The Degree of Master of Science in Electrical Engineering

The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GIcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteases whose activity is known to be responsible for much of the aggrecan degradation seen in human joint diseases. In this study we verified that ManN and GlcN inhibit aggrecanase-mediated degradation induced by IL-1l of the mechanical properties of bovine calf articular cartilage. We then correlated the aggrecanase-generated aggrecan species remaining in the tissue with the results of mechanical testing . After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition (-75-100% inhibition) reached by a concentration of 1.35 mM. Results for GIcN were similar but occured at concentrations five times higher (5-10 mM). Western analysis demonstrated that in tissue which was completely protected by ManN from IL1 -induced degradation in physical properties, aggrecanase cleavage was inhibited at the aggrecanase site at the N-terminal end of the aggrecan core protein (the Glu373 _ Ala374 bond in the interglobular domain). However, the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu' 50' and Glu1 687) was higher than that of controls. This result suggests that the preservation of mechanical properties by hexosamines in explants was primarily due to inhibition of cleavage at the Glu37 3 site. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region. Thesis Supervisor: Alan J. Grodzinsky Title: Professor of Electrical Engineering, Mechanical Engineering, and Bioengineering