An in vitro model of ageing of human articular cartilage sulphated-proteoglycans

Six cases of non-pathological articular cartilage were studied by organ explant culture to assess alterations in tissue sulphated proteoglycans (PGs) as a function of time in culture and donor age. Neosynthesized, 35SO4-labeled and endogenous, or already existing, uronic acid-containing PG populations were studied at several time points over 3-4 weeks. PG extractability did not vary with donor age. The proportion of non-extractable endogenous, but not of neosynthesized, PGs increased with time in culture. Sepharose CL-2B chromatography of neosynthesized and endogenous PGs eluted with associate buffer (0.5 M sodium acetate, pH 5.8) revealed 4 PG subpopulations with Kavs of 0.05, 0.28, 0.68 and 0.9-1.0. With culture time, the percentage distribution of newly synthesized PG subpopulations of large hydrodynamic size increased significantly with a concomitant decrease in the relative amount of smaller PGs. Isopycnic cesium chloride density gradients were performed on pooled Sepharose CL-2B peaks under associative (0.5 M GuHCl) and dissociative (4 M GuHCl) conditions to assess component subclasses of PG aggregates and PG monomers within each PG subpopulation. An analysis of the Kav, 0.05 subpopulation indicated an enrichment in dense PG aggregate (A1) and PG monomer (D1). Both A1 and D1 decreased with in vitro age parallelled by an increase in the respective subclasses of least buoyant density, A4 and D4. Sepharose CL-2B chromatography of D1 fractions within this PG subpopulation indicated a progressive decrease in PG monomer hydrodynamic size with time in culture. In contrast to these age-in-culture related alterations in neosynthesized PGs, the endogenous PGs showed neither a significant change in distribution of PG subpopulations nor PG subclasses over the time period of study. These findings showed the ability of human articular cartilage to alter the profile of neosynthesized PG while maintaining the in situ PG population during in vitro cartilage aging. Such findings suggest that this system may be useful in the elucidation of specific changes in articular cartilage PGs associated with time in culture.