Calcif Tissue Int (1994) 54:399-408 Calcified Tissue International 9 1994 Springer-Verlag New York Inc. Laboratory Investigations Matrix Vesicles Produced by Osteoblast-Like Cells in Culture Become Significantly Enriched in Proteoglycan-Degrading Metalloproteinases after Addition of 13-Glycerophosphate and Ascorbic Acid D. D. Dean, 1 Z. Schwartz, l'a L. Bonewald, 20. E. Muniz, 4 S. Morales, 4 R. Gomez, 1 B. P. Brooks, 1 M. Qiao, 2 D. S. Howell, 4 B. D. Boyan 1 ~Department of Orthopaedics, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7774, USA 2Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7774, USA 3Faculty of Dental Medicine, The Hebrew University/Hadassah, Jerusalem, Israel 4Miami VA Medical Center, Geriatric Research, Education and Clinical Center and Department of Medicine, Arthritis Division, University of Miami School of Medicine, Miami, Florida 33125, USA Received: 14 July 1993 / Accepted: 16 December 1993 Abstract. Matrix vesicles, media vesicles, and plasma mem- branes 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 the extracel- lular matrix. Matrix vesicles were enriched in alkaline phos- phatase specific activity over the plasma membrane and con- tained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collage- nase, lysozyme, hyaluronidase, and tissue inhibitor of me- talloproteinases (TIMP) were not found in any of the mem- brane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 p.g/ml), 13-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcifica- tion. Ascorbate or 13-glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix ves- icles being the most dramatic. This correlated with an in- crease in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an im- portant role in extracellular matrix processing and calcifica- tion in bone. Key words: Matrix vesicles -- Osteoblast-like cells -- Me- talloproteinases -- Ascorbic acid -- [3-Glycerophosphate. Correspondence to: B. D. Boyan Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix [1-3]. It has been hypoth- esized that these organelles are either initial sites of calcifi- cation in vivo or intimately associated with these sites [4, 5]. They have been found to be enriched in alkaline phosphatase [6-11], proteolipids [12], and calcium-phospholipid- phos- phate complexes [13, 14]. In addition, hormones such as vitamin D have been found to regulate matrix vesicle- associated enzymes such as alkaline phosphatase and phos- pholipase A2 [9-11, 15-19]. In the electron microscope, hy- droxyapatite crystals have been observed on the inner leaflet of the matrix vesicle membrane [20]. It is also likely that matrix vesicles are involved in pre- paring the extracellular matrix for mineralization. It has been recognized that proteoglycan aggregates are inhibitors of mineralization and that they must be removed for mineral- ization to begin [21-25]. Hirshman et al. [26] found neutral proteases in matrix vesicles and suggested that they may participate in matrix processing. Katsura and Yamada [27] have made similar observations utilizing matrix vesicles from chick growth plates, and Einhorn et al. [28] have pos- tulated that matrix vesicle-derived proteases may also be involved in fracture callus remodeling. Consistent with these observations, we have recently determined that matrix ves- icles produced by growth zone chondrocytes in culture are enriched in metalloproteinases capable of digesting proteo- glycan [29]. With these observations in mind, we hypothesized that extracellular matrix processing enzymes may also be found in osteoblast-like cell-derived matrix vesicles and that the stage of osteoblast differentiation might also affect the pro- file of enzymes present and/or their activity [30]. In addition, if matrix processing is critical for mineralization, then fac- tors that increase the amount or rate of mineralization should also increase the amount of these enzyme activities found in the matrix vesicles. To explore these questions, matrix ves- icles and plasma membranes from three well-characterized,