Integrative repair of articular cartilage, defined as the healing of cartilage-cartilage or cartilage-tissue interfaces, is required in a number of orthopaedic conditions and rheumatic diseases including osteochondral fractures, osteochondritis dissecans, and osteoarthritis, as well as in articular therapies involving grafts of osteochondral, chondral, or cell-laden synthetic tissues. However, such repair usually does not occur. The broad, long-term objective of this project is to determine the underlying biophysical, cellular, and molecular mechanisms of integrative articular cartilage repair. The detailed understanding of such repair mechanisms and factors that inhibit or promote repair may accelerate the development of medical, surgical, and physical strategies for stimulating cartilage healing, particularly in the aged. Using an in vitro model of cartilage repair, the applicant proposes to determine the role of static mechanical stress in modulating the integrative repair process. The amplitude-dependence of the effects of static mechanical stress, applied normal to the cartilage-cartilage interface, on integrative cartilage repair during up to one month of explant culture will be determined. The function of the repair tissue that forms at the cartilage-cartilage interface will be determined as the adhesive strength, fracture energy, and compressive modulus. The function of the adjacent remodeling cartilage tissue will be determined as tensile and compressive modulus, permeability, and electrokinetic coefficient. Testing of normal cartilage is intended to allow quantitative comparison of the properties of repair tissue to those of normal cartilage. The cellular and physicochemical mechanisms by which static mechanical stress modulates integrative cartilage repair will be assessed. Cell- mediated vs. cell-independent mechanisms, and mechanical vs. physicochemical regulators will be distinguished. The quality of the repair tissue will be correlated with tissue cellularity and phenotypic expression, which are general indices of tissue metabolism. Biomechanical characterization of cartilage before and after integrative repair will include video microscopic methods to allow assessment of tissue inhomogeneity, which is particularly prominent in repair situations, and will provide insight into the physical regulatory mechanisms. The repair process will be further related to the remodeling and deposition of specific cartilage matrix components. The spatial variation in decorin, fibromodulin, and denatured collagen will be determined by immunohistochemistry. By isolating cartilage sections containing the repair tissue and the adjacent cartilage tissue, the density of these matrix components, as well as collagen, collagen crosslinks, and hyaluronan will be quantitated via biochemical assays. The role of lysyl oxidase-mediated collagen crosslinking is determined by inhibition with beta-aminopropionitrile. The role of proteoglycan aggregate is assessed by selective enzyme digestion before mechanical testing. To test and evaluate function-composition-metabolism relationships in integrative repair, the interactive effects of mechanical stress, and IGF-1, TGF-beta 1, and mild trypsin treatment will be studied.
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