1 9 dimethylmethylene blue solution

NM and DM samples were weighed, freeze-dried and weighed again. The weights before and after freeze-drying were recorded to evaluate water content. GAG content was determined using a modified dimethylmethylene blue (DMMB) method as previously described (Farndale et al., 1986 (link)). NM and DM samples were lyophilised to a constant weight and then digested at 60°C in papain buffer (125 mg/mL papain, 5 mM cysteine/HCl, 5 mM disodium EDTA in PBS) for 12 h. The absorbance values of the samples were determined at 525 nm using an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, VT, United States) immediately after the addition of a 1,9-dimethylmethylene blue solution (Sigma-Aldrich). GAG content was calculated using a standard curve, which was made using different concentrations of chondroitin sulfate sodium salt (Sigma-Aldrich). Final values were expressed as μg of GAG per dry weight of sample. Collagen content was determined based on hydroxyproline (HYP) content, which was measured using a spectrophotometric method (Chan et al., 1998 (link)). The amount of HYP in the samples was then determined using a calibration curve prepared using HYP assay (Sigma-Aldrich), and total collagen content per mg dry weight of sample as calculated using a HYP-to-collagen ratio of 1:7.2.