Enhancement by calcium β-glycerophosphate of mineral formation by isolated matrix vesicles in a...
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486 Abstracts from the IV International Conference on Matrix Vesicles cium to phosphorus ratio as hydroxapatite but also displayed a slight trace of magnesium, which suggested they may be similar to Whitlockite. We decided to examine these crystals by scanning electron microscopy (SEM) to be more certain of their shape, habit, and distribution in cartilage. For SEM, lprn sections of conventionally fixed and embedded human articular cartilage were air dried on to stubs and transferred to a low-temperature microincinerator unit. The specimens were ashed at room temperature in an oxygen plasma, coated appropriately, and examined in the SEM. It was found that numerous cuboid crystals were present in articular car- tilage either as a band under the surface or in pericellular regions. Whereas with transmission electron microscopy the size of the cuboid crystals varied from 50 to 250 nm, with SEM we found some crystals larger than the ones we de- scribed before varying from 50 to 650 nm. In addition there were some larger crystals that appeared of indeterminate shape or were spherically rounded. We are now using EPA to determine the Ca:P ratio of different sized cuboid crystals and to compare this with other spheroidal aggregates or needle-shaped apatite crystals. ENHANCEMENT BY CALCIUM &GLYCEROPHOSPHATE OF MINERAL FORMATION BY ISOLATED MATRIX VESICLES IN A FIBRIN CLOT SYSTEM S. Yousuf Ali and Lois Evans Institufe of Orthopaedics, Royal National Orthopaedic Hospital, Sfanmore, Middlesex, UK New methods have been developed for the study of apatite formation by isolated matrix vesicles in vitro. Matrix vesicles obtained from rabbit epiphyseal cartilage are mixed with fibrinogen and with thrombin to form a clot of unit volume. The clot is then incubated in various media containing 45Ca and the amount of calcium incorporated in the clot is de- termined (see page 67 in the Proceedings of the 111 Int. Conf. on Matrix Vesicles. Acenzi, Bonucci, and de Bernard, eds. Wichtig, Milan, 1981). Many phosphorus-containing nucleo- tides and esters act as substrates for the matrix vesicle alkaline phosphatase and promote calcification by contrib- uting phosphorus. More rapid and greater degree of cal- cification can be obtained by using calcium /3-glycerophos- phate as a substrate because it can contribute calcium and phosphorus at the same time for the formation of crystalline apatite. The role of various activators and inhibitors of cal- cification could be studied in this system both biochemically and by electron microscopy. Levamisole appears to inhibit calcification by inhibiting alkaline phosphatase, whereas alu- minum chloride inhibited calcification by inhibiting crystal formation and proliferation. It is possible that calcium gly- cerophosphate-type compounds may be present in chon- drocytes in epiphyseal cartilage and may act as a physio- logic substrate for extracellular alkaline phosphatase in matrix vesicle-mediated calcification. THE INFLUENCE OF ,S-GLYCEROPHOSPHATE ON HUMAN BONE AND MARROW-DERIVED CELLS B.A. Ashton, M. Couch, and M.E. Owen MRC Bone Research Laboratory, Nuffield Orfhopaedic Centre, Oxford and Dept. of Clinical Biochemistry and Human Metabolism, University of Sheffield Medical School, Sheffield, UK Cells derived in vitro as outgrowths from human bone and marrow (BMDC) display many of the characteristics attrib- uted to osteoblast-like cells, including high alkaline phos- phatase (AP) activity, osteocalcin production responsive to 1,25 (OH) 2D,, and increased CAMP production in response to PTH and prostaglandins. The human BMDC display fi- broblastic morphology, secrete a scant matrix, and do not mineralize under regular culture conditions. Following re- ports of successful in vitro mineralization by cells cultured in the presence of &glycerophosphate (PGP) the effect of this agent on human BMDC has been investigated. Primary confluent human BMDC harvested by trypsinization and re- plated at IO5 cells per 35 mm 4 well in 2.5 ml a-MEM containing 20% FBS and buffered with 20 mM HEPES buffer, were subsequently cultured for 7 days in the pres- ence of 0, l-and 5 mM PGP. The cell layers at day 7 were washed, homogenized, and assessed for AP activity, DNA, and inorganic phosphate. Cultures grown in 1 mM /3GP were not significantly different in any respect from the controls. The 5 mM /3GP regimen, however, caused a 50% decrease in the AP activity/well (PC O.OOl), a 30% decrease in the amount of DNA/well (P < 0.001) resulting in a 28% decrease in AP-specific activity (P < 0.005). A linear time-dependent decrease in medium AP was found in the 5 mM PGP cul- tures. The medium osteocalcin levels were not significantly different in the 5 mM j3GP group (83.6+7.9ng/ml, fSEM, n =6) compared with the controls (91.4+10.7ng/ml, n =6). There was no significant difference in the inorganic phos- phate content between the groups.
Transcript of Enhancement by calcium β-glycerophosphate of mineral formation by isolated matrix vesicles in a...
486 Abstracts from the IV International Conference on Matrix Vesicles
cium to phosphorus ratio as hydroxapatite but also displayed a slight trace of magnesium, which suggested they may be similar to Whitlockite. We decided to examine these crystals by scanning electron microscopy (SEM) to be more certain of their shape, habit, and distribution in cartilage. For SEM, lprn sections of conventionally fixed and embedded human articular cartilage were air dried on to stubs and transferred to a low-temperature microincinerator unit. The specimens were ashed at room temperature in an oxygen plasma, coated appropriately, and examined in the SEM. It was found that numerous cuboid crystals were present in articular car- tilage either as a band under the surface or in pericellular regions. Whereas with transmission electron microscopy the size of the cuboid crystals varied from 50 to 250 nm, with SEM we found some crystals larger than the ones we de- scribed before varying from 50 to 650 nm. In addition there were some larger crystals that appeared of indeterminate shape or were spherically rounded. We are now using EPA to determine the Ca:P ratio of different sized cuboid crystals and to compare this with other spheroidal aggregates or needle-shaped apatite crystals.
ENHANCEMENT BY CALCIUM &GLYCEROPHOSPHATE OF MINERAL FORMATION BY ISOLATED MATRIX VESICLES IN A FIBRIN CLOT SYSTEM
S. Yousuf Ali and Lois Evans
Institufe of Orthopaedics, Royal National Orthopaedic Hospital, Sfanmore, Middlesex, UK
New methods have been developed for the study of apatite formation by isolated matrix vesicles in vitro. Matrix vesicles obtained from rabbit epiphyseal cartilage are mixed with fibrinogen and with thrombin to form a clot of unit volume. The clot is then incubated in various media containing 45Ca and the amount of calcium incorporated in the clot is de- termined (see page 67 in the Proceedings of the 111 Int. Conf. on Matrix Vesicles. Acenzi, Bonucci, and de Bernard, eds. Wichtig, Milan, 1981). Many phosphorus-containing nucleo- tides and esters act as substrates for the matrix vesicle alkaline phosphatase and promote calcification by contrib- uting phosphorus. More rapid and greater degree of cal- cification can be obtained by using calcium /3-glycerophos- phate as a substrate because it can contribute calcium and phosphorus at the same time for the formation of crystalline apatite. The role of various activators and inhibitors of cal- cification could be studied in this system both biochemically
and by electron microscopy. Levamisole appears to inhibit calcification by inhibiting alkaline phosphatase, whereas alu- minum chloride inhibited calcification by inhibiting crystal formation and proliferation. It is possible that calcium gly- cerophosphate-type compounds may be present in chon- drocytes in epiphyseal cartilage and may act as a physio- logic substrate for extracellular alkaline phosphatase in matrix vesicle-mediated calcification.
THE INFLUENCE OF ,S-GLYCEROPHOSPHATE ON HUMAN BONE AND MARROW-DERIVED CELLS
B.A. Ashton, M. Couch, and M.E. Owen
MRC Bone Research Laboratory, Nuffield Orfhopaedic Centre, Oxford and Dept. of Clinical Biochemistry and Human Metabolism, University of Sheffield Medical School, Sheffield, UK
Cells derived in vitro as outgrowths from human bone and marrow (BMDC) display many of the characteristics attrib- uted to osteoblast-like cells, including high alkaline phos- phatase (AP) activity, osteocalcin production responsive to 1,25 (OH) 2 D,, and increased CAMP production in response to PTH and prostaglandins. The human BMDC display fi- broblastic morphology, secrete a scant matrix, and do not mineralize under regular culture conditions. Following re- ports of successful in vitro mineralization by cells cultured in the presence of &glycerophosphate (PGP) the effect of this agent on human BMDC has been investigated. Primary confluent human BMDC harvested by trypsinization and re- plated at IO5 cells per 35 mm 4 well in 2.5 ml a-MEM containing 20% FBS and buffered with 20 mM HEPES buffer, were subsequently cultured for 7 days in the pres- ence of 0, l-and 5 mM PGP. The cell layers at day 7 were washed, homogenized, and assessed for AP activity, DNA, and inorganic phosphate. Cultures grown in 1 mM /3GP were not significantly different in any respect from the controls. The 5 mM /3GP regimen, however, caused a 50% decrease in the AP activity/well (PC O.OOl), a 30% decrease in the amount of DNA/well (P < 0.001) resulting in a 28% decrease in AP-specific activity (P < 0.005). A linear time-dependent decrease in medium AP was found in the 5 mM PGP cul- tures. The medium osteocalcin levels were not significantly different in the 5 mM j3GP group (83.6+7.9ng/ml, fSEM, n =6) compared with the controls (91.4+10.7ng/ml, n =6). There was no significant difference in the inorganic phos- phate content between the groups.
