Abstract
This dissertation examined the possible role of membrane proteins of the matrix vesicle in calcification processes of the epiphyseal plate. Matrix vesicles and chondrocytes were isolated by sequential hyaluronidase and collagenase digestion from epiphyseal plates of 24-26 day old New Zealand white rabbits, followed by differential centrifugation. Purity was determined by electron microscopy of the resultant pellets. Matrix vesicles and chondrocytes were isolated ultrastructurally intact and free of contaminant extracellular matrix and intracellular debris. A certain degree of contaminant protease activity in the digestive enzyme solutions was found to be necessary for the adequate release of chondrocytes and vesicles from the extracellular matrix. However, proteolytic damage to isolated vesicles and chondrocytes was discovered to be minimal and reproducible. 1D- and 2D-SDS-PAGE demonstrated that the matrix vesicle contains no unique proteins, as compared to chondrocytes. Quantitative differences do exist, and most major vesicular proteins are only represented as very minor components in the chondrocyte. EDTA extraction of isolated vesicles, however, did result in the 2D-SDS-PAGE visualization of unique proteins. These proteins are exclusively basic and present an electrophoretic appearance characteristic of glycoproteins. Radioiodination of isolated matrix vesicles, 'intact' and osmotically-lysed, and whole chondrocytes demonstrated that a similar array of membrane proteins is accessible to the extracellular environment for each pellet. Furthermore, matrix vesicles are leaky, and the inner surface of its membrane is accessible to the extracellular fluid. Either intravesicular calcification inhibitors must be removed, or calcium and phosphate binding sites on the inner surface of the membrane must become exposed, prior to the initiation of calcification in association with the matrix vesicle. These results suggest that membrane proteins do not play a critical role in the calcification process, as the chondrocyte, a noncalcifying constituent, and the matrix vesicle contain the same membrane proteins, in a similar orientation with respect to the extracellular environment. Furthermore, removal of all vesicular protein by pronase did not affect the calcium and phosphate binding characteristics of vesicles incubated in a simple in vitro calcifying solution. Calcification associated with matrix vesicles may be simply due to its decreased volume, which would allow the local concentration of soluble calcium and phosphate ions to supersaturation level, thus enabling bone mineral precipitation.