Targeted Cleavage of Matrix Molecules in a Neocartilage

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DESCRIPTION (provided by applicant): The goal of this project is to develop a method and model in which a chosen extracellular matrix protein of a tissue grown from cartilage cells (neocartilage) can be cleaved at a specific site. Addition of a translated thrombin protease recognition site will be inserted into the genetic sequence of an extracellular matrix molecule, the genetically modified molecule expressed by chondrocytes, and a neocartilage generated. The genetically modified protein could be cleaved by the addition of thrombin at any point in the development of the tissue. The immediate application is to allow evaluation of candidate structural molecules, molecules that carry load, by comparison of the mechanical properties of the neocartilage before and after cleaving the molecule. If the candidate molecule is a structural molecule, the mechanical properties should be reduced after cleavage of that molecule. The motivation for developing the method is to understand the mechanical failure process in osteoarthritis, where extracellular matrix molecules are digested by lytic enzymes and subsequently the tissue fails under relatively normal loads. Current strategies for disrupting the disease process involve search for agents to block critical molecules in the process. A significant challenge in this approach has been identifying the critical molecules to block. The traditional approach to identifying structural molecules is by digesting candidate molecules with an exogenous enzyme and measuring mechanical properties before and after. The problem with this approach is that there are very few enzymes available that are single molecule specific. The proposed method would allow digestion (cleavage) of a chosen molecule with specificity, greatly enlarging the number of matrix molecules that could be evaluated as candidate structural molecules. The method may have other applications in tissue engineering and drug release. The approach would be a valuable tool in understanding the OA disease process and as a guide for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Osteoarthritis (OA) is the largest cause of disability, but there is poor understanding of the mechanisms of the disease process, particularly how cartilage is degraded. This project will develop methods to identify how load is transmitted through cartilage, and which specific load carrying molecules might be damaged during the OA process. Identification of these molecules would allow development of drugs that could retard the damage and provide a disease modifying treatment to OA.