Aggrecan, the most abundant proteoglycan in articular cartilage, gives the tissue many of its functional properties, including its ability to undergo rapid reversible deformation. Aggrecan subunits interact extracellularly with link protein molecules and with long linear strands of hyaluronan (HA) to form large size aggregates that become immobilized in the matrix. The extracellular matrix of articular cartilage is composed of two compartments: the cell-associated matrix (CM), surrounding each chondrocyte or cell cluster, and the more abundant further removed matrix (FRM) present in the interterritorial areas. During aging, aggrecan molecules in the cartilage matrix exhibit progressive changes in structure and in organization that are still poorly understood. It is not known, for example, if these changes progress at the same rate in the CM, the metabolically-active compartment, as in the less metabolically-active FRM. As the incidence of progressive articular cartilage degeneration, i.e. osteoarthritis (OA), increases markedly during adult life, it is important to learn more about the biochemical basis of the changes which the metabolism of cartilage aggrecan undergoes during normal aging. Articular chondrocytes cultured in alginate beads reestablish a similar matrix composed of two compartments that morphometrically and immunohistochemically closely resemble those present in adult articular cartilage. A unique property of this culture system is that, after solubilization of the beads with chelating agents and mild centrifugation, the cells with their CM can be rapidly separated from molecules in the more loosely organized matrix further removed from the cells. We propose to use this culture system to perform metabolic studies on human and bovine articular chondrocytes from the normal joints of donors of different ages with the goals of further defining the biochemical basis of age-related changes in (i) the rate of formation and composition of these two matrix compartments and (ii) the organization and turnover of aggrecan molecules that become immobilized in each compartment. More specifically, our studies have three major aims: (1) to define the biochemical basis of the age-related decrease in ability of articular chondrocytes to rapidly reform a CM compartment rich in proteoglycan aggregates; (2) to further define age-related changes in the turnover of aggrecan in the metabolically-active CM and less metabolically-active FRM compartments; and (3) to study the effects of interleukin-1alpha (IL-1alpha)- and tumor necrosis factor-alpha (TNF-alpha)-induced chondrocytic chondrolysis on the metabolism of aggrecan, HA and link protein in the CM and FRM compartments. Once normal age-related changes in the metabolism of aggrecan are better understood, the approaches presented here could prove most helpful for identifying disease-related changes in aggrecan metabolism during the early stages of 0A (long-term goal).
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