Mechanical properties, water sorption characteristics, and compound release of grape seed extract-incorporated resins

Abstract Objective This study evaluated the effect of grape seed extract (GSE) incorporation on the mechanical properties, water sorption, solubility, and GSE release from the experimental adhesive resins. Material and Methods An experimental comonomer mixture, consisting of 40% Bis-GMA, 30% Bis MP, 28% HEMA, 0.26% camphorquinone and 1% EDMAB, was used to prepare four GSE-incorporated adhesive resins at concentrations of 0.5, 1, 1.5, and 2 wt%. The neat resin without GSE was used as the control. Six resin beams (25 mm x 2 mm x 2 mm) per group were prepared for flexural strength and modulus of elasticity evaluations using a universal testing machine at a crosshead speed of 1 mm/min. Five disks (6 mm in diameter and 2 mm in thickness) per group were used for microhardness measurements using a Leitz micro-hardness tester with Leica Qgo software. Five disks (7 mm in diameter and 2 mm in thickness) per group were prepared and stored in deionized water for 28 days. Water sorption, solubility, and GSE release in deionized water were calculated for each GSE-incorporated adhesive at the end of 28th day. Data was evaluated using one-way ANOVA and Tukey multiple comparisons. Results Flexural strength, modulus of elasticity and microhardness of GSE-incorporated adhesive decreased significantly with incorporation of 1.5% of GSE (p<0.05). Addition of GSE had no effect on the water sorption of the adhesive resins (p=0.33). The solubility of the resin also increased significantly with incorporation of 1.5% of GSE (p<0.05). Quantities of GSE release increased with increased concentration of GSE in the adhesive resin. Conclusion Up to 1% of GSE can be incorporated into a dental adhesive resin without interfering with the mechanical properties or solubility of the resins.

Mechanical properties, water sorption characteristics, and compound release of grape seed extract-incorporated resins Objective: This study evaluated the effect of grape seed extract (GSE) incorporation on the mechanical properties, water sorption, solubility, and GSE release from the experimental adhesive resins. Material and Methods: An experimental comonomer mixture, consisting of 40% Bis-GMA, 30% Bis MP, 28% HEMA, 0.26% camphorquinone and 1% EDMAB, was used to prepare four GSE-incorporated adhesive resins at concentrations of 0.5, 1, 1.5, and 2 wt%. The neat resin without GSE was used as the control. Six resin beams (25 mm x 2 mm x 2 mm) per group were prepared for flexural strength and modulus of elasticity evaluations using a universal testing machine at a crosshead speed of 1 mm/min. Five disks (6 mm in diameter and 2 mm in thickness) per group were used for microhardness measurements using a Leitz micro-hardness tester with Leica Qgo software. Five disks (7 mm in diameter and 2 mm in thickness) per group were prepared and stored in deionized water for 28 days. Water sorption, solubility, and GSE release in deionized water were calculated for each GSE-incorporated adhesive at the end of 28th day. Data was evaluated using one-way ANOVA and Tukey multiple comparisons. Results: Flexural strength, modulus of elasticity and microhardness of GSE-incorporated adhesive decreased significantly with incorporation of 1.5% of GSE (p<0.05). Addition of GSE had no effect on the water sorption of the adhesive resins (p=0.33). The solubility of the resin also increased significantly with incorporation of 1.5% of GSE (p<0.05).

Introduction
In the modern world, the use of tooth-coloured resin restorations has become very popular, resulting in an increased use of resin adhesives. However, these aesthetic bonded restorations are less durable than the traditional amalgam fillings 8 . The resin-dentine bonded interface is subjected to various mechanical forces, chemical or enzymatic challenges and deteriorates over time 24 . The ability of the bonded interfaces to resist these challenges will increase the longevity of the aesthetic bonded restorations 27 .
Several attempts have been made to increase the durability of bonded restorations either by modifying the resin or the dentine substrate 30 .
Complete infiltration of the demineralized collagen fibrils by adhesive resins is difficult to achieve 19 .
The unprotected collagen fibrils in the hybrid layer are exposed to enzymatic challenges by proteases either from exogenous or endogenous origins 18 .

Various matrix metalloproteinase (MMP) inhibitors
and collagen cross-linkers have been introduced as pre-conditioning agents to preserve the stability of the collagen and enhance long-term durability of the bonded interface 7 .
Recently, grape seed extract (GSE) has shown beneficial effects in the preservation of collagen fibrils.
Grape seed extract contains mostly proanthocyanidins.
Thus, GSE acts as a collagen cross-linker, MMP inhibitor, and remineralizing agent 3,4,22,28 . The use of GSE or proanthocyanidin as a pre-conditioning agent on dentine has improved the resin-dentine bond strength of both sound and caries-affected dentine 1 .
The effects of proanthocyanidin on the mechanical properties of dentine remain high up to three months, but decrease after six months 20 . Proanthocyanidin from GSE has been used as a pre-conditioning agent in various concentrations for durations of 10 minutes to 1 hour 1,3 . The prolonged application time is clinically impractical; hence, proanthocyanidin in the form of GSE has been incorporated in an adhesive resin to simplify the bonding procedures and to allow for a sustained release of GSE in the demineralized dentine over time 6 .
Several studies have incorporated GSE or proanthocyanidin in the adhesive resins and have shown beneficial outcomes 6,9 . The incorporation of 2.5% of GSE in an adhesive preserved the collagen matrix against collagenase stress for 6 days; by contrast, the collagen fibrils in the bonded interfaces formed by an adhesive without GSE were degraded 9 .
Studies have shown that up to 2% of proanthocyanidin from GSE can be incorporated into experimental etch-and-rinse adhesives without adversely affecting bond strength 6 . Therefore, before examining the effect of a GSE-containing adhesive on the durability of resin-dentine bonds, it is necessary to examine whether the incorporation of GSE into the adhesive resin and its subsequent release affects its mechanical properties. Hence, this study examined the effects of incorporation of low concentrations of GSE on the mechanical properties, water sorption, solubility, and GSE release of an experimental adhesive resin. The null hypotheses tested were that GSE incorporation has no effect on (i) the mechanical properties of the adhesive resins; (ii) the water sorption and solubility of the adhesive resins, and (iii) the rate of GSE release from the cured resins. Mechanical properties, water sorption characteristics, and compound release of grape seed extract-incorporated resins for 3x40 s. After curing, the resin specimens were removed carefully from the mold and checked under a stereomicroscope for the presence of air bubbles or cracks. Specimens containing voids or cracks were discarded. The specimens were then polished with 360-grit silicon carbide paper to smooth the rough edges. Six specimens were prepared from each group of dental adhesive resin containing 0, 0.5, 1, 1.5, and 2 wt% of GSE. The specimens were stored in dry conditions at 37°C for 24 h before testing.

Three-point bending test
Three-point bending test was performed using

Results
Modulus of elasticity, flexural strength, and microhardness  Water sorption and solubility Table 2 shows the mean and standard deviation of the water sorption and solubility of GSE-incorporated resins. One way ANOVA and Tukey multiple comparisons showed that GSE concentration had no effect on the water sorption of GSE-incorporated resins (p=0.33), but significantly affected (p=0.000) the solubility of the GSE-incorporated resins (p<0.001).

Proanthocyanidin release
The rates of GSE release from the GSE-incorporated adhesive resins are shown in Figure 1. No release of GSE was observed from the control resin. Two-way ANOVA showed that both "GSE concentration" and "storage time" affected (p=0.000) the GSE release rate (p<0.05). In general, after an initial burst of GSE release for 48 h, the mean release rate declined rapidly and reached a stable plateau after 5 days. The order of GSE release rate is 2%>1.5%>1%>0.5%.
The cumulative GSE release increased with the concentration of GSE in the adhesive resin and was the highest in the 2% GSE-incorporated adhesive ( Figure 2).

Discussion
The addition of a therapeutic material into dental adhesive resin can disturb its polymerization and affect the mechanical properties of the polymerized resin 2 .
Grape seed extract is a source of proanthocyanidins and, during our study, we have used GSE containing 90-95% of proanthocyanidins. Since our aim was to use the proanthocyanidin from GSE, we have used the GSE available with the highest concentration of proanthocyanidin for the laboratory studies. As GSE is a free radical scavenger, it is necessary to find an optimum GSE concentration for incorporation into dental resins, so as to maximize its cross-linking and protease inhibitory properties, with minimal adverse effects on resin polymerization properties.
However, it was difficult to select the appropriate GSE concentrations, since proanthocyanidin from GSE has been used in previous studies at a higher concentration as a pre-treatment agent 3   This study aimed to examine the possibility of incorporation of GSE into an adhesive resin to enhance the biomechanical properties of the dentine by providing sustained release of GSE over time.
Therefore, the quantity of GSE release from the GSE-incorporated adhesive resins over 28 days was Mechanical properties, water sorption characteristics, and compound release of grape seed extract-incorporated resins examined. Our results showed that incorporation of 2% of GSE into the resins has the highest amount of GSE release when compared to 0.5-1.5%. Thus, the third null hypothesis that GSE incorporation had no effect on the rate of GSE release from the cured resins was rejected. All the tested adhesives showed an initial burst of GSE release in the first 24 h, and this was previously reported to be due to the presence of microvoids and surface-bound drugs 14

Conclusions
Within the limits of this study, it may be concluded that: The incorporation of 0.5 to 1% grape seed extract into an adhesive resin had no adverse effect on its mechanical properties, water sorption, and solubility.
The rate of grape seed extract release increased with the concentration of grape seed extract incorporated into the adhesive resin.