The primary hypothesis is that during disc degeneration there is a loss of normal phenotype by the cells of nucleus pulposus or anulus fibrosus and expression of a phenotype characterized macroscopically as a """"""""fibrotic replacement"""""""". This may be reversible, with changes in the mechanical and/or biochemical environment resulting in loss of phenotype, or the changes may be an irreversible commitment to a fibroblast-like phenotype. Understanding the mechanism of initial fibrotic changes is key to developing strategies of intervention. Degeneration will be evaluated by quantitative biochemical and histochemical methods at specific locations in the disc. Because proteoglycans (PGs) represent a major extracellular matrix component, show changes that are highly correlated with aging and degeneration, and have turnover rapid enough to reflect short-term changes in the matrix, as opposed to very stable collagens, PGs and their associated link proteins will be used as markers. The presence of functional hyaluronic add-binding regions, the cleavage of the protein core in the chondroitin sulfate (CS) or keratan sulfate (KS) rich regions, and the presence and ratios of link proteins 1, 2, and 3 will be examined. The human disc will be the main focus. Cells will be isolated from regions of the disc by digestion with proteases and cultured in alginate beads in media that allows the in vitro reexpression of the normal chondrocyte-like phenotype and preserves viable fibroblasts. Matrix production will be assessed histochemically by alcian blue staining, CS detected by the monoclonal antibodies to chondroitinase ABC produced neoepitopes, and highly sulfated epitopes of KS will be detected by the monoclonal antibody 5D4. Fibrotic replacement would be characterized by fibroblast-like cells which do not re-express an alcian blue stainable matrix. Fibrocartilage will produce CS-rich matrices with a decrease in KS while cells from both the anulus and nucleus that reproduce their phenotype will produce CS and highly KS-positive matrices. These assays can be done on as few as several thousand cells. Evidence for or against this hypothesis would be collected in human discs of different grades. Several models which reflect various aspects of the human condition will also be evaluated and include: A) Proteolytic injection which allows regrowth of disc, B) multiple protease injected discs which produces fibrotic replacement, C) modest diskectomy with no disruption of the endplates and only a small hole through the anulus which allows anulus regrowth, D) extensive diskectomy including the removal of the anulus which produces fibrotic ingrowth, and E) chondrodystrophoid dogs with natural aging and degeneration. The human disc cells grown on defined matrices encapsulated in alginate to investigate the role of various components in maintaining phenotypic stability will also be investigated. This study will provide key information on the degenerative process.
