or NaOCl was not statistically different regarding SBS to dentin (p= 0.790).
CONCLUSION: 3% bromelain enzyme can be as effective as TiF4 and NaOCl and phosphoric acid 37% in terms of the SBS of composite resin to dentin. Copyright: © Shiraz University of Medical Sciences.

Introduction

Hybridization is a process in which a hybrid layer is formed on dentin surface through bonding phenomenon[1]. Hybrid layer is formed through demineralization of dentin as well as infiltration of resin monomers into collagen fibrils, and emerged after dentin demineralization[2]. To enable effective hybrid layer formation, smear layer should be removed prior to dentin surface bonding[3]. Different materials have been introduced for dentin pretreatment before adhesive application[4].

Irrigating solutions used during the bonding procedure may lead to alteration in the chemical and physical properties of dentin[5]. These materials react with organic tissue leading to change of the chemical structure and mechanical properties of dentin[6]. Sodium hypochlorite (NaOCl) has been used on dentin as a non-specific deproteinizing agent[7]. Given that NaOCl is cytotoxic, scientists seek for a new and ideal substance to deproteinize dentin[8-9]. It has been reported that application of 5% NaOCl causes a significant decrease in the shear bond strength (SBS) of nano-filled composite resins, and NaOCl has negative effects on bond strength[4]. Another study found that, there was not any statistically significant difference between control group and NaOCl-treated group[8].

Fluoridated materials can enhance dentin remineralization and decrease secondary caries formation[10]. Fluoride mainly affects longevity of restorations by inducing degradation of unprotected collagen. Titanium tetrafluoride (TiF4), a fluoridated product, has been suggested as an effective factor against dental caries formation[11-13]. It has been indicated that a tenacious titanium-rich coating is readily formed after application of TiF4 solution to enamel and cementum[14]. However, the use of TiF4 solution as a smear layer-modifying agent is a recent development, and further research is needed to approve its feasibility[15]. Different studies using TiF4 as pretreatment have reported different results [4,7,16]. It has been concluded that application of TiF4 on dentin does not significantly affect the micro-tensile and SBS of the composite resin[10,13].

Deproteinizing enzymes such as collagenase and bromelain enzyme are newer techniques to remove collagen network[17]. Bromelain is a proteolytic enzyme commercially obtained from the fruit or stem of pineapple. The function of proteases is to catalyze the hydrolysis of proteins to amino acids[18]. Bromelain is commonly used in medical supplements. Bromelain can act similarly to non-steroidal anti-inflammatory drugs and has been demonstrated to have fewer side effects. In Europe, it is orally or topically used for post-surgery wounds, trauma, and deep burns[19]. One study revealed that removal of unsupported collagen fiber with bromelain enzyme, after acid etching, improves bond strength[8].

There is insufficient information about the effect of 4% TiF4 and 3% bromelain enzyme[20] on the SBS of composite resins, and there was no comparison between these agents and 5% NaOCl. Therefore, we conducted this study to compare the SBS of restorative composites to dentin deproteinized by 5% NaOCl, 4% TiF4, and 3% bromelain enzyme.

Materials and Method

Sample

In this experimental study, forty intact extracted human maxillary premolars were selected and used with the approval of the Ethics Committee of Shiraz University of Medical Sciences, Shiraz, Iran (IR.SUMS. REC. 1395.S1191). They were completely sound premolars without any caries, cracks, and fractures. The teeth were hand-scaled and stored in 0.5 % thymol solution (Merck-Germany). Middle dentin was exposed by sectioning the crowns at a depth of 2 mm from dentinoenamel junction (DEJ) parallel to the occlusal surface, under water coolant, using a diamond disk (SP, Brazil)[21].

The roots of the sectioned teeth were embedded in acrylic resins of 1.5 cm diameter and 2.5 cm height, 1.0 mm below the cementoenamel junction. Then, the occlusal surfaces of the teeth were polished with a 600-grit silicon carbide paper (Starcke, Germany) under constant water spray to homogenize the dentin surface[21-22]. The teeth were randomly assigned to four groups of ten teeth (n=10) based on the dentin deproteinizing method:

For group 1, samples were etched for 15 seconds with 37% phosphoric acid gel (condac37; FGM, Brazil), rinsed for 15 seconds with water, and gently air dried so that a surface with a brilliant appearance to achieve wet-bonding remained on the dentin. Two consecutive coats of Adper single bond (3M, ESPE, St.Paul, USA) was gently applied on the dentin surface by a disposable micro-brush. Subsequently, it was gently dried for 2-5 seconds and light-cured for 10 seconds (according to the manufacturer’s instructions). Next, the nanofilled composite resin (Z350; 3M, ESPE, USA) was embedded in a Teflon mold of 3 mm diameter and 2 mm height over the central part of the prepared dentin. Later, we put a transparent matrix band on the occlusal surface of the composite and finger pressed it. Finally, additional composite was removed by the explorer.

For group 2, the procedure was similar to group 1 except that in this group, after acid application, 5% NaOCl (Merck, Germany) was actively applied by syringe for 2 minutes on the prepared surface. Next, the NaOCl-treated surface was thoroughly rinsed for 60 seconds. The rest of the procedure continued in the same way as group1.

For group 3, the procedure was similar to group 1; however, after acid application, 4% TiF4 (wt/v; PH: 1.2) (sigma Aldrich-USA) achieved by dissolving TiF4 in deionized distilled water by chemist, was actively applied by a disposable micro-brush for 1 minute. We then thoroughly rinsed the TiF4 treated surface for 30 seconds.

Finally, for group 4, the procedure was similar to group 1; however, after acid application, 3% bromelain enzyme (Salamat Parmoon Amin manufacturer, Iran) achieved by dissolving bromelain powder in deionized distilled water by chemist, was applied by a disposable micro-brush for 1 minute. Then, we thoroughly rinsed the bromelain treated surface for 30 seconds. The rest of the procedure continued in the same way as group 1.

In each specimen, the prepared composites were cured for 40 seconds (according to the manufacturer’s recommendation) by a halogen light polymerizing unit (Monitex Blue Luxcer M855) with the light intensity of 600 mW/cm2 as the device tip was perpendicular to composite surface.

The specimens were then stored in distilled water at room temperature for 7 days before being subjected to the SBS test (MPa). The shear bond test was performed at a cross-head speed of 1 mm/min and calculated in MPa, using universal testing machine (Zwick-Roell; Z020, Germany) (Figure 1). The results were analyzed using one-way ANOVA and Tukey test.

Figure1

A sample in universal testing machine

Statistical analysis

Statistical analyses were conducted using the SPSS software (Version 19, Chicago, IL, USA). Values are presented as mean ± SD, and group differences were calculated by the parametric one-way ANOVA test (p< 0.05). The p value was 0.790 in our result, and there were no statistically significant differences between our groups; therefore, based on the statistical rules, there was no need for Tukey HSD test for the two-by-two comparisons of the groups.

Results

Table 1 demonstrates the mean and standard deviation as well as p-value of the SBS of composites to dentin, and Figure 2 shows mean±SD. One-way ANOVA test showed no statistically significant differences between th e groups (p= 0.790±SD). Therefore, bromelain, despite its low concentration, is as effective as the other three groups and the SBS was statistically similar in all the groups.

Table 1

Mean and SD of SBS (MPa) of all four tested groups and p-value between groups (n=10)

Groups	Mean±SD	p Value
37% H3PO4	15.45±3.55	0.790
37% H3PO4+ NaOCl	15.35±4.49
37% H3PO4+TiF4	17.01±4.85
37% H3PO4+Bromelain Enzyme	16.27±3.53

Figure2

Maximum and minimum of SBS of all groups

Discussion

In our study, NaOCl was used prior to bonding procedures. Previous studies indicated that after using it in the treatment of dentin, an increase in wettability was expected due to the hydrophilic surface produced by collagen removal[23-24].

It has been indicated that dehydration of collagen fibrils exposed by acid etching is associated with decrease in dentin matrix. These results are owing to incomplete infiltration of resin monomers into dentin. In other words, reduction occurs in dentin bond strength[25] Thus to preserve the collagen fibrils expanded prior to adhesive application, it is necessary to maintain the dentin moist[26].

Sharafeddin et al.[4] reported that using 5% NaOCl caused a significant decrease in the SBS of dentin to both nano-filled composite resin and silorane-based composite resin. Previous studies demonstrated that the use of 5% NaOCl significantly decreased the SBS of single bond and silorane adhesive to dentin[4,27-28]. In our study, the application of NaOCl was prior to etching. Moreover, in the previous study, the adhesive system for silorane-based composite resin was self-etch, but we used the total etches adhesive system. Decrease of bond strength using NaOCl in the previous study can be explained by production of oxygen after NaOCl decomposition into NaCl and O2[29-30]. The released oxygen in this chemical reaction prevents the polymerization of adhesive agents. These reactive residual free radicals in the NaOCl-treated dentin compete with the propagation of the vinyl-free radicals generated during light activation of the adhesive system, causing minimum infiltration of resin monomers and incomplete hybrid layer formation[4]. There is another possible explanation of lower bond strength, which is based on the deep or surface dentin treatment. Uceda-Gomez et al.[7] reported that the lower bond strength values were obtained in deep dentin rather than surface dentin. They attributed this phenomenon to smaller amount of intertubular dentin in deep dentin. In our study, deep occlusal substrate had much less intertubular dentin to penetration by resin tag; therefore, there is not a firm bonding to the tubule wall in this location. Hence, bonding efficacy was not seen in deep dentin.

The present study indicated that pretreatment with NaOCl resulted in the statistically similar SBS of the composites compared to the control group. Our result was in agreement with one study by Chauhan et al.[8]. They reported that the teeth, etched with 37% phosphoric acid for 15 seconds, rinsed with water, blot dried, and deproteinized with 5% NaOCl did not have statistically significant differences in SBS compared to the similar group without 5% NaOCl treatment.

One study conducted by Cecchin et al.[31] indicated that the use of 1% NaOCl alone, compared to dentin, led to higher tensile bond strength of the adhesive system. NaOCl (1%) was applied to dentin every 5 minutes for 1 hour. However, the solution concentration and the application time were different from those of our study, which may have caused different results.

It seems that NaOCl may affect bond strength differently, based on the chemical structure of the adhesive system, NaOCl concentration, and the type of the initiator in the adhesive system used.

Application of TiF4, as a dentin-pretreatment, was previously proposed in some studies[16,30]. In the study conducted by Bridi et al.[13], dentin was treated with a 2.5% TiF4 solution for 1 minute. They evaluated the micro-tensile bond strength of one- or two-step self-etching adhesive systems, and results demonstrated that pretreatment of dentin with a TiF4 solution did not affect the bond strength of either of the adhesive systems.

Application of TiF4 in the present study resulted in similar SBS to the control group. Our findings were in line with the study conducted by Devabhaktuni and Manjunath[10]. They applied TiF4 to dentin before and after phosphoric acid conditioning, and application of TiF4 had no influence on bond strength to dentin.

In addition, in the study conducted by Sharafeddin et al.[4], the authors concluded that the effect of 4% TiF4 on the nano-filled composite’s SBS to dentin was not statistically significant compared to the control group.

It is well known that low pH (about 1.2) of TiF4 is suitable to attach titanium to oxygen atom of the phosphate group[5]. This process causes formation of a titanium dioxide glaze-like layer on the surface to produce a globular titanium-rich surface layer[29,32]. This layer protects dentin demineralization from highly acidic pH (approximately 1.2) of the TiF4 solution. TiF4 was actively applied with a brush for 60 seconds[15]. One of the erosions inhibiting characteristics of TiF4 depends on its method of application on dentin. It seems that application with a micro-brush leads to surface wear rather than allowing the formation of the glaze-like surface layer[25]. This is the reason why 4% TiF4 pretreatment had no effect on SBS.

The application of 3% bromelain enzyme resulted in similar SBS to the control group. This result differs from the result of the study by Chauhan et al.[8]. They used the same procedure to determine effectiveness of bromelain enzyme pretreatment composite resin on SBS; however, there were some differences in type of incubation and bromelain enzyme concentration. Firstly, they used the teeth occlusal surfaces. Secondly, they did not mention the amount of enzyme concentration. In our study, however, we sectioned the teeth occlusal surfaces at a depth of 2 mm from DEJ. Moreover, we used 3% bromelain enzyme by a disposable micro-brush.

In another study, Dayem and Tameesh[17] assessed the deproteinizing effect of bromelain enzyme. Application of bromelain enzyme led to collagen network removal and significantly decreased the global leakage scores of the adhesive system. This is due to the ability of bromelain enzyme to efficiently remove the collagen network from acid etched dentin. This, subsequently, will lead to an increase of diffusion potential of the monomer to the intact dentin minimizing nano-leakage. The difference of their results from those of our study is due to some differences in methodology. Firstly, their method of evaluation was scanning electron microscopy. Secondly, dentin cavity preparation was on buccal and lingual surfaces. Moreover, they stored the teeth in 50% ethanol at 8ºc for 1 month.

There were only few studies assessing the effect of bromelain enzyme on the bond strength of composites to dentin since bromelain is a new material in restorative dentistry and is under investigation. Although application of these three solutions (NaOCl, TiF4 and bromelain enzyme) did not result in destruction of the etched dentin surface, it did not have any effect on the increase of SBS. It seems that preparation steps, type of adhesive systems, deproteinization with TiF4, and bromelain enzyme affect the SBS of composite resins. Further studies should be conducted to evaluate the effect of different concentrations of bromelain enzyme and TiF4 on deep and surface dentin. Moreover, Future studies should be considered with no acid etching.

Conclusion

Considering the limitations of this in vitro study, it can be concluded that, the pretreatment of premolar dentin with NaOCl, TiF4, and bromelain enzyme leads to equal SBS of composite resins. In other words, bromelain enzyme, a safe solution without any side effects, can successfully remove collagen network as effective as TiF4 or NaOCl, after acid etching.