This application addresses the mechanism(s) by which collagen fibril growth is regulated during morphogenesis and growth of the chicken metatarsal tendons. We have demonstrated that collagen fibrils are initially assembled and deposited by fibroblasts as discrete segments (10-20 umm long). As development proceeds a maturation process occurs which transforms the fibril segments within the immature tissue into both longer and larger diameter fibrils. This results in a functional tendon with appropriate mechanical properties. Our hypothesis is that fibril segment fusion at later stages of tendon development requires changes in the surface properties of the fibril segments permitting lateral association and growth. We will determine the role of fibril-associated decorin in the regulation of fibril growth using biochemical, immunochemical and molecular genetic approaches to correlate decorin expression/content as well as it fibrillar localization with specific stages in tendon development (i.e. 10-14 days versus 17-20 days). In addition, we will investigate the role(s) of matrix metalloproteinases, specifically stromelysins and gelatinases in fibril growth. We will identify matrix metalloproteinases that may be involved in the removal of a specific component utilizing substrate gel electrophoresis of metatarsal tendon extracts from different stages of limb development. Inhibitor activity will be correlated with stage in tendon development using reverse zymography. In addition, gene and protein expression will be correlated with specific stages in tendon morphogenesis (i.e. 10-14 days versus 17-20 days of development) using cDNA probes and antibodies for candidate metalloproteinases/inhibitors. We hypothesize that the precipitous increase in fibril growth seen at 17-18 days of development is associated with changes in gene expression. These changes are translated into an alteration in the synthesis of specific macromolecules and/or turnover resulting in a modification of the surface properties in immature fibril segments. A program of changes in gene expression would ultimately result in alterations which permit fibril fusions thereby yielding in mature, mechanically competent tendon. Accordingly, we will elucidate the changes in gene expression associated with the distinct stages in fibril growth (14 versus 18 day of development) employing differential screening and subtraction cloning methods. The elucidation of the mechanism(s) regulating fibril growth is important to our understanding of normal limb development, growth and repair.
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