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 the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase 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 vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 micrograms/ml), beta-glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or beta-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 vesicles being the most dramatic. This correlated with an increase 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 important role in extracellular matrix processing and calcification in bone.

This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and beta-glucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and beta-glucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

A new microassay method with precision of *5% was developed to quantitate pyrophosphate in synovial fluid and plasma with starting volumes of 0.5 ml. A highly significant elevation of synovial fluid pyrophosphate concentration was found in some patients with osteoarthritis and gouty arthritis in addition to the well-established elevation in synovial fluid of subjects with pseudogout. No gradient of pyrophosphate between plasma and synovial fluid of patients with rheumatoid arthritis or in normal subjects was detectable. Studies of various parameters reflecting inflammation failed to show a correlation with the concentration of pyrophosphate in patients with pseudogout.Chondrocalcinosis polyarticularis familiaris was described in 1958 by Zitnan and Sitaj (1) in Czechoslovakia. In their study radiodense bands in the cartilage constituted the major diagnostic feature with the menisci of the knee joints most commonly involved. They postulated that there existed one phenotypic form which seems characterized by polyarthritis de- Submitted for publication May 22, 1972; accepted Nov 16, 1972. veloping at a young age with rapid and progressive involvement of multiple joints and a second form developing at an advanced age (2, 3) with progressive affliction of a few joints.McCarty et a1 described patients with clinically quite different characteristics but with similar radiologic findings (4). The acute polyarthritis in their patients was linked to the presence in their synovial fluid of crystals of calcium pyrophosphate dihydrate (CaPPi) (5) and the probable role of these crystals to initiate inflammation was elaborated upon in subsequent papers from McCarty's laboratory ( 6 , 7 ) . The unique deposition of CaPPi, also, led to an examination of pyrophosphate (PPi) metabolism in man (8'9). Pseudogout resembled urate-gout in respect to intraarticular and intrasynovial crystal deposition, but PPi concentrations in the plasma of patients with pseudogout have not been found elevated, nor has the urinary output of PPi been increased (8,10). These results and the finding of apparently normal red cell pyrophosphatase activity in patients with pseudogout (ll)-failed to indict a systemic disorder of PPi metabolism. In regard to a possible local articular disorder, precipitation of CaPPi from synovial fluid, supersaturated in respect of Ca