D. Amir

Changes in extracellular matrix vesicles during healing of rat tibial bone: A morphometric and biochemical study

Primary mineral formation in woven bone has been associated with the production of extracellular matrix vesicles. Previous studies have demonstrated an increase in phospholipid: Ca:Pi complexes (CPLX) immediately prior to hydroxyapatite formation. Since matrix vesicles are enriched in phosphatidylserine and PS is the major phospholipid in CPLX, the present study examined whether the morphologic appearance of matrix vesicles and initial formation of crystals within them could be correlated to changes in their phospholipid composition and metabolism. Ablation of the tibial marrow in rats was used as the model since this procedure induces endosteal repair with primary mineralization. The morphologic appearance of the matrix vesicles was assessed by morphometric analysis at the electron microscopic level. Matrix vesicles were divided into 4 categories: empty, amorphic, crystal, and rupture. There was time dependent decrease in the number of empty and amorphic matrix vesicles which correlated with an increase in crystal and rupture type. Distance from the calcification front decreased as more rupture-type vesicles were noted. In a parallel set of experiments, matrix vesicle-enriched membranes (MVEM) were isolated from homogenates of endosteal tissue removed from the treated tibia as well as from the contralateral control. There was an increase at 6 days in MVEM alkaline phosphatase and phospholipase A2 specific activities in both limbs, the magnitude of response being significantly greater in the treated legs. The phospholipid composition of the MVEM changed with time. SPH was highest at day 3, PS was detectable only in day 6 and 14 samples, and PC exhibited a time dependent decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of extracellular matrix vesicles in healing of rat tibial bone three days after intramedullary injury

SummaryThe 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 vesicles membrane. The maturation process is accompanied by an increase of the vesicular diameter and its approximation to the calcifying front.

The relationship between extracellular matrix vesicles and the calcifying front on the 21st day after injury to rat tibial bone

The relationship between extracellular matrix vesicles and the calcifying fronts was examined by studying vesicular diameters and types. Transmission electron microscopy combined with computerized morphometry three weeks after injury to the tibial bone in rats was used. The different vesicle types were defined as: (1) vesicles with electron lucent contents referred to as empty; (2) vesicles with amorphous electron opaque contents, called amorphic; (3) vesicles containing crystalline depositions, called crystalline; and (4) vesicles containing crystalline structures with ruptured membranes, referred to as ruptured. The diameters of most vesicles ranged between 0.07 and 0.17 micron. More than 95% of the vesicles were located less than 2 micron from the calcified front. The vesicles were distributed among the categories as follows: empty, 9.6%; amorphic, 19.3%; crystal, 39.2%; and ruptured, 31.9%, respectively. The diameters of the crystalline and ruptured vesicles were significantly larger than those of the empty and amorphic types. The ruptured type had the largest diameters. The sequence of distances from the calcified front was recorded as follows: ruptured, crystalline, amorphic, and empty, with the ruptured and crystalline types being the closest to the front. This study supports the accepted theory on matrix vesicle mineralization. The cell is responsible for secretion of empty vesicles that accumulate amorphous Ca and Pi to form a hydroxyapatite crystal. This is followed by rupture of the vesicular membrane. The propagation of the process is accompanied by an increase in the vesicular diameter and its approximation to the calcifying front.