The main hypothesis of the proposal is that after insult by an exogenous protease, such as chymopapain or alternate enzymes such as protease K or pronase, the nucleus pulposus has the ability to regenerate. This hypothesis will be tested by examining biochemical and histologic evidence or recovery using as a model system, dog intervertebral disc injected with protease (0.2 to 1 mg/level) and followed from 2 weeks to 1 year to determine the ability of the nucleus and annulus to recover. Proteoglycan synthesis will be used as a measure of matrix formation and studied using 35S-sulfate with in vitro labeling and the biochemical characterization including the chondrointin sulfate chain lengths, % chondrointin and keratan sulfate. This will be done using enzymes and analysis of the glucosamine/galactosamine ratio by high pressure liquid chromatography. Histologically, the samples will be followed at the light level after Safranin-O fast green staining and by immunofluorescent techniques with antibodies to matrix molecules such as fibronectin: The samples will be analyzed for changes in structure by morphometric analysis. The changes in the adjacent facet joints will also be followed for possible degenerative changes. This will be done with a biochemical analysis of the cartilage for water changes and changes in 35S-sulfate uptake and matrix protein alterations. The histology of the cartilage and bone will be followed by prelabeling in the animals with tetracycline analogs and using both light & fluorescent microscopy. In addition to remodeling after chymopapain injection, the proteases elaborated by the nucleus during early remodeling will be followed using specific substrates and inhibibors.
