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PubMed2001

[Actin is located in the nucleus and nuclear matrix of HeLa cells].

HeLa cell nuclei were isolated and the nuclear matrix specimens were prepared. After labelled with an anti-actin antibody and FITC-conjugated secondary antibody, both the nuclei and nuclear matrix specimens were observed to emanate specific yellow-green fluorescence. The fluorescent signals in the nuclear matrix were much stronger than that in the nuclei, and the signals in former were widespread throughout the whole structure while that in the latter were mainly defined to their peripheral regions. A 43 kD band was revealed in nuclei and nuclear matrix specimens by SDS-PAGE, and was then proved to be actin by Western blot, confirming the immuno-fluorescence observations. When stained with TRICT-conjugated phalloidin, both the nuclei and nuclear matrix specimens were found to give off specific, red fluorescent signals which represent the location of F-actin (filamentous actin), and the pattern of TRITC signals distribution in the nuclei and nuclear matrix showed similarity with that of FITC signals. The existence and significance of actin and F-actin in the nuclei and nuclear matrix were discussed.

Actins↗
PubMed2001

Extracellular fibrillar matrix architecture of human placental villi at term.

The spatial organisation of the extracellular fibrillar matrix of normal human placental villi at term can be directly visualised by scanning electron microscopy after 2N-NaOH maceration technique. By these methods, the extracellular fibrillar matrix of placental villi appears as a continuous network of isolated collagen fibrils and/or small fibrillar bundles interwoven each other. This sort of "collagenous fibrillar skeleton" forms the axis of chorionic villi and connects them with the basal plates running through the whole villous system of the placenta. Significant variations in the spatial arrangement as well as in the quantity of the extracellular matrix is observed at different levels of the villous ramification. Within the stem villi, the fibrillar extracellular matrix are abundant and, whereas the fibrils near the villous surface run parallel to the longitudinal axis of the villous (outer fibrils), those located in the inner core of the villous are arranged circularly around the wall of the fetal vessels (inner fibrils). In mature intermediate and terminal villi, viceversa, the extracellular fibrillar matrix is significandy reduced and the fibrils are mainly organised in a thin circular layer around the capillaries and sinusoids. The present study demonstrated the existence of a diverse spatial architecture of the extracellular matrix that results to be peculiar to the various levels of the ramification of the villous tree. Therefore, these morphological data strongly suggest a "compartmentalisation" of the villous tree as suggested by previous immunohistochemical study. Such a highly organised "collagenous fibrillar skeleton" stresses the important mechanical role of the extracellular matrix in sustaining the chorionic fetal vessels and the trophoblastic layer. Furthermore, the fine reticular-meshed network observed within the terminal villi suggests that at this level an additional role ensuring a favourable milieu for active feto-maternal exchanges may exist.

Adult↗
PubMed1992

Effect of matrix systems and polymerization techniques on microleakage of Class II resin composite restorations.

This study investigated the effect of different sized light curing tips (2 mm and 11 mm) with curing techniques associated with different matrix systems (Mylar with reflective wedge and metal with wooden wedge) on microleakage of a posterior resin composite material (P-50) placed using a bonding agent (Scotchbond 2). Eighty Class II mesial and distal slot preparations (40 with enamel gingival margins and 40 with dentin gingival margins) were randomly divided into four equal groups: 1) metal matrix, large tip; 2) Mylar matrix, light emitting wedge, large tip; 3) Mylar matrix, light emitting wedge, small tip; 4) metal matrix, small tip. Three-way ANOVA indicated no statistical differences among groups using small and large curing tips. The results showed significantly more leakage (alpha = 0.05) in the dentin gingival cavosurface margin groups than the enamel cavosurface margin groups. The results also showed significantly greater leakage in the polymerization procedures associated with the metal matrix groups (alpha = 0.05) compared to polymerization procedures associated with the Mylar matrix groups.

Analysis of Variance↗
PubMed1992

Impaired attachment of hepatocytes to extracellular matrix components after chronic ethanol administration.

BACKGROUND: Previous studies have shown that the assembly and properties of the hepatocyte plasma membrane are altered by ethanol administration, indicating possible changes in the receptor-mediated binding of the plasma membrane to extracellular matrix substrates. In the present study, the effects of chronic ethanol consumption on the ability of hepatocytes to attach to various components of the extracellular matrix were investigated. EXPERIMENTAL DESIGN: Rats were pair-fed for 5 weeks with a liquid diet containing either ethanol (as 36% of total calories) or isocaloric carbohydrate. The effects of ethanol treatment on hepatocyte-extracellular matrix interactions was ascertained by determining the ability of isolated hepatocytes to attach to various extracellular matrix substrates. RESULTS: The attachment of hepatocytes, isolated from the ethanol-fed rats, to laminin-coated plates was significantly decreased compared with hepatocytes from chow-fed or pair-fed controls. Greater decreases in attachment were seen when higher numbers of hepatocytes were seeded in the plates. Similar inhibitions of attachment were also observed when fibronectin or type I collagen were used as matrices. Time-course cell attachment assays indicated that the maximum extent of attachment rather than the rate of attachment was primarily altered by chronic ethanol feeding. Hepatocytes from the ethanol-fed rats also detached more readily from the matrix-coated plates than those from the controls. A reduced number of functional surface receptors for matrix components is likely the most important factor that accounts for the ethanol-induced impairment of hepatocyte attachment. CONCLUSIONS: These results indicate that chronic ethanol administration impairs the interactions of hepatocytes with their extracellular matrix and that this defect could lead to alterations of hepatocyte structure and function.

Animals↗
PubMed2004

Reconstruction of rabbit urethra using urethral extracellular matrix.

BACKGROUND: Urethral reconstruction for both congenital and acquired etiologies remains a challenge for most urologic surgeons. Tissue engineering has been proposed as a strategy for urethral reconstruction. The purpose of this study was to determine whether a naturally derived extracellular matrix substitute developed for urethral reconstruction would be suitable for urethral repair in an animal model. METHODS: A urethral segmental defect was created in 20 male rabbits. The urethral extracellular matrix, obtained and processed from rabbit urethral tissue, was trimmed and transplanted to repair the urethral defect. Then, the regenerated segment was studied histologically by haematoxylin-eosin staining and Van Gieson staining at 10 days, 3 weeks, 6 weeks, and 24 weeks postoperation. Retrograde urethrography was used to evaluate the function of the regenerated urethras of 4 rabbits 10 and 24 weeks after the operation. The urodynamics of 4 rabbits from the experimental group and control group I were assessed and compared. In addition, 4 experimental group rabbits were examined by a urethroscope 24 weeks after the operation. RESULTS: At 10 days after operation, epithelial cells had migrated from each side, and small vessels were observed in the extracellular matrix. The matrix and adjacent areas of the host tissue were infiltrated with inflammatory cells. The epithelium covered the extracellular matrix fully at 3 weeks postoperation. Well-formed smooth-muscle cells were first confirmed after 6 weeks, at which point the inflammatory cells had disappeared. At 24 weeks postoperation, the regenerated tissue was equivalent to the normal urethra. Urethrography and urodynamic evaluations showed that there was no difference between normal tissue and regenerated tissue. CONCLUSIONS: Urethral extracellular matrix appears to be a useful material for urethral repair in rabbits. The matrix can be processed easily and has good characteristics for tissue handling and urethral function.

Animals↗
PubMed2004

Albumin is not present in the murine interphotoreceptor matrix, or in that of transgenic mice lacking IRBP.

PURPOSE: The interphotoreceptor matrix mediates interactions between the retinal pigment epithelium, photoreceptors, and Muller cells. Each of these cells contributes to specific proteoglycans, proteins, and growth factors in the interphotoreceptor matrix. Some components, such as interphotoreceptor retinoid binding protein (IRBP), are virtually unique to the interphotoreceptor matrix. It has been proposed that serum albumin, thought to be present in the interphotoreceptor matrix, could act as a surrogate retinoid binding protein within the subretinal space of transgenic mice lacking interphotoreceptor retinoid binding protein. To address this question, we sought to determine whether albumin is present in the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice. METHODS: We examined the distribution of albumin in IRBP-/- mice and IRBP+/+ mice using immunofluorescence and immunoperoxidase histochemistry. RESULTS: The distribution of albumin within the corneal stroma, sclera, and capillaries is consistent with previous work. Serum albumin could not be detected in the interphotoreceptor matrix. The distribution of albumin in IRBP-/- mice was similar to that of their wildtype counterparts. CONCLUSIONS: Serum albumin is not a component of the interphotoreceptor matrix of IRBP+/+ mice or IRBP-/- mice.

Animals↗
PubMed2005

[Repair of thyroid cartilage defects with chondrocyte-allogenous acellular cartilaginous matrix composite in rabbits].

OBJECTIVE: To investigate the feasibility of repairing thyroid cartilage defects by implantation of chondrocyte-allogenous acellular cartilaginous matrix (chondrocyte-ACM) composite in rabbits. METHODS: The thyroid chondrocytes were isolated and co-cultured in vitro with allogenous acellular cartilaginous matrix (ACM) to form the chondrocyte-ACM composite. The composite was analyzed histologically and was used to repair defects of thyroid cartilage. Eighteen New Zealand adult rabbits were made the defect models of thyroid cartilage at the two sides and divided into three groups. The defects were repaired with chondrocyte-ACM composite in the experimental group (n= 6), with simple ACM in the ACM group (n=6) and without any material in the control group (n=6). The animals were sacrificed at 8 weeks after operation. The specimens were evaluated histologically. RESULTS: In vitro, the growth of chondrocytes was observed on the surface of allogenous acellular cartilaginous matrix and no chondrocytes grew inside the matrix. The defect filled with muscle and connective tissues in control group; the lymphocyte infiltration was observed in the matrix and no new cartilage formation occurred at 8 weeks after operation in simple ACM group and experimental group. So the defect repair of rabbits thyroid cartilage failed. CONCLUSION: The allogenous acellular cartilaginous matrix failed to serve as a scaffold for chondrocytes both in vitro and in vivo. The allogenous acellular cartilaginous matrix should be improved.

Animals↗
PubMed1991

Nuclear matrix targets for anticancer agents.

The nuclear matrix of eukaryotic cells comprises a dynamic framework on which DNA is organized into discrete functional units of replication and transcription. There is growing evidence that matrix-associated DNA and proteins are direct targets of a wide range of clinically active anticancer agents. DNA associated with matrix-bound replication and transcription sites has a relatively open conformation and is preferentially damaged by ionizing radiation and certain alkylating agents. Fludarabine phosphate, a purine antimetabolite, inhibits DNA replication by blocking the synthesis of matrix-associated primer RNA and RNA-primed Okazaki fragments. VM-26 and m-AMSA appear to interact specifically with nuclear matrix topoisomerase II, and one mechanism of cellular resistance to these agents is associated with depletion of the matrix enzyme. Studies of the interactions of anticancer agents with targets in the nuclear matrix should provide further insight into the mechanisms by which these agents exert their therapeutic effects.

Alkylating Agents↗
PubMed1991

Reactivity of synthetic peptide analogs of adhesive proteins in regard to the interaction of human endothelial cells with extracellular matrix.

Vascular endothelial cells, providing a nonthrombogenic surface to the lumenal aspect of blood vessels, are anchored to matrix adhesion molecules in the subendothelium through their respective receptors belonging to a superfamily of integrins. We analyzed the reactivity of synthetic peptide analogs of adhesive proteins toward human umbilical vein endothelial cells (HUVEC), assaying their detachment from extracellular matrix and attachment to extracellular matrix components in vitro. Synthetic peptide analogs Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP), Arg-Gly-Asp-Val (RGDV), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Asp-Phe (RGDF), which are analogous to "cell adhesion sites" of fibronectin, vitronectin, von Willebrand factor, and alpha-chain of human fibrinogen, respectively, caused significant detachment of HUVEC from the extracellular matrix in vitro at the concentrations ranging from 0.5 to 1.5 mmol/L. They also interfered with attachment of HUVEC to surfaces coated with subendothelial extracellular matrix or its components. The synthetic peptide analog of HHLGGAKQAGDV, which is homologous to the gamma-chain of human fibrinogen sequence 400-411, did not cause any measurable effect on the integrity of HUVEC monolayers (detachment and attachment). "Hybrid" peptides bearing salient features of both sequences, ie, Ala-Lys-Gln-Arg-Gly-Asp-Phe (AKQRGDF) and Lys-Gln-Arg-Gly-Asp-Phe (KQRGDF), had an attenuated effect on the detachment of HUVEC from extracellular matrix. Thus, the integrity of the human endothelial cell monolayer anchored to the extracellular matrix, as measured in detachment and attachment assays, is disturbed by peptides containing RGD sequence whereas the synthetic peptide His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) is nonreactive.

Amino Acid Sequence↗
PubMed1992

Xyloside-induced disruption of interphotoreceptor matrix proteoglycans results in retinal detachment.

Unique domains of the retinal interphotoreceptor matrix (IPM), termed cone matrix sheaths, are composed largely of chondroitin 6-sulfate proteoglycan in most higher mammalian species. Recent investigations suggest that cone matrix sheaths participate in the maintenance of normal retinal attachment. To investigate the potential functional roles of IPM proteoglycans further, the synthesis of cone matrix sheath chondroitin 6-sulfate proteoglycan was perturbed in vivo. Intravitreal injections of p-nitrophenyl-beta-D-xylopyranoside (xyloside), a sugar that inhibits chondroitin sulfate proteoglycan synthesis, were administered to Yucatan micropigs. Their eyes were examined funduscopically and electroretinographically. At selected times, the eyes were enucleated and examined histochemically and immunohistochemically with various probes directed against cone photoreceptor cells and cone matrix sheaths. The IPM was affected selectively after xyloside administration; no inner retinal pathology or dysfunction was detected morphologically or electroretinographically. The degree of xyloside-induced perturbation was dependent on the duration of xyloside exposure and dose. It was classified into three stages, based on morphologic and histochemical criteria. Although all three stages could be observed in a given retina, a single stage typically predominated, depending on the particular dosage regimen. The early stage was characterized by IPM disruption, as evidenced by disorganization of chondroitin 6-sulfate and peanut agglutinin (PNA)-binding glycoconjugates. Cone photoreceptor cell outer segment degeneration and markedly decreased chondroitin 6-sulfate immunoreactivity distinguished the middle stage. During the late stage, there was a near complete absence of both immunoreactive chondroitin 6-sulfate and PNA-binding glycoconjugates in the IPM. Shallow retinal detachments that appeared funduscopically as patches of retinal whitening frequently were observed after moderate durations of xyloside exposure; these progressed peripherally with continued xyloside exposure. Histologically, the areas of retinal whitening corresponded to regions in which cone matrix sheaths were split transversely (ie, in a plane perpendicular to the longitudinal axes of the photoreceptor cell outer segments) or were separated completely from cone outer segments. Similar effects were not observed in control eyes. These results suggest that adhesion between the neural retina and retinal pigmented epithelium may be dependent, in part, on continuous synthesis of cone matrix sheath-associated proteoglycans and, potentially, other IPM proteoglycans. In addition, these proteoglycans appear to be necessary for the maintenance of cone photoreceptor cell outer segment integrity.

Animals↗
PubMed1991

Activation of the coagulation mechanism on tumor necrosis factor-stimulated cultured endothelial cells and their extracellular matrix. The role of flow and factor IX/IXa.

Infusion of tumor necrosis factor (TNF) into tumor-bearing mice led to intravascular clot formation with fibrin deposition in microvessels in the tumor bed in close association with the vessel wall, which could be prevented by active site-blocked factor IXa (IXai). This observation prompted us to examine the role of the intrinsic system in activation of the coagulation mechanism on TNF-stimulated human endothelial cell monolayers and endothelial-derived matrix during exposure to purified coagulation factors or flowing blood. Treatment of endothelial cells in intact monolayers with TNF induced expression of the procoagulant cofactor tissue factor (TF) in a dose-dependent manner, and after removal of the cells, TF was present in the matrix. TNF-treated endothelial cell monolayers exposed to blood anticoagulated with low molecular weight heparin induced activation of coagulation. Addition of IXai blocked the procoagulant response on TNF-treated endothelial cells, and consistent with this, the presence of factor IX/VIIIa enhanced endothelial TF/factor VII(a) factor X activation over a wide range of cytokine concentrations (0-600 pM). When TF-dependent factor X activation on endothelial cells was compared with preparations of subendothelium, the extracellular matrix was 10-20 times more effective. IXai blocked TF/factor VII(a) mediated activated coagulation on matrix, but only at lower concentration of TNF (less than 50 pM). Similarly, enhancement of factor Xa formation on matrix by factors IX/VIIIa was most evident at lower TNF concentrations. When anticoagulated whole blood flowing with a shear of 300 s-1 was exposed to matrices from TNF-treated endothelial cells, but not matrices from control cells, fibrinopeptide A (FPA) generation, fibrin deposition, and platelet aggregate formation were observed. FPA generation could be prevented by a blocking antibody to TF and by active site-blocked factor Xa (Xai) over a wide range of TNF concentrations (0-600 pM), whereas IXai only blocked FPA generation at lower TNF concentrations (less than 50 pM). Activation of coagulation on matrix from TNF-stimulated endothelial cells was dependent on the presence of platelets, indicating the important role of platelets in propagating the reactions leading to fibrin formation. These observations demonstrate the potential of cytokine-stimulated endothelium and their matrix to activate coagulation and suggest the importance of the intrinsic system in factor Xa formation on cellular surfaces.

Animals↗
PubMed1988

Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization.

Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. Core (protein) interactions seem to be responsible for the association of the proteoglycan with the extracellular matrix.

Antibodies, Monoclonal↗
PubMed1988

Energy dispersive X-ray microanalysis of sulfated glycosaminoglycans in cartilage matrix stained with alcian blue 8GX.

X-ray spectra were recorded from 400-700 nm matrix areas of 0.5 micron sections prepared from the articular cartilages of 15- and 23-year-old human cadavers. The X-ray microanalysis was carried out (i) on untreated material; (ii) after removing sulfate group by a methylation procedure; (iii) after staining with a copper containing cationic phatolcyanin dye, alcian blue 8GX, preceded by carboxymethylation. K alpha peaks of sulphur could be detected in methylated (i.e. desulfated) samples. These peaks probably indicated the presence of sulphur-containing amino acids in different matrix proteins. Consequently, the measurements of sulphur despite its general use cannot be recommended for the X-ray microanalysis of sulfated glycosaminoglycans of cartilage matrix. K alpha peaks of copper could be identified after carboxymethylation and staining with alcian blue. After carboxymethylation, alcian blue can only be bound to the dissociated sulfate groups of glycosaminoglycans in the cartilage matrix. According to our spectrophotometric studies, approximately one molecule of alcian blue combined with one sulfate group. These data suggested that this technique could be used for semiquantitative estimation of sulfated glycosaminoglycans in small areas of the cartilage matrix. Using this method, we found a higher occurrence of sulfated glycosaminoglycans in the territorial matrix than in the interterritorial matrix of the intermediate and deep zones of the human articular cartilage.

Adolescent↗
PubMed1987

Heterogeneity in the production of collagens and fibronectin by morphologically distinct clones of a human tumor cell line: evidence for intratumoral diversity in matrix protein biosynthesis.

Recent studies of murine tumor models and certain human tumor cell lines have provided evidence for intratumor heterogeneity in expression of extracellular matrix receptors and in the elaboration of matrix-degrading enzymes. However, little is known about possible intratumoral heterogeneity in the production of matrix macromolecules. We have, therefore, examined the biosynthesis and secretion of matrix proteins by cells derived from a polyclonal human cell line (JH-17) established from a large cell undifferentiated carcinoma of the lung. For the present studies, we focused on the production of collagens and structural glycoproteins by two phenotypically different aneuploid clones, designated C13 and C22. These clones were distinctive in their inability to grow in soft agar or to form tumors in nude mice and had identical DNA contents. Tumor cells were labeled with [3H]proline and the newly synthesized proteins accumulating in the culture medium were identified using biochemical and immunologic techniques. Clone C13 secreted at least three genetically distinct collagens, including type V procollagen (PC), type IV procollagen, and a type VIII-like collagen. By contrast, the clone C22 synthesized fibronectin, and a single bacterial collagenase-sensitive and pepsin-resistant component consistent with type I trimer. These studies emphasize the potential diversity of matrix proteins synthesized by neoplastic cells and suggest that there is intratumoral heterogeneity in matrix protein biosynthesis in vivo. These studies further suggest that tumor-derived matrix may be altered during tumor progression or cell selection in vivo.

Carcinoma, Small Cell↗
PubMed1988

Factor XIII cross-linking of fibronectin at cellular matrix assembly sites.

We describe the effect of activated Factor XIII (Factor XIIIa, plasma transglutaminase) on the incorporation of plasma fibronectin into extracellular matrix by cultured human fibroblasts. In the absence of added Factor XIIIa, fibronectin binds to cultured fibroblast cell layers and is assembled into disulfide-bonded multimers of the extracellular matrix. When Factor XIIIa was included in the binding medium of skin fibroblasts, accumulation of 125I-fibronectin in the deoxycholate-insoluble matrix was increased. Fibronectin accumulating in the cell layer was cross-linked into nonreducible high molecular weight aggregates. The 70-kDa amino-terminal fragment of fibronectin inhibited the binding and cross-linking of 125I-fibronectin to cell layers, whereas fibrinogen had little effect. When 125I-fibronectin was incubated with isolated matrices or with cell layers pretreated with cytochalasin B, it did not bind and could not be cross-linked by Factor XIIIa into the matrix. HT-1080 human fibrosarcoma cells bound exogenous fibronectin following treatment with dexamethasone; Factor XIIIa cross-linked the bound fibronectin and caused its efficient transfer to the deoxycholate-insoluble matrix. These results indicate that exogenous fibronectin is susceptible to Factor XIIIa-catalyzed cross-linking at cellular sites of matrix assembly. Thus, Factor XIIIa-mediated fibronectin cross-linking complements disulfide-bonded multimer formation in the stabilization of assembling fibronectin molecules and thus enhances the formation of extracellular matrix.

Cell Line↗
PubMed1986

[Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles].

Matrix vesicles are present in the calcifying front and in the site of callus formation of fracture heeling. In calcifying process, matrix vesicles have important roles. The metalloprotease was isolated from matrix vesicles and subsequently characterized. Matrix vesicles obtained from chicken epiphysial cartilage by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles and isolated by Sephadex G-150 gel filtration. Disc electrophoresis of the enzyme gave a single protein band. The matrix vesicle protease had a MW of 33,000 daltons, an optimal pH of 7.2, and was inhibited 100% by 0.1 mM EDTA and 0.2 mM o-Phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by o-phenanthroline was reversed by Co2+, Zn2+, Fe2+ and Cu2+. The protease released from the matrix vesicle at the calcifying front could degrade non-collagenous protein moieties which inhibit precipitation of minerals in the extra-vesicular matrix and thus facilitate mineralization.

Animals↗
PubMed1988

The role of mineralization in experimental models of osteogenetic induction with decalcified bone matrix.

The role of mineralization was studied in an experimental model of osteoinduction consisting of the implant of decalcified bone matrix in the rat muscle. After vascular connective tissue invasion of the matrix, the first phenomena is the appearance of hydroxyapatite crystals in the area of chondroid metaplasia from where the deposit of calcium-phosphate is extended to the surrounding decalcified matrix. The recalcified areas act as a substratum for the neo-osteogenesis. The appearances observed reproduced those of enchondral ossification, where the first osteoblasts differentiated on the calcified cartilaginous matrix and began to supply new bone matrix. Non-decalcified bone matrix implants were surrounded by a connective tissue capsule with features identical to those of a foreign body reaction and with total absence of vascular invasion. The absence of induction in this type of implant may be related either to masking of the osteoinducing protein in the calcified matrix, or to the absence of cells with osteogenic differentiation potential due to the lack of cellular invasion of the implant.

Animals↗
PubMed1984

Mineralization by matrix vesicles.

Matrix vesicles are widely regarded as the initial site of calcification in epiphyseal growth plate cartilage, in growing bone and in predentin. This opinion has recently been challenged on grounds that the early aqueous methods used for electron microscopic tissue preparation may have produced an erroneous picture by causing mineral dislocation. However, this argument has now been refuted by multiple investigators throughout the world using a variety of anhydrous methods coupled with electron probe analysis to show convincingly that matrix vesicles are, indeed, associated with initial mineral. Matrix vesicles appear to mineralize by concentrating calcium and phosphate at a protected site close to the inner leaflet of the vesicle membrane. Calcium may be attracted by its affinity for acidic phospholipids of the vesicle membrane, and phosphate may be concentrated by the action of transmembrane phosphatases of the matrix vesicle membrane. Evidence is accumulating to suggest that alkaline phosphatase of the matrix vesicle membrane functions as a phosphotransferase or phosphate vector, transporting PO4 across the vesicle membrane. The mechanism(s) of matrix vesicle biogenesis are discussed including budding from the plasma membrane (for which there is much support), cell degeneration (for which there is gathering support), extrusion of intracytoplasmic vesicles (for which there is weak support), and extracellular subunit self-assembly (for which there is little support). It is suggested that none of these mechanisms is necessarily exclusive, thus more than one mechanism may function in the same tissue. Finally, it is noted that in many calcific diseases, ranging from arthritis to atherosclerosis, mineralization is initiated by extracellular membrane-invested vesicles which are probably analogous to the matrix vesicles of skeletal tissues.

Animals↗