Biochemical Quantification of Accumulated Tissue
After harvest, constructs were cut out of the culture inserts, blotted dry, weighed (wet weight), digested by papain (40 µg/mL in 20 mM ammonium acetate, 1 mM EDTA, and 2 mM DTT) for 48 hours at 65°C, and stored at -20°C until analysis. Aliquots of the digest were assayed separately for proteoglycan, collagen and DNA contents. The proteoglycan content was estimated by quantifying the amount of sulphated glycosaminoglycans using the dimethylmethylene blue dye binding assay (Goldberg & Kolibas, 1990). Collagen content was estimated from the determination of the hydroxyproline content. Aliquots of the papain digest were hydrolyzed in 6 N HCl at 110ºC for 18 hours and the hydroxyproline content of the hydrolyzate was then determined using chloramine-T/Ehrlich’s reagent assay (Woessner, 1961). Collagen content was estimated assuming hydroxyproline accounts for 10% of the total collagen mass in cartilage (Heinegard et al. , 1998). The DNA content was also determined from the papain digest using the Hoechst dye 33258 assay (Kim et al. , 1988).