Mechanical-physicochemical properties and biocompatibility of catechin-incorporated adhesive resins

Abstract Several anti-proteolytic dentin therapies are being exhaustively studied in an attempt to reduce dentin bond degradation and improve clinical performance and longevity of adhesive restorations. Objectives This study assessed the effect of epigallocatechin-3-gallate (EGCG) on long-term bond strength when incorporated into adhesives. Material and Methods Adhesive systems were formulated with EGCG concentrations of 0 wt%: (no EGCG; control); 0.5 wt% EGCG; 1.0 wt% EGCG, and 1.5 wt% EGCG. Flexural strength (FS), modulus of elasticity (ME), modulus of resilience (MR), compressive strength (CS), degree of conversion (DC), polymerization shrinkage (PS), percentage of water sorption (%WS), percentage of water solubility (%WL) and cytotoxicity properties were tested. Dentin microtensile bond strength (µTBS) was evaluated after 24 h and again after 6 months of water storage. The adhesive interface was analyzed using scanning electron microscopy (SEM). Results No significant differences were found among the groups in terms of FS, ME, MR, CS and PS. EGCG-doped adhesives increased the DC relative to the control group. EGCG concentrations of 1.0 wt% and 0.5 wt% decreased the WS of adhesives. WL decreased in all cases in which EGCG was added to adhesives, regardless of the concentration. EGCG concentrations of 1.0 wt% and 0.5 wt% reduced cytotoxicity. EGCG concentrations of 1.0 wt% and 0.5 wt% preserved µTBS after 6 months of storage, while 1.5 wt% EGCG significantly decreased µTBS. SEM: the integrity of the hybrid layer was maintained in the 0.5 wt% and 1.0 wt% EGCG groups. Conclusion EGCG concentrations of 1.0 wt% and 0.5 wt% showed better biological and mechanical performance, preserved bond strength and adhesive interface, and reduced cytotoxicity.


Introduction
Immediate adhesive interface in dentin bond seems to be effective in dental restorations 1 , long-term bond strength values have been found to decrease significantly 2 . This decrease is due to the imperfect and degradable hybrid layer created by the current adhesive systems 3 . This degradation can be caused by factors, such as: the hydrophilic characteristics of infiltrated resin monomers 4 , and/or incomplete polymerization of infiltrated monomers, which can affect the chemical and mechanical stability of the hybrid layer 5 .

EGCG has antioxidant and anti-inflammatory
properties and is effective in inhibiting acid production in dental plaque bacteria; it also exhibits antimicrobial activity against Streptococcus mutans 11 . EGCG engages in hydrophobic interactions with collagenases and gelatinases and can modify the secondary structure of MMPs by inhibiting their activity 11,14 . In addition to its anti-proteolytic activity, EGCG is also known to promote collagen cross-linking through hydrogen bonding, thus improving collagen properties such as modulus of elasticity 9 . A hydrogen bonding interaction between EGCG and Bis-GMA hydroxyl groups may also occur. At concentrations higher than 2% w/w, EGCG has been shown to impair the degree of conversion of monomers 10 . EGCG has been shown to be a promising agent in the maintenance of long-term dentin bond strength 10, 13 . The incorporation of EGCG into adhesive systems is one of several clinical strategies that seek to preserve the longevity of composite restorations.

Microtensile bond strength (µTBS)
The study was approved by the Local Review Board (nº 11.794). Eighty sound human molars that had been extracted for therapeutic reasons were used in this study. Flat mid-coronal dentin surfaces were exposed by using water-cooled 450-grit aluminum Half of the teeth from each group was stored in distilled water at 37°C for 6 months before testing.
The other half was tested after 24 h of water storage to determine µTBS. The samples were sectioned into dentin-resin sticks (sections measuring approximately 1 mm), which produced 5 testing sticks per tooth. (25% for 20 min, 50% for 20 min, 75% for 20 min, 95% for 30 min and 100% for 60 min). Next, they were immersed in hexamethyldisilazane (Fluka) in a gas exhaust hood for 10 min. They were then placed on a filter paper under a glass bell for 30 min at room temperature to complete the dehydration process 8 Interactions were also significant (F=7.10; p=0.000).  The viability curves (in percentages) of FP7 cells in serial dilutions of the adhesives tested are presented in Figure 1. There were statistically significant differences between the cytotoxicity results of the adhesives tested (p=0.005). The 0.5 wt% EGCG and 1.0 wt% EGCG adhesives presented significantly higher cell viability when compared to the control adhesive in the case of the medium with 1% dilution ( Table 3).
The SEM analysis showed that all experimental adhesives were able to produce a hybrid layer with some resin tags inside dentinal tubules and a continuous thin layer of adhesive ( Figure 2). After 6 months of water storage, a crack was observed in the control group, and a gap was observed between the adhesive layer and the hybrid layer in the 1.5 wt% EGCG group. The 0.5 wt% EGCG and 1.0 wt% EGCG groups maintained the integrity of the hybrid layer with no failures or cracks (Figure 3).    Restorative material that is highly resilient can change, deform or flex dissipating incoming voltages and is, therefore, better able to help preserve the adhesive bond between the tooth and the restoration 31 .

Control
This dissipation preserves the adhesive interface and can support distortions that occur due to microscopic movements of dental substrates 32 , causing them to behave as a single body. Adhesives in which EGCG was incorporated did not differ statistically from the control sample in terms of modulus of resilience. In other words, the incorporation of EGCG did not affect the ability of the material to bend or deform, nor did it affect its ability to dissipate tensions occurring in the interface.  and also with collagen fibrils, avoiding bound water into collagen fibrils due to its lower hydrophilicity 24 .
The cytotoxicity analysis in this study showed that the 0.5 wt% EGCG and 1.0 wt% EGCG concentrations are less cytotoxic than the control adhesive.
Biologically, the hybrid layer can seal the toothrestoration interface and protect pulpal tissue 34 .
However, adhesives can release compounds, which can diffuse through the subjacent dentinal tubules and reach the pulpal tissue, a process which can have biological effects with toxic potential. Figure 1 shows that, at 10% dilution of the adhesives into the culture media, cell growth for all adhesives was less than 10% (0.1 cell viability).
However, at 1.0% dilution of adhesives into the culture media, 0.5 wt% EGCG and 1.0 wt% EGCG adhesives enabled more than 50% fibroblast cell growth, a result which is suggestive of low cytotoxicity for these adhesives. The high cell viability values for the 0.5 wt% EGCG and 1.0 wt% EGCG concentrations relative to the control group could be explained by the fact that, at certain levels, catechins have been found to have excellent biocompatible and chemopreventive properties; for example, they are able to protect normal cells against genotoxic effects 34 . On the other hand, the 1.5 wt% EGCG adhesive was incapable of providing the positive effects on fibroblasts observed in the 0.5 wt% EGCG and 1.0 wt% EGCG adhesives; its results were similar to those of the control sample. was not cytotoxicity to odontoblast-like cells and retained its anti-proteolytic activity after extraction from a dental copolymer, results which are consistent with those of this study. In another study 26 , EGCG was found to modulate secretion of various inflammatory and anti-inflammatory mediators in odontoblastic cells.
The authors analyzed smaller EGCG concentrations (2.5 to 160 µM) in cytotoxicity tests than those used in this study (0.5 wt%=10.9 mM; 1.0 wt%=21.8 mM; 1.5 wt%=32.7 mM). However, even at high concentrations, it should be argued that the EGCG does not show a relevantly cytotoxic behavior.

Conclusions
According to the results obtained, it can be concluded that: the incorporation of EGCG in experimental adhesive systems at concentrations of 0.5 wt% and 1.0 wt% produced adhesives with better biological and mechanical performance and that EGCG is therefore a potentially useful component in adhesives that offer long-term bond integrity.