Matrix metalloproteinase activity and inhibition in articular cartilage: Effects on composition and biophysical properties and relevance to osteoarthritis

The objectives of this thesis were threefold: (1) to characterize the ability of matrix metalloproteinases (MMPs) to degrade cartilage matrix constituents of intact cartilage; (2) to quantify the effects of this degradation on the functional biophysical properties of cartilage; and (3) to evaluate the ability of synthetic and natural MMP inhibitors to modulate these changes in composition and biophysical properties.

Cartilage from rabbit stifle joints which were exposed to stromelysin for 1 hour in vivo showed little difference in proteoglycan content from cartilage from contralateral control joints, while synovial fluid proteoglycan content from stromelysin treated joints was significantly higher than that of controls. Histological examination revealed that loss of proteoglycan, as indicated by both toluidine blue and FVDIPEN antibody staining, occurred primarily in a narrow region of the tissue near the articular surface. Consequently, the mechanical properties, such as equilibrium modulus and dynamic stiffness, of cartilage samples from treated joints did not differ significantly from those of control joints, while electromechanical properties, such as streaming potential and electrokinetic coupling coefficient, of cartilage from treated joints were degraded significantly compared to controls. Systemic administration of a synthetic MMP inhibitor significantly decreased release of proteoglycan fragments to synovial fluid, inhibited loss of proteoglycan as indicated by toluidine blue and FVDIPEN antibody staining, and prevented changes in cartilage electromechanical properties due to intraarticular injection of stromelysin.

The degradation of cartilage matrix by endogenous MMPs was characterized by treatment of explants with 4-aminophenylmercuric acetate (APMA) to activate native MMPs or by treatment with interleukin-1$\beta$ (IL-1$\beta$) or retinoic acid (RA), which upregulate production of MMPs by chondrocytes, and may increase levels of other endogenous proteinases. Treatment with APMA resulted in significant loss of proteoglycan by 1 day in culture and in significant swelling response by 3 days in culture. Matrix degradation was manifest in changes in sample mechanical and electromechanical properties. Both a synthetic MMP inhibitor and the native MMP inhibitor, TIMP, significantly inhibited proteoglycan loss, swelling response, and changes in physical properties due to APMA treatment. Treatment with IL-1$\beta$ and RA induced significant loss of proteoglycan after 4 days in culture. Treatment with IL-1$\beta$ resulted in an increased swelling response after 8 days in culture, while RA treatment did not. Both IL-1$\beta$ and RA treatments resulted in significant changes in tissue mechanical and electromechanical properties which were partially modulated by addition of TIMP to the culture media. Use of synthetic inhibitors also partially modulated proteoglycan loss from IL-1$\beta$ and RA treated tissue. Analysis of fragments in the culture media from these samples indicated that cleavage of proteoglycan did not occur at the VDIPEN-FFGV site associated with MMP activity, but rather at the NITEGE-ARGSVL site on the aggrecan core protein. This was the only proteinase activity observed against aggrecan in this system, and it was partially inhibited by the addition of a synthetic MMP inhibitor to the culture media. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.) (Abstract shortened by UMI.)