Salivary Esterases Dramatically Reduce Collagen Biostability Promoted by Galloylated Polyphenols

Objectives: To evaluate the effect of salivary esterases on dentin collagen biostability promoted by galloylated polyphenols.
Methods: Human third molars were sectioned into 6-micron dentin films which were completely demineralized (10% phosphoric acid). The resulting collagen films (n=6 x 12 films) were treated by 2% crosslinkers: galloylated natural polyphenols (epigallocatechin gallate [EGCG] and tannic acid (TAC)], non-galloylated polyphenol (epigallocatechin [EGC]) and chemical crosslinker N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride with N-hydroxysuccinimide (EDC/NHS-60s). Films were treated for 60 s followed by extended rinse (EtOH for 30 min) to remove the unbound crosslinker. One EDC/NHS group for 1h treatment was also tested (EDC/NHS-1h). Half of the films were submitted to incubation in human saliva for 48 h to induce hydrolysis by salivary esterases. Then, all films were subject to collagenase degradation (n=6 x 6 films). The collagen biostability against collagenase was assessed by weight loss % (WL) and hydroxyproline release in µg/mg film (HYP). Endogenous MMPs’ activity was assessed by confocal laser scanning microscopy. Data were analyzed via two-way ANOVA and Games Howell’ test (α=0.05).
Results: The galloylated polyphenols exerted higher collagen biostability without saliva incubation (TAC: WL 9.4±2.8 and HYP 4.3±1.5; EGCG: WL 41.4±5.9 and HYP 6.32±0.6) as compared to untreated control and other crosslinkers (p < 0.001). However, once exposed to saliva incubation a dramatically reduced collagen biostability was detected (TAC: WL 40.0±5.5 and HYP 9.52±1.4; EGCG 67.6±5.1 and HYP 41.43 ± 10.2), p < 0.05. Similar results were observed in MMPs activity (p < 0.001).
Conclusions: Hydrolysis of ester bonds in galloyl motifs by salivary esterases reduce collagen crosslinking and biostability enhanced by galloylated polyphenols.