Collagenase from clostridium hystoliticum

Dentin specimens were prepared as described above. After GAGs removal, dentin was washed and incubated with 100 μg/ml of collagenase from Clostridium hystoliticum (Sigma-Aldrich) in 0.2 M ammonium bicarbonate buffer (pH 7.9) for 24 hours at 37°C under agitation (Bedran-Russo, Castellan, Shinohara, Hassan, & Antunes, 2011 (link)) (n = 10). Collagenase solution was replaced after 1, 2, 4, 8 and 24 hours, and every day until complete degradation of specimens (up to 7 days of incubation). Collagen solubilization was estimated by hydroxyproline (HYP) release at each time point using a method previously described (Reddy & Enwemeka, 1996 (link)) with minor modifications. In brief, lyophilized collagenase solution was re-suspended in 10 μl of DW and 40 μl of 2 M sodium hydroxide and hydrolyzed by incubation at 120 °C for 1 hour. Then, solutions were incubated with chloramine T reagent for 25 min at room temperature followed by 1M Erlich´s reagent for 40 min at 65°C. Standard curve was done using 2 to 25 μg/ml of trans-4-hydroxy-L-proline (Sigma-Aldrich). Absorbance was read at 550 nm in a spectrophotometer (Spectramax Plus). HYP release was calculated according to dry weight of the specimens and expressed in μg/ml/mg dentin. Total HYP release was statistically analyzed by one-way ANOVA and Scheffe post-hoc test (α = 0.05).