Papain type 3

Collagen and GAG content analysis was performed on all the tissue specimens. Collagen quantifications of the tissue samples were conducted according to the manufacturer protocol (Sigma, USA). The samples (n = 3 per processing group) were homogenized in 100 μl of water and then added to 100 μl of 12 N HCl and hydrolyzed at 120 °C for 3 hr. A 50 μl of the hydrolyzed sample was placed on a 96‐well plate and dried at 60 °C. The samples were incubated at room temperature for 5 min after a 100 μl chloramine T/oxidation buffer mixture (94:6) was added. The samples were incubated again at 60 °C for 90 min after 100 μl of diluted DAMB reagent was added. Once the samples reached room temperature, their absorbance was measured at 560 nm and compared with the reference curve obtained from hydroxyproline solution. Collagen quantification data were expressed in terms of hydroxyproline. To quantify the GAG content, a GAG assay kit (Blyscan, Biocolor, USA) was utilized following the manufacturer's recommendations. The samples (n = 4 per processing group) were lyophilized and digested in papainase buffer containing papain type III (Worthington Biochemical, USA). Supernatants were collected by centrifugation. The sample was evaluated at 656 nm absorbance and compared with the reference curve using known GAG standard solutions.