2.6.5. Glycosaminoglycan (GAG) Quantification

GAG quantification was performed using papain digestion. Triplicates of each condition collected at each previously established time point were tested. The samples were collected and stored in eppendorf tubes at −80 ◦C until further analysis. Digestion solution was prepared by adding papain (Sigma Aldrich, Saint Louis, MO, USA) and *N*-acetyl cysteine (Sigma Aldrich) at concentrations of 0.05% and 0.096%, respectively, to 50 mL of digestion buffer (200 mM of phosphate buffer containing 1 mM EDTA (Sigma Aldrich), pH 6.8). Each specimen was incubated with 600 μL of digestion buffer, overnight at 60 ◦C. After a 10-min centrifugation at 1300 rpm, the supernatant was collected. Dimethymethylene Blue (DMB) stock solution was prepared dissolving 16 mg of DMB powder in 900 mL of distilled water containing 3.04 g of glycine and 2.73 g of NaCl. pH was adjusted to 3.0 with HCl and the volume adjusted to 1 L. The solution was stored at RT covered by aluminum foil. Chondroitin sulphate (Sigma, C8529) solution was prepared in water in a 5 mg/mL stock solution and kept refrigerated. Standards were prepared from serial dilutions of this solution. Three samples per condition were considered per time point, and the absorbance of each sample was measured at 530 nm using a microplate reader.
