Purification and further characterization of isolated matrix vesicles from rat alveolar bone

Abstract

Extracellular matrix vesicles from rat alveolar bone were isolated by collagenase digestion and differential centrifugation. Further purification was performed by discontinuous sucrose density gradient centrifugation. Control tissues, kidney and liver, were processed according to the same procedures. Sucrose density gradient centrifugation of bone matrix vesicles revealed two peaks of enzymatic activity: “light” and “heavy” vesicle-enriched fractions. Electron micrographs revealed a higher degree of purification of the “light” rather than the “heavy” vesicle-enriched fraction. This coincided with the high levels of enzymatic activity detected in this fraction. Preparations obtained from kidney and liver had significantly lower levels of activity of alkaline phosphatase and ATPase as compared to the bone matrix vesicle fractions. There were also differences in the positions of enzyme activity peaks in the sucrose gradiant fractions from the three tissues studied. Electron microscopic examination of kidney and liver fractions revealed structures larger than the purified bone matrix vesicles. In addition no electron-dense material was found within organelles from kidney and liver and they were studded with numerous ribosomes. Our observations indicate that the present method of isolation and purification yields fractions of matrix vesicles which are specific to bone and are significantly different from those obtained from kidney and liver.