This project is a consolidation of Project 3 and Project 4 of the previous Program Project. The major goal of this project will be to establish a detailed mechanistic understanding of how different mutations in two cartilage-specific collagen genes (COL2A and COL9A2) result in chondrodysplasia and premature OA.
The aim will be to utilize in vitro and in vivo systems to gain a full understanding of the effects of these mutations on the biochemistry and structure of the cartilage matrix, on embryonic skeletal and joint development and finally on adult articular cartilage. To achieve this, we will exploit the poly 2-hydroxyethyl methacrylate (poly-HEMA) chondrocyte culture system we developed during the previous funding periods which allows the long-term preservation of the cartilage-specific phenotype of chondrocytes. Second, we will establish lines of knock-in mice that harbor the same mutations in cartilage-specific collagen genes that cause chondrodysplasia and premature OA in humans. Third, we will create transgenic mouse lines in which the mutated collagen molecules can be expressed in a cartilage specific and conditional manner. The resulting animal lines will be examined in detail employing morphologic, histologic, and histopathological assessments and will be made available to our collaborators and to other investigators to study the functional effects of the mutations in vivo. Furthermore, articular cartilage from these mice will be examined biomechanically employing a highly sensitive osmotic loading method to answer the functional impact of the mutations on the biomaterial properties of the tissue and attempt to establish structure/function relationships that may be of direct relevance to the pathogenesis of osteoarthritis. Fourth, we will utilize purified collagen molecules and in vitro fibril formation and protein interaction assays to obtain a better understanding of the effects of these mutations on collagen monomer structure, fibril assembly and structure, and molecular interactions between collagens II and IX. The overall goal of this project will be achieved by pursuing the following Specific Aims: (1) To determine the effects of expression of mutated extracellular matrix genes on the phenotype of chondrocytes in culture and the functional characteristics of the resulting cartilaginous matrix; (2) To generate authentic mouse models of the human chrondrodysplasias caused by mutations in the human COL2A1 and COL9A2 genes by introducing (knocking-in) the analogous human mutations in the corresponding mouse genes; (3) To determine the effects of conditional tissue-specific expression of mutant collagens in the articular of adult mice; and (4) To study the effects of Arg75, Arg519 and Arg789 to Cys mutations in type II collagen on collagen fibril assembly and to study interactions between wild type and mutant collagen II and collagen IX.
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