Col-GAG and MC-GAG scaffolds were prepared using lyophilization, as described previously (3739). Briefly, microfibrillar, type I collagen (Collagen Matrix, Oakland, NJ) and chondroitin-6-sulfate (Sigma-Aldrich, St. Louis, MO) were combined in suspension in the absence and presence of calcium salts (calcium nitrate hydrate, Ca(NO3)2·4H2O; calcium hydroxide, Ca(OH)2; Sigma-Aldrich, St. Louis, MO) in an acetic acid (Col-GAG) or phosphoric acid (MC-GAG) solution. Using a constant cooling rate technique at a rate of 1°C/min, the solution was frozen from room temperature to −10°C using a freeze dryer (Genesis, VirTis). Following sublimation of the ice phase, scaffolds were sterilized via ethylene oxide and cut into 8-mm disks in diameter and 4 mm in height for culture.

Cross-linking of scaffolds was performed after rehydration in phosphate-buffered saline (PBS) for 4 hours using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC; Sigma-Aldrich) and N-hydroxysuccinimide (NHS; Sigma-Aldrich) at a molar ratio of 5:2:1 EDAC:NHS:COOH, where COOH represents the amount of collagen in the scaffold as we previously described (40). Scaffolds were washed with PBS to remove any of the residual chemical.