Physicochemical Characterization of the Nucleational Core of Matrix Vesicles

Abstract

While previous studies revealed that matrix vesicles (MV) contain a nucleational core (NC) that converts to apatite when incubated with synthetic cartilage lymph, the initial mineral phase present in MV is not well characterized. This study explored the physicochemical nature of this Ca 2؉ and P i -rich NC. MV, isolated from growth plate cartilage, were analyzed directly by solidstate 31 P NMR, or incubated with hydrazine or NaOCl to remove organic constituents. Other samples of MV were subjected to sequential treatments with enzymes, salt solutions, and detergents to expose the NC. We examined the NC using transmission electron microscopy, energy-dispersive analysis with x-rays, and electron and x-ray diffraction, Fourier transform-infrared spectroscopy, high performance thin-layer chromatographic analysis, and SDS-polyacrylamide gel electrophoresis. We found that most of the MV proteins and lipids could be removed without destroying the NC; however, NaOCl treatment annihilated its activity. SDS-polyacrylamide gel electrophoresis showed that annexin V, a phosphatidylserine (PS)-dependent Ca 2؉ -binding protein, was the major protein in the NC; high performance thin-layer chromatographic analysis revealed that the detergents removed the majority of the polar lipids, but left significant free cholesterol and fatty acids, and small but critical amounts of PS. Transmission electron microscopy showed that the NC was composed of clusters of ϳ1.0 nm subunits, which energy-dispersive analysis with x-rays revealed contained Ca and P i with a Ca/P ratio of 1.06 ؎ 0.01. Electron diffraction, x-ray diffraction, and Fourier transform-infrared analysis all indicated that the NC was noncrystalline.

Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: Indications for an amorphous precursor phase

“…Several studies reported the presence of transient precursor calcium phosphate phases involved in the deposition of carbonated hydroxyapatite in different in vivo systems. ACP was identified in vesicles associated with cartilage ossification (23), and the presence of an OCP-like precursor phase was detected in mouse calvaria bone mineralization (14). ACP was shown to form as a precursor phase of carbonated hydroxyapatite in chiton teeth (2).…”

“…Recently, collagen mineralization via a transient amorphous calcium phosphate phase was shown in vitro to produce aligned intrafibrillar carbonated apatite crystals (15). Identification of the initial mineral phase deposited in vivo during bone mineralization has, however, proved to be a challenging task (7,9,11,14,20,22,23). This is attributed to the very small size of even the mature biomineral particles deposited within a dense organic matrix, their highly substituted structure and poorly crystalline nature, and the difficulty of separating newly formed mineral from the mature crystallites.…”

Biophysical aspects of biomineralization

“…The MV lumen contains a NC that consists mostly of AnxA5, amorphous calcium phosphate complexes and PS and is capable of inducing mineral formation (Wu et al 1997(Wu et al , 1993. These findings contributed to the design of liposome models containing PS, Ca 2+ and P i complexes.…”

“…Various experiments have revealed the presence of HA crystals both inside and outside collagen fibrils in the ECM (McNally et al 2012) and also within the lumen of matrix vesicles (MVs) (Ali et al 1970;Anderson 1969;Bonucci 1970). MVs are extracellular vesicles with a diameter ranging from approximately 100 to 300 nm (Anderson 2003;Anderson et al 2010;Cui et al 2016;Golub 2009;Wuthier and Lipscomb 2011) which harbor the biochemical machinery needed to establish the appropriate inorganic pyrophosphate (PP i ) to inorganic phosphate (P i ) ratio (Ciancaglini et al 2006Harmey et al 2004;Hessle et al 2002;Johnson et al 2000;Roberts et al 2007;Yadav et al 2011;Zhou et al 2012) required to initiate the synthesis of apatitic mineral (Bechkoff et al 2008;Thouverey et al 2009;Wu et al 1997), which will subsequently be propagated onto the collagenous ECM. These vesicles are released from the hypertrophic chondrocytes and mature osteoblasts, the cells responsible for endochondral and membranous ossification.…”

“…Different localizations and ways of interaction with MV membrane indicate that AnxA6 has distinct functions during the mineralization process (Theobald et al 1994;Wu et al 1997). Expression of AnxA6 in cells reduces cell proliferation (Theobald et al 1994;Wu et al 1997), suggesting that AnxA6 may play a role in growth plate cartilage in the zones of maturation and hypertrophy where cell division stops. Many studies have reported that VSMC-derived MVs are enriched in AnxA6 and AnxA2 and that these annexins can stimulate mineralization when associated with type I, but not type II, collagen (Chen et al 2008).…”