Disc degeneration which causes significant disability and loss of the quality of life, involves cellular, nutritional and mechanical components. The investigators hypothesize that cellular changes occur early and will test two hypotheses. The first is that there is a loss of normal matrix producing phenotype of cells in the nucleus pulposus and an increase of a fibrotic phenotype. This may be reversible as a result of changes in environment or irreversible. This phenotype may also be more sensitive to catabolic cytokines. The second hypothesis is that notochordal cells, which are critical to the development of nucleus pulposus, also play a role either directly by synthesis of matrix components or indirectly by influencing the other cells in the nucleus delaying early changes; and their loss accelerates degeneration.In order to test the first hypothesis, which considerable preliminary data supports, both matrix producing cells (MPC) and nonmatrix producing cells (NMPC) will be isolated from human disc and characterized from normal and degenerative discs. Sensitivity to IGF-1, TGF-B1, il-1B, and TNF-a will be examined.Co-culture experiments will be used to determine if interactions between the two cell populations occur. 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, PGs and their associated link proteins will be used as indicator molecules. Presence of hyaluronic acid-binding regions, the cleavage of the protein core in chondroitin sulfate (CS) or keratan sulfate (KS) rich regions, and the presence and ratios of link proteins 1, 2, and 3 will be examined. Collagen types will also be determine. To test hypothesis two, notochordal cells will be isolated from nonchondrodystrophoid canine nucleus pulposus, a unique source of large amounts of these cells, and their characteristics determined with respect to matrix synthesis, turnover, and response to the growth factors and cytokines. Co-cultures with MPC and/or NMPC will determine if there are interactions between the different cell types. Key observations will be confirmed with human notochordal cells.
