Isolation and Characterization of Calcifying Matrix Vesicles from Epiphyseal Cartilage

TLDR

Matrix vesicles linked to early cartilage calcification were isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase and fractionated into seven groups by differential centrifugation. The isolated extracellular vesicles, enriched in alkaline phosphatase, pyrophosphatase, ATPase, and 5′‑AMPase, contain enzymes that raise orthophosphate to promote hydroxyapatite formation, while cellular fractions hold most DNA and acid phosphatase, marking the first isolation of membrane‑bounded extracellular particles from normal tissue.

Abstract

Matrix vesicles, associated with initial calcification in cartilage, have been isolated from bovine fetal epiphyseal cartilage. Cartilage was digested with collagenase, then partitioned into seven fractions by differential centrifugation. The cellular fractions contained over 80% of the DNA in the digest. The extracellular fraction that contained matrix vesicles, in which apatite crystals were often seen on electron microscopy, also displayed the highest specific activity for alkaline phosphatase, pyrophosphatase, ATPase, and 5′-AMPase (EC 3.1.3.1., 3.6.1.1, 3.6.1.3, and 3.1.3.5, respectively). Most of the acid phosphatase (EC 3.1.3.2) activity, on the other hand, was found in the cellular fractions, indicating that matrix vesicles are quite distinct from lysosomes. This appears to be the first instance of isolation of membrane-bounded extracellular particles from any normal tissue. The matrix vesicles possess enzymes that can increase the local concentration of orthophosphate and thus could lead to the formation of hydroxyapatite. The membrane-bounded matrix vesicles may also provide a mechanism for ATP-dependent transport of calcium or phosphate into the lumen of the vesicles with resultant mineralization.