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Ca uptake by chondrocyte membrane-phosphatidyl serine hybrid vesicles
476
Abstracts from the IV International Conference on Matrix Vesicles
Cap+ acquisition necessary for the initial stages of dentine calcification. In this study the fine structure of the changes taking place in the differentiating preameloblast and in the extracellular matrix are reported in relation to dentine calcification in 2-day-old hamster tooth germs. Cellular changes leading to a differentiated odontoblast and ameloblast in the hamster were in general comparable to those reported for other rodent species. Initial mineral deposits were observed in the predentine only when the basal lamina was completely removed or in very advanced stages of removal. The initial dentine calcification was mostly related to collagen fibers in the vicinity of the preameloblast rather than to matrix vesicles However, structures resembling matrix vesicles were observed in the predentine mainly before basal lamina removal. Conclusive association of matrix vesicles with mineralization was not demonstrated. Enamel matrix secretion concided with the initial dentine calcification. Enamel matrix droplets also mineralized in the mantle dentine. The appearence of high Ca*+ binding activity in the apical plasma membranes of the preameloblast has also been demonstrated to coincide with mantle dentine calcification. From these results we tentatively conclude that apart from preodontoblasts, preameloblasts are involved in the initial stages of dentine calcification. Subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).
Growth cartilage cells were isolated from the ribs of weanling rabbits and cultured at high cell density. During 7 days, proteoglycans and type II collagen were synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured with labeled sulfate in thier proteoglycans, bone marrow cells caused release of material containing labeled sulfate into the medium. Electron micrographs of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells removed proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was inhibited. Chondrocytes were isolated from fetal bovine limb cartilage and cultured in vitro. The shape of these cells was not polygonal, as growth cartilage cells of rabbits, but elongated. Metachromasia was not found in these cells, indicating that these cells did not synthesize proteoglycans. However, when cultured with parathyroid hormone or hydrocortisone, these cells gained intense metachromasia. Electron micrographs of the extracellular matrix of these cultured cells under normal conditions showed that needles of mineral were deposited as clusters. Furthermore, matrix vesicles that contained needle-like crystals were seen. The elemental composition of the crystalline matrix appeared to be primarily composed of calcium and phosphorus. Therefore, these two types of cultured cells seem to be good systems for studies on endochondral ossification.
CaUPTAKEBYCHONDROCYTEMEMBRANEPHOSPHATIDYL SERINE HYBRID VESICLES E. Golub and I. Shapiro Univ. Penn. Sch. Dent. Med., Philadelphia,
PA, USA
Matrix vesicles (MV) initiate apatite formation. This function may result from their ability to accumulate Ca and phosphate ions. MV differ in content from chondrocyte membranes in that they are enriched in alkaline phosphatase (AP) and acidic phospholipids, including phosphatidyl serine (PS). To examine the importance of this lipid component, we constructed hybrid vesicles by fusing chondrocyte plasma membranes fragments with PS liposomes. For this experiment, cells were isolated from avian growth cartilage, and membrane fragments were isolated and purified by isopycnic density gradient centrifugation. Liposomes were prepared by sonication of a freeze-thawed PS suspension (5 mM HEPES, 100 mM NaCI, pH 7) followed by membrane filtration (0.45 pm) and gel filtration through Sephadex G-75. Fusion was carried out at room temperature for 16 h. Hybrids were isolated by isopynic density gradient centrifugation. Peak AP fractions were pooled and assayed for 45Ca uptake activity. Calcium accumulation by hybrid vesicles was much greater than that of the parent membranes. Moreover, the extent of calcium uptake was directly related to their PS content. These data therefore support the hypothesis that Ca accumulation by MV may result from the high PS content of their membranes. Support: DE-06533, DE-02623, AM-344 11,
SIMULATION OF CHONDROGENESIS AND PREOSTEOGENESIS IN VITRO Fuji0 Suzuki Department of Biochemistry, Facuffy of Dentistry, Osaka University, Suita, Osaka, Japan
ROLE OF ACTIN MICROFILAMENT DEPOLYMERIZATION IN MATRIX VESICLE FORMATION BY PRIMARY CULTURES OF EPIPHYSEAL GROWTH PLATE CHONDROCYTES John E. Hale and Roy E. Wuthier Department SC, USA
of Chemistry, Univ. of S. Carolina, Columbia,
The mechanism by which matrix vesicles (MV) form remains to be fully elucidated. In this study we explored the role of microfilaments in this process because these cytoskeletal elements are known to be involved in vesicle formation in other systems (e.g. erythrocyte and intestinal brush-border vesicles). To study this vesiculation phenomenon, we perturbed the cytoskeleton of MV-releasing cultured chondrocytes using two fungal metabolites, cytochalasin-D and phalloidin, both of which are known to affect microfilament function. Cytochalasin-D induces breakdown of actin microfilaments; phalloidin stabilizes microfilaments, preventing their breakdown. Our results show that when cytochalasinD (IO to 80 ng/ml) is added to postconfluent cultures it greatly inhibits both cell division (expressed as 3-H-thymidine uptake) and cellular alkaline phosphatase (AP) activity but had little affect on MV AP levels. When expressed as a pecentage of cellular AP, release of MV AP was found to be stimulated by cytochalasin-D. In contrast, phalloidin (100 to 500 ng/ml) mildly stimulated both cell division and cellular AP but decreased MV AP levels. Further, when expressed as a percentage of cellular AP, phalloidin was shown to inhibit MV release by the cultured cells. These findings suggest that release of MV requires breakdown of actin microfilaments in a manner similar to that occurring during intestinal brush-border vesiculation.
Abstracts from the IV International Conference on Matrix Vesicles
Cap+ acquisition necessary for the initial stages of dentine calcification. In this study the fine structure of the changes taking place in the differentiating preameloblast and in the extracellular matrix are reported in relation to dentine calcification in 2-day-old hamster tooth germs. Cellular changes leading to a differentiated odontoblast and ameloblast in the hamster were in general comparable to those reported for other rodent species. Initial mineral deposits were observed in the predentine only when the basal lamina was completely removed or in very advanced stages of removal. The initial dentine calcification was mostly related to collagen fibers in the vicinity of the preameloblast rather than to matrix vesicles However, structures resembling matrix vesicles were observed in the predentine mainly before basal lamina removal. Conclusive association of matrix vesicles with mineralization was not demonstrated. Enamel matrix secretion concided with the initial dentine calcification. Enamel matrix droplets also mineralized in the mantle dentine. The appearence of high Ca*+ binding activity in the apical plasma membranes of the preameloblast has also been demonstrated to coincide with mantle dentine calcification. From these results we tentatively conclude that apart from preodontoblasts, preameloblasts are involved in the initial stages of dentine calcification. Subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO).
Growth cartilage cells were isolated from the ribs of weanling rabbits and cultured at high cell density. During 7 days, proteoglycans and type II collagen were synthesized and secreted, forming a metachromatic matrix. When cultured together with growth cartilage cells precultured with labeled sulfate in thier proteoglycans, bone marrow cells caused release of material containing labeled sulfate into the medium. Electron micrographs of the extracellular matrix of growth cartilage cells cocultured with bone marrow cells showed that needles of mineral were deposited within and in close apposition to the surfaces of matrix vesicles. These findings suggest that enzymes released from bone marrow cells removed proteoglycans, which may be inhibitors of mineral growth, and consequently mineralization was inhibited. Chondrocytes were isolated from fetal bovine limb cartilage and cultured in vitro. The shape of these cells was not polygonal, as growth cartilage cells of rabbits, but elongated. Metachromasia was not found in these cells, indicating that these cells did not synthesize proteoglycans. However, when cultured with parathyroid hormone or hydrocortisone, these cells gained intense metachromasia. Electron micrographs of the extracellular matrix of these cultured cells under normal conditions showed that needles of mineral were deposited as clusters. Furthermore, matrix vesicles that contained needle-like crystals were seen. The elemental composition of the crystalline matrix appeared to be primarily composed of calcium and phosphorus. Therefore, these two types of cultured cells seem to be good systems for studies on endochondral ossification.
CaUPTAKEBYCHONDROCYTEMEMBRANEPHOSPHATIDYL SERINE HYBRID VESICLES E. Golub and I. Shapiro Univ. Penn. Sch. Dent. Med., Philadelphia,
PA, USA
Matrix vesicles (MV) initiate apatite formation. This function may result from their ability to accumulate Ca and phosphate ions. MV differ in content from chondrocyte membranes in that they are enriched in alkaline phosphatase (AP) and acidic phospholipids, including phosphatidyl serine (PS). To examine the importance of this lipid component, we constructed hybrid vesicles by fusing chondrocyte plasma membranes fragments with PS liposomes. For this experiment, cells were isolated from avian growth cartilage, and membrane fragments were isolated and purified by isopycnic density gradient centrifugation. Liposomes were prepared by sonication of a freeze-thawed PS suspension (5 mM HEPES, 100 mM NaCI, pH 7) followed by membrane filtration (0.45 pm) and gel filtration through Sephadex G-75. Fusion was carried out at room temperature for 16 h. Hybrids were isolated by isopynic density gradient centrifugation. Peak AP fractions were pooled and assayed for 45Ca uptake activity. Calcium accumulation by hybrid vesicles was much greater than that of the parent membranes. Moreover, the extent of calcium uptake was directly related to their PS content. These data therefore support the hypothesis that Ca accumulation by MV may result from the high PS content of their membranes. Support: DE-06533, DE-02623, AM-344 11,
SIMULATION OF CHONDROGENESIS AND PREOSTEOGENESIS IN VITRO Fuji0 Suzuki Department of Biochemistry, Facuffy of Dentistry, Osaka University, Suita, Osaka, Japan
ROLE OF ACTIN MICROFILAMENT DEPOLYMERIZATION IN MATRIX VESICLE FORMATION BY PRIMARY CULTURES OF EPIPHYSEAL GROWTH PLATE CHONDROCYTES John E. Hale and Roy E. Wuthier Department SC, USA
of Chemistry, Univ. of S. Carolina, Columbia,
The mechanism by which matrix vesicles (MV) form remains to be fully elucidated. In this study we explored the role of microfilaments in this process because these cytoskeletal elements are known to be involved in vesicle formation in other systems (e.g. erythrocyte and intestinal brush-border vesicles). To study this vesiculation phenomenon, we perturbed the cytoskeleton of MV-releasing cultured chondrocytes using two fungal metabolites, cytochalasin-D and phalloidin, both of which are known to affect microfilament function. Cytochalasin-D induces breakdown of actin microfilaments; phalloidin stabilizes microfilaments, preventing their breakdown. Our results show that when cytochalasinD (IO to 80 ng/ml) is added to postconfluent cultures it greatly inhibits both cell division (expressed as 3-H-thymidine uptake) and cellular alkaline phosphatase (AP) activity but had little affect on MV AP levels. When expressed as a pecentage of cellular AP, release of MV AP was found to be stimulated by cytochalasin-D. In contrast, phalloidin (100 to 500 ng/ml) mildly stimulated both cell division and cellular AP but decreased MV AP levels. Further, when expressed as a percentage of cellular AP, phalloidin was shown to inhibit MV release by the cultured cells. These findings suggest that release of MV requires breakdown of actin microfilaments in a manner similar to that occurring during intestinal brush-border vesiculation.