Ultrastructural Tissue Morphometry of the Distribution of Extracellular Matrix Vesicles in Remodeling Rat Tibial Bone Six Days after Injury

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Abstract
A study of the distribution of extracellular matrix vesicles on the 6th day of bone healing was performed by methods of transmission electron microscopy combined with computerized morphometry. The vesicles were detected on the electron micrographs and grouped according to their diameters, distance from the calcified front and type. The different types were selected as follows: (a) vesicles with electron-lucent contents, i.e. ‘empty’; (b) vesicles with amorphous electron-opaque contents, i.e., ‘amorphous’; (c) vesicles containing crystalline depositions, i.e., ‘crystal’, and (d) vesicles containing crystalline structures with ruptured membranes, i.e. ‘rupture’. Most of the vesicles were concentrated between diameters of 0.02 and 0.22 µm. Most of the vesicles were found within a distance of less than 3 µm from the calcified front. The vesicles were distributed according to their types: ‘empty’, ‘amorphous’, ‘crystal’ and ‘rupture’ in 14, 39, 34 and 13%, respectively. The diameters of the ‘crystal’ and ‘rupture’ vesicles were significantly larger than those of the ‘empty’ and ‘amorphous’ types. The sequence of distances from the calcified front was recorded as follows: ‘rupture’, ‘crystal’, ‘amorphous’ and ‘empty’, the ‘rupture’ type being the closest to the front. The results of the present study confirm the accepted hypothesis on calcification via extracellular matrix vesicles. It is thought that the cell secretes ‘empty’ vesicles that accumulate amorphous Ca and P to form a hydroxyl-apatite crystal. This is followed by rupture of the vesicular membrane. The propagation of the process is accompanied by increase in the vesicular diameter and its approximation to the calcifying front.