The effect of bone injury on extracellular matrix vesicle proliferation and mineral formation

Removal of tibial bone marrow in rats is followed by primary bone formation, resorption and marrow restitution. The first week of healing is characterized by partially calcified trabeculae. After 2 weeks, a higher degree of calcification and partial resorption are observed. The third week is characterized by massive resorption of the trabeculae, which are replaced in the fourth week by new bone marrow tissue. This model was used to study primary calcification. Transmission electron micrographs of the young bone revealed osteoblasts, matrix vesicles and calcified fronts. The different vesicular types were defined as 'empty', 'amorphous', 'crystal', and 'rupture'. The vesicles were studied on days 3, 6, 8, 12, 14, 18, 21, 23 and 28 after injury. The mean diameters of most vesicles ranged between 100.3 and 121.9 nm, and their mean distance from the calcified front was less than 976.6 nm. Vesicular density, calculated as number per 10 m2, increased on the eighth day and decreased from the fourteenth day onwards. Highest diameter values were recorded on the sixth day, and decreased onward. Vesicular distance from the calcified front decreased continuously. Distribution of vesicle number, diameter, and distance in each class showed that numbers of empty and amorphous vesicles decreased and of crystal and rupture increased throughout the experiment. Distances from the calcified front and vesicular diameters varied as follows: 'rupture', 'crystal', amorphous', and 'empty', the 'rupture' type being the closest to the front and of the largest diameter. The results confirm the hypothesis that the cell is responsible for the secretion of electron lucent vesicles that accumulate Ca and Pi to form amorphous calcium phosphate complexes that convert to hydroxyapatite. Crystal growth is followed by membrane rupture.