MATRIX VESICLES PRODUCED BY OSTEOBLAST-LIKE CELLS IN CULTURE BECOME SIGNIFICANTLY ENRICHED IN PROTEOGLYCAN-DEGRADING METALLOPROTEINASES AFTER ADDITION OF BETA-GLYCEROPHOSPHATE AND ASCORBIC-ACID

Matrix vesicles, media vesicles, and plasma membranes from three well- characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1 ) were evaluated for their content of enzymes capable of processing th e extracellular matrix. Matrix vesicles were enriched in alkaline phos phatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix v esicle enrichment in neutral metalloproteinase varied with the cell li ne, whereas collage nase, lysozyme, hyaluronidase, and tissue inhibito r of metalloproteinases (TIMP) were not found in any of the membrane f ractions examined. MC-3T3-E1 cells were cultured for 32 days in the pr esence of ascorbic acid (100 mu g/ml), beta-glycerophosphate (5 mM), o r a combination of the two, to assess changes in matrix vesicle enzyme s during calcification. Ascorbate or beta-glycerophosphate alone had n o effect, but in combination produced significant increases in both ac tive and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dra matic. This correlated with an increase in the formation of von Kossa- positive nodules. The results of the present study indicate that osteo blast-like cells produce matrix vesicles enriched in proteoglycan-degr ading metalloproteinases. In addition, the observation that matrix ves icles contain significantly increased metalloproteinases under conditi ons favorable for mineralization in vitro lends support to the hypothe sis that matrix vesicles play an important role in extracellular matri x processing and calcification in bone.