Effect of Protease Inhibitor Specificity on Dentin Matrix Properties

Objective: 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.

Methods: Pulverized dentin powder obtained from human dentin disks (0.5 mm thickness) completely demineralized with 10% HPO 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 and CTX) 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%).

Results: Telopeptide assays showed significantly lower CTX values after treatment with E-64 and Odanacatib. E-64 and Odanacatib at all tested concentrations significantly reduced the release of ICTP. 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.

Significance: 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.