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).