Calcification of isolated matrix vesicles and reconstituted vesicles from fetal bovine cartilage

Ca deposition by isolated matrix vesicles from fetal calf growth plate cartilage and by a deoxycholate extract from matrix vesicles that included their phosphatase was studied under defined in vitro conditions. Electron microscopy showed that after removal of deoxycholate and lyophilization of the vesicle extract, new vesicles were reconstituted, often with multiple membrane layers. Both intact calf vesicles and reconstituted vesicles initiated Ca deposition maximally when supplied with ATP, GTP, CTP, or UTP. Only nucleoside triphosphates supported Ca deposition well; mono- and diphosphoesters, although hydrolyzed, were ineffective as substrates. Nucleoside triphosphates supported Ca deposition even if the final [Ca] X [P] reached in the reaction mixture was below a metastable level (3.5 mM2), suggesting that matrix vesicles or reconstituted vesicles promote calcification by localizing Ca or PO4 or both. ATP or GTP supported Ca deposition readily at concentrations ranging from 0.25 to 1.0 mM but, at 2.5 and 5.0 mM, Ca deposition was inhibited. The ATPase of intact matrix vesicles and reconstituted vesicles was stimulated by addition of Ca2+ and Mg2+. Ca deposition did not require additional Mg2+. These results lend support to the hypothesis that matrix vesicles and their phosphatases play an important role in mineralization.