I n the process of bone formation, osteoblasts mineralize the matrix by promoting the seeding of basic calcium phosphate crystals of hydroxyapatite in the sheltered interior of shed membrane-limited matrix vesicles (MVs) and by propagating hydroxyapatite mineral onto the collagenous extracellular matrix (osteoid; refs. 1 and 2). Tissue-nonspecific alkaline phosphatase (TNAP), an isozyme of a family of four homologous human alkaline phosphatase genes (3), plays a role in bone matrix mineralization. Deactivating mutations in the TNAP gene causes the inborn error of metabolism known as hypophosphatasia (4), characterized by poorly mineralized cartilage (rickets) and bones (osteomalacia), spontaneous bone fractures, and elevated extracellular inorganic pyrophosphate (PP i ) concentrations (5). The severity and expressivity of hypophosphatasia depends on the nature of the TNAP mutation (6). TNAP is present in MVs (7), and it has been proposed that the inorganic phosphate (P i )-generating activity of TNAP is required to generate the P i needed for hydroxyapatite crystallization (8-10). However, the ability of TNAP to hydrolyze PP i also has been hypothesized to be important to promote osteoblastic mineralization (11, 12), because PP i suppresses the formation and growth of hydroxyapatite crystals (13). In fact, heritable extracellular PP i deficiencies are models of ectopic calcification such as ankylosing spinal hyperostosis and pathologic soft-tissue ossification (14-16). PP i is produced by the nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity of a family of isozymes that include PC-1, B10͞PDNP3, and autotaxin (17-19). However, PC-1 seems to be the only NTPPPH present in MVs (20). TNAP knockout (KO) mice (21-23) recapitulate the heritable metabolic disease hypophosphatasia (5), whereas PC-1-null mice display hypermineralization abnormalities similar to cartilage calcification in osteoarthritis (14) and ossification of the posterior longitudinal ligament of the spine (15).We previously identified PC-1 as the likely NTPPPH isozyme to act on the same pathway with TNAP as antagonistic regulators of extracellular PP i concentrations (20). In this paper, we have tested the hypothesis that bone abnormalities caused by the lack of TNAP could be counterbalanced by the removal of PC-1 and vice versa. We show that bone mineralization in double-KO mice lacking both TNAP and PC-1 is essentially normal, providing evidence that TNAP and PC-1 are key regulators of bone mineralization by determining the normal steady-state levels of PP i . Our work suggests that TNAP and PC-1 may be useful therapeutic targets for the treatment of bone mineralization abnormalities. Materials and MethodsAkp2 and Enpp1 KO Mice. The generation and characterization of the Akp2 KO mice has been reported (22,23). These Akp2 KO mice were hybrids of C57BL͞6 ϫ 129͞J mouse strains. The generation of the Enpp1 KO mice has been reported briefly (24). These Enpp1 KO mice were hybrids of C57BL͞6 ϫ 129͞SvTerJ mouse strains. Akp2͞Enpp1 double-heterozy...

Matrix vesicles (MVs) are extracellular, 100 nM in diameter, membrane-invested particles selectively located at sites of initial calcification in cartilage, bone, and predentin. The first crystals of apatitic bone mineral are formed within MVs close to the inner surfaces of their investing membranes. Matrix vesicle biogenesis occurs by polarized budding and pinching-off of vesicles from specific regions of the outer plasma membranes of differentiating growth plate chondrocytes, osteoblasts, and odontoblasts. Polarized release of MVs into selected areas of developing matrix determines the nonrandom distribution of calcification. Initiation of the first mineral crystals, within MVs (phase 1), is augmented by the activity of MV phosphatases (eg, alkaline phosphatase, adenosine triphosphatase and pyrophosphatase) plus calcium-binding molecules (eg, annexin I and phosphatidyl serine), all of which are concentrated in or near the MV membrane. Phase 2 of biologic mineralization begins with crystal release through the MV membrane, exposing preformed hydroxyapatite crystals to the extracellular fluid. The extracellular fluid normally contains sufficient Ca2+ and PO4(3-) to support continuous crystal proliferation, with preformed crystals serving as nuclei (templates) for the formation of new crystals by a process of homologous nucleation. In diseases such as osteoarthritis, crystal deposition arthritis, and atherosclerosis, MVs initiate pathologic calcification, which, in turn, augments disease progression.

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Vesicles have been identified within the cartilage matrix of the upper tibial epiphyseal plate of normal mice . They were seen at all levels within the plate and usually did not appear to be in contact with cartilage cells . Vesicles were concentrated within the matrix of the longitudinal septa from the proliferative zone downward . They varied considerably in size (-300 A to --I µ) and in shape . They were bounded by unit membranes, and contained materials of varying density including, rarely, ribosomes. A close association was demonstrated between matrix vesicles and calcification : in the lower hypertrophic and calcifying zones of the epiphysis, vesicles were found in juxtaposition to needle-like structures removed by demineralization with ethylenediaminetetraacetate and identified by electron diffraction as hydroxyapatite and/or fluorapatite crystal structure--the former being indistinguishable from the latter for most cases in which electron diffraction methods are employed . Decalcification also revealed electron-opaque, partially membrane-bounded structures within previously calcified cartilage of the epiphyseal plate and underlying metaphysis which corresponded in size and distribution to matrix vesicles . It is suggested that matrix vesicles are derived from cells and that they may play a role in initiating calcification at the epiphysis .

The presence of skeletal hypomineralization was confirmed in mice lacking the gene for bone alkaline phosphatase, ie, the tissue-non-specific isozyme of alkaline phosphatase (TNAP). In this study, a detailed characterization of the ultrastructural localization, the relative amount and ultrastructural morphology of bone mineral was carried out in tibial growth plates and in subjacent metaphyseal bone of 10-day-old TNAP knockout mice. Alizarin red staining, microcomputerized tomography (micro CT), and FTIR imaging spectroscopy (FT-IRIS) confirmed a significant overall decrease of mineral density in the cartilage and bone matrix of TNAP-deficient mice. Transmission electron microscopy (TEM) showed diminished mineral in growth plate cartilage and in newly formed bone matrix. High resolution TEM indicated that mineral crystals were initiated, as is normal, within matrix vesicles (MVs) of the growth plate and bone of TNAP-deficient mice. However, mineral crystal proliferation and growth was inhibited in the matrix surrounding MVs, as is the case in the hereditary human disease hypophosphatasia. These data suggest that hypomineralization in TNAP-deficient mice results primarily from an inability of initial mineral crystals within MVs to self-nucleate and to proliferate beyond the protective confines of the MV membrane. This failure of the second stage of mineral formation may be caused by an excess of the mineral inhibitor pyrophosphate (PPi) in the extracellular fluid around MVs. In normal circumstances, PPi is hydrolyzed by the TNAP of MVs' outer membrane yielding monophosphate ions (Pi) for incorporation into bone mineral. Thus, with TNAP deficiency a buildup of mineral-inhibiting PPi would be expected at the perimeter of MVs.

Skeletal cells control the initiation of mineralization in vivo and determine the selective distribution pattern of mineralization by releasing calcification-initiating, submicroscopic, extracellular matrix vesicles (MVs) at selected sites in the extracellular matrix. The overall objective of this review is to outline what is currently known about the mechanisms of MV biogenesis and mineral initiation, while emphasizing recent observations that enhance our understanding of these mechanisms. Data from studies on the general mechanism of biogenesis of outer membrane vesicles and the formation and function of non-skeletal matrix vesicles is presented to stimulate thought concerning the possible biological functions that these structures may share with MVs.

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

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