Effect of Tannic Acid Application on Durability of Bond of Etch and Rinse Adhesive Resins

Introduction

Use of adhesive resin systems is a mandatory step for restoring teeth with tooth-colored restorative materials. Although improvement in adhesive systems has established effective bond to tooth structure, the quality of bond becomes unstable and degrades over time, particularly in dentin (1). The main reasons for this reduction in bond include: hydrolytic degradation via water sorption, incomplete penetration of resin monomers, collagenolytic activity of matrix metalloproteinase (MMPs), Cystein and Cathepsin enzymes (2,3). MMPs are proteolytic enzymes with cellular origin consisting of 26 members. Dentin possesses endogenous MMPs which regulate its physiologic and pathologic metabolism (4,5). Since resin cannot penetrate to the whole depth of acid etched dentin, some collagen fibrils at the base of the hybrid layer remain exposed. These fibrils can be degraded by MMPs activity and hydrolysis process, so bond durability may decrease (6,7). Three main strategies have been suggested in order to inactivate MMPs or improve bond durability including application of MMP inhibitors (8,9), collagen cross linkers (10,11) and drying agents (12). Inhibitors decrease aging of bonding substrates and improve stability of collagen fibrils in the hybrid layer, thereby improving bond durability (7,13). They can act via binding to the active site of catalytic domain and changing the pro-enzyme molecular structure, inhibiting enzymes’ catalytic activity or through Ca and Zn ions chelating (cation chelating mechanism), like tetracycline and doxycycline (14-16).  One of the most important MMP inhibitors is chlorhexidine which is well known as a disinfectant and serves as a great MMP inhibitor as well (13,17). Cross linking agents such as Glutaraldehyde, Proanthocyanidin and tannic acid improve mechanical properties and increase modulus of elasticity and tensile strength of demineralized dentin by inducing more exogenous cross links (10). They change collagen fibers’ structure and stabilize them against degradation (2,18). However, some disadvantages have been mentioned for cross linkers. Synthetic cross linkers like Glutaraldehyde or Carbodiimide have high levels of cytotoxicity. Moreover, their long term stability is indistinct. On the other hand, natural cross linkers have fewer shortcomings (2). Tannic acid (TA) is a commercial form of Tannin, a polyphenol with weak acidity. TA was previously used as a vasoconstrictor to inhibit bleeding of gingival tissue and also as a dentin acid conditioner. However, it was only capable of removing smear layer and not demineralizing dentin. TA binds to collagen amid groups via its hydroxyl group, rendering fibrils more stable and resistant against MMPs (19,20).

The aim of this study was to evaluate the effect of different concentrations and application times of TA on bond strength and bond durability of a total etch adhesive resin. The null hypothesis of this study is that different concentrations and application times of tannic acid (TA) have no significant effect on immediate and two-month bond strengths.

Methods and Materials

180 human third molar teeth without caries and cracks were selected. After debridement, the teeth were stored in normal saline solution (Shafa, Iran) for 1-2 weeks in standard room temperature (25 ^c). The solution was renewed every week. Then the samples were vertically mounted in self-cure acrylic resin blocks (Acropars, Iran) from 1 mm below CEJ. The enamel of occlusal surfaces were removed and then dentin surfaces were gently ground flat with 400 and 600 grit silicon carbide sand papers under running water. The samples were randomly divided into 10 groups of 18. In the first group (control), dentin surfaces were etched by 15% phosphoric acid (Ultradent, USA) for 15 seconds and washed out for 15 seconds. Then, adhesive (PQ1, Ultradent, USA) was applied according to the manufacturer’s instructions and polymerized using a LED curing unit (Blue Phase C8, IvoclarVivadent, Schaan, Lichtenstien) at 800mW/cm² for 20s. Consequently, one transparent plastic tube with 1.5 mm diameter and 4mm length was carefully placed on the dentin surface. Resin composite build-up was accomplished using one increment of 1mm and two 2-mm increments with Z250 (Shade A1, 3M ESPE; St Paul, MN, USA), each increment being light cured for 40s. Specimens were post-cured from each surface for 40s. In other experimental groups, all procedures were the same as the control group except application of the solution before using adhesives. In the second group (positive control), aqueous solution of 2% CHX (Consepsis, Ultradent, USA) was applied on the dentinal surface with micro brush (Pegasus, Astek Innovations, England) removing the remnants after 30 seconds with a clean micro-brush.  (2, 18). In the third (TA20) and forth (TA30), 20% and 30% TA solutions were applied respectively. These groups were divided into four subgroups according to time of TA applications (30 s, 1 min, 3 min and 5 min). The remnants of TA were removed with a clear micro-brush.

In half of the samples of each experimental group, the shear bond strength test (SBS) was performed after 24 hour storage in 100% humidity by using Universal Testing Machine (Zwick/Z250, Type KAP-Z, Zwick Roell Group; Ulm, Germany( at crosshead speed of 1 mm/min. According to the time of evaluating bond strength samples were divided into 2 subgroups.  Micro tensile bond strength test was done on half of the specimens after 24 hours from preparation. In other samples, SBS was evaluated after two month storage in collagenase solution (Cholestridium Histoliticum, 125 U/mg, Sigma Aldrich, USA). The solution was renewed every week. Failure modes were determined at 40X magnification using a stereomicroscope (LEO, 1450 UP, Zeiss; Oberkochen, Germany) and recorded as adhesive, cohesive in enamel, cohesive in composite or mixed. Data were processed using SPSS version 11.5 software (SPSS Inc. Chicago, IL). Normal distribution of the data was confirmed using Kolmogorov-Smirnov test, and data were analyzed using One-way ANOVA and Duncan tests with the level of significance set at 0.05.

Results

The mean values of SBS are presented in Table 1. There was no significant difference in immediate bond strength among experimental groups (P-value= 0.2). However, after 2 months storage in collagenase solution, a significant difference was found among groups.  Bond strengths of CHX and 20% TA applied for 3 minute, were significantly higher than the control group. Failure assessment is summarized in Table 2. Adhesive failure was the most observed failure mode in all experimental groups.

Table 1. The mean value of SBS of experimental groups (MPa). Values with different uppercase letters denote statistically-significant difference at P < 0.05.

Table 2. Distribution of Failure modes in experimental groups