Collagen production by chondrocytes in alginate bead culture

The thesis addresses the question "Do alginate beads provide an environment similar to that of cartilage for the culture of chick chondrocytes?" and answers were obtained using a range of biochemical and other techniques to compare the ECM constituents produced in alginate to those observed in vivo. The results show that, by most criteria, the alginate system provides an environment similar to that in vivo: cartilage-specific collagens (II, IX, X and XI) were produced, with ratios analogous to those in vivo, and collagen IX present in both proteoglycan and non-proteoglycan forms. Moreover, sulphated glycosaminoglycans were retained in the cell-associated matrix over a 14 day culture period, an observation that suggests proteoglycan aggregation. Two major differences were however noted between alginate culture and cartilage. First, collagen failed to assemble into fibrils, but instead formed segment-long-spacing (SLS) crystallites, a form normally found when collagen assembles under low pH conditions, and here the failure in assembly was shown to be due to the presence of alginate. Second, a substantial proportion of procollagen was not fully processed to collagen, leaving high amounts of a precursor which retains the N-propeptide (pN II). Substitution of 10% fetal bovine serum (FBS), which contains proteinase inhibitors, with insulin in the culture medium decreased pN II concentrations (relative to collagen II) but also decreased total collagen production. The enzyme procollagen N-proteinase (107 kDa), which cleaves N-propeptides from pN-collagen I, was active on pN II when a mixture of ECM components was present but was not active on a purified pN II substrate. This suggested that the enzyme requires a co-factor for activity on pN II, but preliminary investigations failed to identify it. This thesis thus shows that alginate beads provide a good environment for studying chondrocyte differentiation and ECM production, but not for morphological studies.