Development of model in vitro culture systems for studying cartilage metabolism in health and disease

The generation of neocartilage grafts ex vivo shows promise for the repair of articular cartilage defects. This thesis describes (1) the metabolism and macromolecular organisation of grafts produced utilising a Transwell culture system at biochemical and microscopic levels, (2) the effect of chondrocyte de-differentiation, induced by monolayer expansion, on resultant graft tissue architecture, and (3) how addition of exogenous hyaluronan of varying molecular weights, during and post graft generation effect chondrocyte metabolism, with a view to explaining effects of hyaluronan administration in viscosupplementation procedures. Grafts generated from primary chondrogenic sources were of hyaline nature with distinct zonal stratification as found in native articular cartilage evidenced by cell morphology, matrix organisation and immunohistochemical composition. Graft tissue produced from immature sources was more synthetically active than those produced from mature sources. Passage expansion of chondrocytes in monolayer culture caused progressive reduction in graft thickness, loss of zonal architecture, and a more fibrocartilaginous tissue histology, consistent with a de-differentiating chondrocyte phenotype. Grafts subjected to exogenous hyaluronan of smaller molecular weight than that typically found in native aggrecan aggregates (500kDa-1000kDa) had an increased release of proteoglycan from graft tissue. Addition of hyaluronan with significantly higher molecular weight than endogenous hyaluronan resulted in an increased retention of proteoglycan within graft tissue, suggesting that high molecular weight hyaluronan in viscosupplementation procedures may be facilitating the retention of newly synthesised 'repair aggrecan' in pathological cartilage, slowing down the rate of cartilage destruction through loss of proteoglycan from the tissue. Futhermore, the addition of 490kDa (similar molecular weight to endogenous hyaluronan) showed a marked increase in the amount of aggrecan synthesised. Collectively, this data suggests that viscosupplementation procedures should incorporate a mixture of hyaluronan ranging between 490kDa-3000+kDa in order to provide optimal benefits of aggrecan retention and biosynthesis to osteoarthritic patients receiving this treatment modality