Effect of protease inhibitor specificity on dentin matrix properties.

Abstract

To evaluate protease activity of dentin matrices subjected to treatment with non-specific (chlorhexidine - CHX), cysteine cathepsin specific (E-64), and cysteine cathepsin-K (CT-K) specific (Odanacatib - ODN) inhibitors. Pulverized dentin powder obtained from human dentin disks (0.5 mm thickness) completely demineralized with 10% H₃PO₄ were challenged in 1 mL lactic acid (LA) (0.1M, pH 5.5) or stored in deionized water for 30 min. Aliquots of dentin powder were then immersed in 1 mL of CHX (2%), E-64 (10 μM and 20 μM) or Odanacatib (0.2 nM and 1 μM) for 30min. Degradation of dentin collagen was determined by telopeptide assays measuring the sub-product release of C-terminal cross-linked telopeptides (ICTP) and C-terminal peptide (CTX) in incubation media, which correlates with matrix metalloproteinases (MMP) and CT-K activities respectively (n = 3). The ICTP and CTX data were normalized to concentration of total protein (ICTP_tp and CTX_tp) in the media, measured by bicinchoninic acid assay. Dentin matrix properties were also measured by gravimetric change (n = 8) and ultimate tensile strength (UTS) (n = 10). Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test and independent t-test (α = 5%). Telopeptide assays showed significantly lower CTX_tp values after treatment with E-64 and Odanacatib. E-64 and Odanacatib at all tested concentrations significantly reduced the release of ICTP_tp. Gravimetric analysis showed no significant difference between the tested inhibitors and control except for CHX after lactic acid challenge. UTS results showed significantly higher values for E-64 (20 μM) and Odanacatib (0.2 nM and 1 μM) groups in deionized water. Dentin therapies targeting enzymes such as CT-K by specific inhibitors may provide superior pharmacokinetics and optimum efficacy due to precise protein binding, consequently limiting collagen degradation directly or indirectly by enzyme related pathways.