Matrix vesicles isolated from apical pulp of rat incisors: Crystal formation in low Ca×Pi ion-product medium containing β-glycerophosphate

Abstract

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.