Membrane-bound Matrix Vesicles Research Articles

Biological stenciling of mineralization in the skeleton: Local enzymatic removal of inhibitors in the extracellular matrix.

Biomineralization is remarkably diverse and provides myriad functions across many organismal systems. Biomineralization processes typically produce hardened, hierarchically organized structures usually having nanostructured mineral assemblies that are formed through inorganic-organic (usually protein) interactions. Calcium‑carbonate biomineral predominates in structures of small invertebrate organisms abundant in marine environments, particularly in shells (remarkably it is also found in the inner ear otoconia of vertebrates), whereas calcium-phosphate biomineral predominates in the skeletons and dentitions of both marine and terrestrial vertebrates, including humans. Reconciliation of the interplay between organic moieties and inorganic crystals in bones and teeth is a cornerstone of biomineralization research. Key molecular determinants of skeletal and dental mineralization have been identified in health and disease, and in pathologic ectopic calcification, ranging from small molecules such as pyrophosphate, to small membrane-bounded matrix vesicles shed from cells, and to noncollagenous extracellular matrix proteins such as osteopontin and their derived bioactive peptides. Beyond partly knowing the regulatory role of the direct actions of inhibitors on vertebrate mineralization, more recently the importance of their enzymatic removal from the extracellular matrix has become increasingly understood. Great progress has been made in deciphering the relationship between mineralization inhibitors and the enzymes that degrade them, and how adverse changes in this physiologic pathway (such as gene mutations causing disease) result in mineralization defects. Two examples of this are rare skeletal diseases having osteomalacia/odontomalacia (soft bones and teeth) - namely hypophosphatasia (HPP) and X-linked hypophosphatemia (XLH) - where inactivating mutations occur in the gene for the enzymes tissue-nonspecific alkaline phosphatase (TNAP, TNSALP, ALPL) and phosphate-regulating endopeptidase homolog X-linked (PHEX), respectively. Here, we review and provide a concept for how existing and new information now comes together to describe the dual nature of regulation of mineralization - through systemic mineral ion homeostasis involving circulating factors, coupled with molecular determinants operating at the local level in the extracellular matrix. For the local mineralization events in the extracellular matrix, we present a focused concept in skeletal mineralization biology called the Stenciling Principle - a principle (building upon seminal work by Neuman and Fleisch) describing how the action of enzymes to remove tissue-resident inhibitors defines with precision the location and progression of mineralization.

Compounded PHOSPHO1/ALPL Deficiencies Reduce Dentin Mineralization

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1-/- mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1-/-;Alpl+/-). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1-/- mice, and the almost complete absence of matrix vesicles in the Phospho1-/-;Alpl+/- mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.

192 MODULATED EXOSOME SECRETION BY VASCULAR SMOOTH MUSCLE CELLS IS A NOVEL REGULATORY MECHANISM OF VASCULAR CALCIFICATION

Vascular calcification is a regulated pathological process similar to bone formation which is mediated by vascular smooth muscle cells (VSMCs) undergoing osteogenic transdifferentiation. Initiation of vascular calcification occurs in small membrane-bound matrix vesicles (MVs), secreted by VSMCs into the extracellular matrix however the mechanisms regulating MV biogenesis and secretion are unclear. Fetuin-A, a circulating protein abundant in MVs was used to trace the origin of VSMC-derived MVs. Alexa488-labelled fetuin-A was rapidly uptaken by human VSMCs and appeared in the late endosomal system and multivesicular bodies (MVBs) indicating that MVs are secreted from the endosomal compartment similar to exosomes; extracellular vesicles originating from MVBs and secreted by the range of cells. Biochemical analysis of MVs showed that they were enriched with exosomal markers, CD9 and CD63. Furthermore, inhibition of sphingomyelin phosphodiesterase 3 (SMPD3), which in involved in exosome biogenesis abrogated MV secretion by VSMCs confirming that MVs represent exosomes. Calcifying conditions induced exosome secretion by VSMCs and this was accompanied by elevated SMPD3 expression. Importantly, an inhibition of SMPD3 prevented VSMC calcification. Phenotypic modulation of VSMCs and loss of the contractile phenotype resulted in elevated exosome secretion while contractile VSMCs secreted significantly less exosomes. Notably, elevated extracellular calcium rapidly induced calcification of synthetic VSMCs whilst contractile VSMCs did not calcify. In agreement with this data, abundant immunohistochemical staining for CD63 was observed only in atherosclerotic human aorta in close association with calcified areas with little staining detected in the healthy vessel wall. Taken together this study demonstrates that MVs originate from MVBs and are secreted via the exosomal pathway. Loss of the contractile phenotype and mineral imbalance promote VSMC calcification by enhanced exosome secretion. Targeting the mechanisms of VSMC exosome secretion and/or their loading with calcification inhibitors may provide novel therapeutic interventions aimed for the prevention of vascular calcification.

Expression of Dynamin II in Odontoblast During Mouse Tooth Development

Odontoblasts secrete a collagen-based matrix and release numerous membrane-bound matrix vesicles, which are involved in dentin formation during tooth development. Dynamin II is a GTPase protein that contributes a variety of vesicular budding events, such as endocytotic membrane fission, caveolae internalization and protein trafficking in the Golgi apparatus. However, the expression and function of dynamin II in odontoblasts has not been reported. Therefore, this study examined the expression and possible role of dynamin II in odontoblasts during tooth development and mineralization. The levels of mRNA and protein expression in MDPC23 cells were significantly high at the early stages of differentiation and then decreased gradually thereafter. Immunohistochemistry showed that dynamin II was not expressed near the region of the odontoblasts at embryonic day 17 (E17) and E21. However, dynamin II was expressed strongly in the odontoblast layer at postnatal day 1 (PN1) and decreased gradually at PN3 and PN5. In addition, at PN15 in the functional stage, the dynamin II protein was also expressed in the odontoblast process as well as adjacent to the nuclear region. In conclusion, dynamin II may be involved in the transport of vesicles containing collageneous and non-collageneous proteins for dentin formation in odontoblast, suggesting that it is a good nanomolecule as a candidate to regulate the secretion of collagen on the bone and other nano material.

Extracellular matrix calcification in chronic kidney disease

Extracellular matrix (ECM) mineralization occurring in soft tissues such as blood vessels is highly prevalent in patients with chronic kidney disease. This problem is now recognized as an active and regulated process involving a complex interaction of inducers and inhibitors. This review outlines new findings on calcification at the level of the ECM, with a focus on recent studies evaluating the pathogenesis of calcification of the vasculature. Mechanisms promoting vascular calcification include dysregulation of mineral metabolism, especially high levels of phosphate and calcium, release of membrane-bound matrix vesicles from vascular smooth muscle cells (VSMCs), and formation of apoptotic bodies. Subsequent changes in the phenotype of VSMCs to osteoblast-like cells follow, inducing ECM formation and attracting local factors involved in the mineralization process (similar to the process of bone formation). Along with the loss of various calcification inhibitors, there is also likely a role for elastin and elastin-degrading enzymes, sodium-phosphate co-transporters, and the transmembrane protein klotho, as potential key regulators of ECM calcification. Mechanisms of ECM calcification are not completely understood, although likely to be multifactorial. Increasing insight provides potential therapeutic options to reduce the burden of ectopic calcification and subsequent cardiovascular morbidity and mortality.

Arteriosclerosis, vascular calcifications and cardiovascular disease in uremia

Arterial calcification in chronic kidney disease (CKD) is associated with increased cardiovascular risk. The mechanisms responsible for arterial calcification include alterations of mineral metabolism and expression of mineral-regulating proteins. Arterial calcification is similar to bone formation, involving differentiation of vascular smooth muscle cells (VSMCs) into phenotypically distinct osteoblast-like cells. Elevated phosphate and/or calcium trigger a concentration-dependent increase of calcium precipitates in VSMC in vitro. The calcification is initiated by VSMC release of membrane-bound matrix vesicles and formation of apoptotic bodies. The presence of serum prevents these changes, indicating the presence of calcification inhibitors. Arterial calcification occurs in two sites: the tunica intima and tunica media. Intimal calcification is a marker of atherosclerotic disease and is associated with arterial stenotic lesions. Medial calcification influences outcome by promoting arterial stiffening whose principal consequences are left-ventricular hypertrophy and altered coronary perfusion. Aortic stiffness is an independent predictor of all-cause and cardiovascular mortality in CKD patients. Age, duration of dialysis, smoking and diabetes are risk factors for the development of arterial calcification in end-stage renal disease. Oversuppression of parathyroid hormone and low bone turnover potentiate the development of arterial calcification. Arterial disease in CKD patients is characterized by extensive calcification. Evidence has accumulated pointing to the active and regulated nature of the calcification process. Elevated phosphate and calcium may stimulate sodium-dependent phosphate cotransport involving osteoblast-like changes in cellular gene expression. Arterial calcification is responsible for stiffening of the arteries with increased left-ventricular afterload and abnormal coronary perfusion as the principal clinical consequences.

Human Vascular Smooth Muscle Cells Undergo Vesicle-Mediated Calcification in Response to Changes in Extracellular Calcium and Phosphate Concentrations

Patients with ESRD have a high circulating calcium (Ca) x phosphate (P) product and develop extensive vascular calcification that may contribute to their high cardiovascular morbidity. However, the cellular mechanisms underlying vascular calcification in this context are poorly understood. In an in vitro model, elevated Ca or P induced human vascular smooth muscle cell (VSMC) calcification independently and synergistically, a process that was potently inhibited by serum. Calcification was initiated by release from living VSMC of membrane-bound matrix vesicles (MV) and also by apoptotic bodies from dying cells. Vesicles released by VSMC after prolonged exposure to Ca and P contained preformed basic calcium phosphate and calcified extensively. However, vesicles released in the presence of serum did not contain basic calcium phosphate, co-purified with the mineralization inhibitor fetuin-A and calcified minimally. Importantly, MV released under normal physiologic conditions did not calcify, and VSMC were also able to inhibit the spontaneous precipitation of Ca and P in solution. The potent mineralization inhibitor matrix Gla protein was found to be present in MV, and pretreatment of VSMC with warfarin markedly enhanced vesicle calcification. These data suggest that in the context of raised Ca and P, vascular calcification is a modifiable, cell-mediated process regulated by vesicle release. These vesicles contain mineralization inhibitors derived from VSMC and serum, and perturbation of the production or function of these inhibitors would lead to accelerated vascular calcification.

Ultrastructural modifications of the extracellular matrix upon calcification of growth plate cartilage as revealed by quick-freeze deep etching technique.

The ultrastructural changes in cartilage matrix that occur during calcification have been examined in chick epiphyseal growth plate cartilage prepared by quick-frozen, deep-etched, and rotary shadowed replicas. The extracellular cartilage matrix contains a reticular network closely associated with an extensive network of collagen. The components of the reticular network, including thick and thin filaments, are attached directly to the cell membrane, matrix vesicle membrane, and collagen fibrils. This network, which interconnects the matrix vesicles and collagen, fills the extracellular matrix. The dimensions of the reticular network seem to remain almost constant in size from the reserve and proliferative zones to the calcifying zone. The collagen fibrils seem to consist of subfibrillar structures that branch and anastomose. In optimally quick-frozen, deep-etched, prepared collagen, a cross-banding pattern was exposed. Globular structures stud the collagen fibrils, which gradually diminish in number from the reserve zone down to the calcifying zone. The matrix vesicles, when fractured, showed a granular appearance. In most cases, the fracture plane passed through the bilayer of the matrix vesicle membrane. The true surface of the matrix vesicle membrane, therefore, was exposed after deep etching. At the calcifying zone, crystal deposition had occurred in needle-like and/or plate-like form within the membrane-bound matrix vesicles. The reticular network was still intact in the vicinity of the calcified matrix, but in the intercrystalline space, neither the reticular structure nor the globular structure was detectable. Within the calcified matrix, both reticular and granular structures had disappeared from the interfibrillar space of the collagen fibrils.

Matrix vesicles isolated from apical pulp of rat incisors: Crystal formation in low Ca×Pi ion-product medium containing β-glycerophosphate

The ultrastructure of crystal formation in vitro associated with extracellular membrane-bound matrix vesicles (MV) isolated from rat incisor pulp was studied in Dulbecco's modified Eagle's medium (DMEM) supplemented with an organic phosphate, Na-beta-glycerophosphate (BGP). Matrix vesicles were isolated from basal regions of the pulps using a collagenase digestion and ultra-centrifugation method. Isolated MV contained alkaline phosphatase (ALP) activity and had diameters of 30-200 nm. Membrane structures of the isolated MV were well preserved. Incubation of MV in DMEM in the presence of BGP caused the development of bilaminar electron densities associated with the vesicle membrane. These preceded crystal deposition which was observed in the culture medium after 3 days. Both heat-inactivated MV incubated with BGP, and fresh MV incubated in the absence of BGP failed to show crystal formation, even after 3 days. Staining of demineralized sections of mineralized MV with uranyl acetate and lead citrate, revealed numerous needle-like structures similar in shape to the untreated crystals. Electron diffraction patterns of the newly formed crystals revealed a pattern consistent with hydroxyapatite. The requirement of BGP for mineralization of these MV and the long lag time before crystal formation is probably due to the low calcium (Ca) x inorganic phosphate (Pi) ion product in the original medium. The requirement of ALP activity which would cause hydrolysis of BGP and a rise in Pi would favor the precipitation of biologic apatite from the culture medium.

Ultrastructure of matrix vesicles in chick growth plate as revealed by quick freezing and freeze substitution.

The ultrastructure of extracellular membrane-bound matrix vesicles (MVs), their biogenesis, and the surrounding matrix in chick tibial growth plate were studied after quick freezing and freeze substitution (FS) in an organic solvent. There were several notable differences in the ultrastructural preservation of cartilage when FS was used as compared with conventional fixation. The ultrastructural appearance of MVs after FS was extremely variable. Within the MVs, intravesicular filaments, amorphous material, and membrane-associated undercoat structures were observed. Intravesicular filaments, similar in diameter to microfilaments seen in the cytoplasm, were attached to the inside of MV membranes. This observation indicates the similarity of MV membranes and the plasma membrane. In some MVs in the proliferative zone an electron-dense material was present along the inner side of the MV membrane. In the prehypertrophic zone, crystalline material often appeared within the electron-dense material, which may be a precursor form of hydroxyapatite. The earliest crystals observed were in MVs but not in the extracellular matrix. Regarding MV formation, in addition to budding from cell surfaces and to cellular disintegration, this study also indicates that a sequential process of extrusion of preformed cytoplasmic structures may occur. Also, small MVs measuring 25-40 nm seem to arise from the disruption of large MVs. This is a previously unreported observation on MV biogenesis. FS preserves proteoglycans in the cartilage matrix as a fine, filamentous network. Initial extracellular calcification was not associated with this network.

Ultrastructural observations on chick bone processed by quick-freezing and freeze-substitution.

The ultrastructure of newly formed bone was examined with the use of quick-freezing followed by freeze-substitution. Osteoblasts and young osteocytes were characterized by a smooth cell contour, whereas old osteocytes were irregular in shape. The plasma and intracytoplasmic membranes were clearly identifiable as trilaminar substructures. With the method described herein the tissue is handled in the anhydrous state. Thus mitochondrial granules could be demonstrated in all samples, since their preservation is not affected by non-aqueous solutions. The matrices of intact mitochondria were densely stained with post-staining. The contents of the Golgi complex, rough-surfaced endoplasmic reticulum (RER), nuclear envelope, vesicles, and vacuoles were stained to various degrees. Lacunar spaces were always filled with flocculent and filamentous materials, and the plasma membrane was in direct contact with them. Membrane-bounded matrix vesicles were clearly visible within the osteoid extracellular matrix which was the initial site of mineral crystal deposition. In heavily mineralized bone matrix, the periodic pattern of collagen fibrils was retained, and the electron density of mineralized matrix in freeze-substituted and unstained sections which had been floated on ethylene glycol was greater than that encountered in sections processed in aqueous reagents.

The origin of psammoma bodies in the human arachnoid villi.

The fine structure of the human arachnoid villi was studied to clarify the origin of psammoma bodies. Within the villous surface layer, collagen fibrils and fine granular material clustered forming microcores of variable caliber measuring up to 10 microns. An early stage of psammoma body formation was seen more frequently in these villous microcores than in the meningocytic whorls. The villous microcores contained a large number of membrane-free matrix granules as well as a small number of membrane-bound matrix vesicles and matrix minerals. The matrix granules were irregularly oval structures with electron-lucent halo, measuring 0.05-0.70 micron in diameter. Hydroxyapatite crystals were frequently precipitated within and around the matrix granules which aggregated with calcifying matrix vesicles and matrix minerals. Numerous calcifying matrix granules were present within and around enlarging psammoma bodies. The matrix granules may serve as the principal calcification nidi of psammoma bodies in the human arachnoid villi. The possible mechanisms of matrix granule biogenesis are extrusion of preformed arachnoid cell structures or secretion of fine granular material with its extracellular assemblage.

Ultrastructural study of the formation of psammoma bodies in fibroblastic meningioma.

The fine structure of psammoma bodies was examined in four cases of fibroblastic meningioma. In general, large numbers of various-sized calcified bodies (psammoma bodies) were scattered among the interstitial fibers. In these bodies, the smallest calcific site was found in the extracellular membrane-bound matrix vesicles, which measured approximately 0.1 to 0.2 mu in diameter. In addition, extracellular "matrix giant bodies," with or without hydroxyapatite aggregates and measuring up to 3 mu in diameter, were frequently encountered. These bodies were apparently invested with single, double, or multiple concentric walls averaging nearly 0.1 mu thick. The presumably originated from the neoplastic cells as a consequence of cytoplasmic residuals associated with cellular degeneration or necrotic cell processes. Hydroxyapatite crystals precipitated repeatedly within the bodies. The precipitate may gradually aggregate within the bodies, and gather in clusters, resulting in a large psammoma body. Finally, collagen fibers around the calcified giant bodies accrued deposits of apatite crystals to make a huge psammoma body. These findings suggest that both matrix giant bodies and matrix vesicles may serve as initial nidus of calcification of psammoma bodies in fibroblastic meningioma. Consequently, this mineralization process may represent a certain dystrophic calcification of meningocytic cells.

The morphology of the calcification front in articular cartilage. Its significance in joint function.

Biochemical and histochemical studies have indicated that there is specific cellular activity in the region of the calcification front of articular cartilage implying that a regulation process takes place there. Using scanning and transmission electron microscopy and light microscopy to examine tissue sections of both undecalcified and decalcified articular cartilage in the region of the calcification front, we have looked at its morphology with particular reference to its cellular control. Our observations show that physiological calcification is an active process under cellular control and is related to the presence of extracellular membrane-bound matrix vesicles.

Electron optical and analytical observations of rat growth plate cartilage prepared by ultracryomicrotomy: The failure to detect a mineral phase in matrix vesicles and the identification of heterodispersed particles as the initial solid phase of calcium phosphate deposited in the extracellular matrix

Electron microscopy, electron diffraction, and high-spatial-resolution electron probe X-ray microanalysis have been used to characterize certain ultrastructural components observed in undecalcified, unfixed, and unstained thin sections of epiphyseal growth plate cartilage from tibiae of normal 4-week-old rats. The tissue was prepared anhydrously by dry ultracryomicrotomy. Electron microscopy revealed electron-dense mitochondrial granules (500–1000 Å in diameter) within chondrocytes of proliferative and hypertrophic cartilage zones. The mitochondrial granules in frozen thin sections generated no distinct reflections on electron diffraction. X-ray spectra from individual granules showed both calcium and phosphorus with a molar Ca/P ∼0.8–1.1. There was no apparent decrease in the number of mitochondrial granules as extracellular mineralization increased toward distal portions of the growth plate. Electron microscopy of the extracellular regions of proliferative and hypertrophic cartilage zones showed matrix vesicles (1000–2500 Å in diameter) and small, electron-dense particles (200–800 Å in diameter). No mineral particles within or associated with the membrane-bound matrix vesicles could be observed by electron microscopy in any region of the growth plate examined, and only phosphorus could be detected in individual vesicles on X-ray microanalysis. The small, electron-dense particles were not membrane limited; they were distributed in a heterodispersed fashion and found in the same cartilage regions in which matrix vesicles were present, as well as in regions proximal to those in which matrix vesicles occurred. These small, heterodispersed structures were not apparent in sections of rat cartilage fixed and stained by aqueous methods. The electron-dense, heterodispersed particles in frozen sections produced no coherent pattern on electron diffraction and generated calcium and phosphorus with a molar Ca/P ∼0.9–1.0 following X-ray microanalysis. The electron optical and analytical data are consistent with the conclusion that the small heterodispersed particles represent the first solid phase of calcium phosphate deposited in the extracellular matrix of rat epiphyseal growth plate cartilage. When the same tissue was examined after preparation by conventional aqueous techniques, matrix vesicles were observed containing distinct and relatively large crystals characterized by weak calcium and phosphorus on electron probe microanalysis. The comparative results obtained from completely unfixed tissue and from tissue exposed to aqueous solvents raise serious doubts as to whether matrix vesicles contain a solid phase of calcium phosphate in vivo and consequently question the proposed role of the calcification of matrix vesicles in tissue mineralization.

Ultrastructural features of the epithelial-mesenchymal interface in an ameloblastic fibro-odontoma.

The ultrastructural features of the epithelial-mesenchymal interface in a case of an ameloblastic fibro-odontoma was studied with special reference to possible signs of "inductive" processes. In most parts of the tumor, the odontogenic epithelium was separated from the connective tissue by a thick rim of a finely filamentous meshwork in which a basal lamina was occasionally observed. Mesenchymal cells were seen to touch the filamentous meshwork but no membrane bound matrix vesicles were recorded. Small areas of dentin-like tissue were found in the juxtaepithelial connective tissue while enamel-like areas and spherical calcified masses were encountered in epithelial islands. The organic matrix in relation to the enamel-like tissue consisted of either tubular fibers or a fine-granular material. It was assumed that the tubular matrix component directed the formation of long enamel-like crystals, and that the fine-granular matrix was degraded tubular fibers in which spherical calcified masses might arise. Spherical calcified masses could be found in separate follicles also where they were related to a fine-fibrillar matrix or collagenous material. The cell layers forming the wall of the islands had a great resemblance to those of an enamel organ, but the findings of dentin-free, enamel-like areas are not compatible with the inductive theory of normal odontogenesis.

Ultrastructural studies on the origin and structure of matrix vesicles in bone of young rats.

Tibiae of young rats were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in order to study the origin and structure of matrix vesicles in periosteal bone. SEM studies have shown that periosteal osteoblasts have elongated processes with globular structures of 0.1 micron in diameter attached to the cell surface and processes. Similar structures were found to cover the inner surface of osteoblastic lacunae. The SEM studies have further shown that in the periosteal surface (forming bone) the above-described globules once mineralized, aggregated to form larger, nonhomogeneous mineralized spherules in which, by proper treatment with NaOCl, hydroxyapatite crystals could be exposed. Endosteal osteoblasts had fewer processes, devoid of the globular structures. Similarly, osteocytic and osteoclastic processes, although elongated and numberous, were not covered by the globular structures. In the matrix, collagen fibers of forming bone were randomly orientated, while in the deeper areas of bone they formed bundles with a longitudinal orientation. TEM studies have shown that the structures found on the osteoblastic surface and in the matrix are membrane-bound matrix vesicles which seem to be formed by budding from cell processes. Preformed membrane-bound vesicles were also observed by TEM inside sections of osteoblastic processes. These vesicles resembled the extracellular matrix vesicles in size and shape, thus giving the impression that at least some of the matrix vesicles are preformed cellular structures. While comparing SEM with TEM, it can be conducted that in bone, as in cartilage, matrix vesicles which probably serve as the initial locus of calcification, are formed directly by osteoblasts.

Pyrophosphate stimulation of calcium uptake into cultured embryonic bones. Fine structure of matrix vesicles and their role in calcification

The onset of mineralization in embryonic chick femurs was studied as a model for the initiation of biological calcification. Electron microscopy confirmed the presence of calcifying matrix vesicles within newly formed bone, and showed that these vesicles were the initial site of crystal deposition. Matrix vesicles were first seen on day 6 of embryonic development, and already were present in considerable numbers on day 7, at which time mineral deposition was just beginning. As a reflection of initial mineralization the uptake of 45Ca and 40Ca into 7-day-old bones was studied during 2 days in organ culture. A control level of uptake was established using a defined culture medium, P-6. Addition of inorganic pyrophosphate (PP i) to this medium caused a marked increase in calcium uptake into areas of matrix which normally calcify in vivo. The maximal 45Ca uptake, greater than 4-fold, was achieved with 4 μg of P per milliliter of PP i and was partially heat-inhibitable. Since the matrix vesicles are known to be rich in inorganic pyrophosphatase, it is proposed that mineralization is promoted in vesicles by the enzymatic hydrolysis of pyrophosphate. The membrane-bounded matrix vesicles appear to provide the necessary enzymes and environment to concentrate calcium and phosphate for initiating crystal formation.

Electron microscopic studies on guinea pig rib cartilage. Structural heterogeneity and effects of extraction with guanidine-HCl.

The guinea-pig rib cartilage consists of chondrocytes dispersed in an intercellular substance composed of collagen fibrils, often characteristically cross-striated, and polygonal granules. Electron-dense membrane-bounded matrix vesicles are also observed intercellularly, especially in the central, partly calcified zone of the cartilage. With respect to their location in a cross-section of the rib, the chondrocytes differ in size, shape and intracellular fine structure. Thus, three separate types of cells are recognized. Peripheral chondrocytes have a flattened shape and are largely occupied by the nucleus. In the cytoplasm, the granular endoplasmic reticulum is the most extensive organelle. Intermediate chondrocytes are oval or round in shape. The endoplasmic reticulum and the Golgi complex are both prominent. Mitochondria and membrane-bounded cytoplasmic dense bodies are more numerous than in the peripheral cells. The ground cytoplasm often contains a few lipid droplets. In the central chondrocytes, such droplets sometimes fill the entire cytoplasm. Concomitantly, the nucleus is usually completely heterochromatic and the cells are therefore regarded as being metabolically inert.

Isolation and characterization of calcifying matrix vesicles from epiphyseal cartilage.

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.