Dose and time effects of genipin on dentin collagen

Genipin (GE) is a naturally occurring cross-linker that can stabilize collagen matrix in various tissues. Recently, it has been shown that the GE treatment of dentin collagen improves dentin-resin bond strength. However, the optimal conditions of GE-induced cross-linking are not well established. Objective: To determine the effects of dose and time of GE treatment on stability, amino acids and lysyl oxidase (LOX)-mediated cross-links of dentin collagen. Method: Bovine dentin was pulverized, demineralized and the insoluble collagen was collected. The samples were then treated with 0.01%, 0.1% and 0.5% of GE for 30min, 1hr, 4hrs, 8hrs, 12hrs and 24hrs. After the treatment, an aliquot of each sample was digested with bacterial collagenase and the digestibility was assessed by hydroxyproline analysis. The rest of the samples were reduced with NaB³H₄, hydrolyzed with 6N HCl and subjected to amino acid and cross-link analysis by HPLC. Result: The results demonstrated that GE-treated collagen became insoluble against collagenase digestion in a dose- and time-dependent manner. The major amino acids that were diminished with GE treatment were lysine and hydroxylysine residues and the decreases were dose- and time-dependent. The cross-link analysis showed that GE treatment generated NaB³H₄, reducible compounds but the LOX-mediated collagen cross-links including both immature and mature cross-links were not affected. Conclusion: The GE treatment stabilizes dentin collagen likely by forming lysine/hydroxylsyine-based cross-links some of which are reducible with NaB³H₄.