The distribution of extracellular matrix vesicles on the third day of bone healing was studied by morphometric analysis of transmission electron micrographs. Detection and grouping of the vesicles was performed according to type, diameter, and distance from the calcified front. The different types were selected as follows: vesicles with electron-lucent contents ("empty"), vesicles with amorphous electron-opaque contents ("amorphic"), vesicles containing crystalline depositions ("crystal"), and vesicles containing crystalline structures with ruptured membranes ("rupture"). The majority of vesicles were between 0.07 μm and 0.12 μm in diameter and were located at less than 3 μm from the calcified front. The distribution of the "empty", "amorphic", "crystal", and "rupture" vesicles was 23.2%, 74%, 2.5%, and 0.3% respectively. Their sequence of arrangement according to diameter was as follows: "empty", "amorphic", "crystal", and "rupture", the empty vesicles constituting the smallest and the "rupture" the largest type. Distances from the calcified front were similar for the "empty", "amorphic", and "crystal" vesicles, while the "rupture" type was located nearest to the front. The present observations support the widely acknowledged hypothesis on the role of extracellular matrix vesicles in mineralization. It is thought that the secretion of "empty" vesicles from the cell is followed by intravscular accumulation of amorphous Ca and Pi to form a hydroxyapatite crystal that, in turn, ruptures the vesicle's membrane. The maturation process is accompanied by an increase of the vesicular diameter and its approximation to the calcifying front.
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