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At least 973 records · Page 54Linked to original sources
PubMed1992

Immunohistochemical localization of matrix proteins in the femoral joint cartilage of growing commercial pigs.

The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular-epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycan was more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

Direct correlation of collagen matrix deformation with focal adhesion dynamics in living corneal fibroblasts.

The purpose of this study was to develop and apply a new model for investigating how the organization and movement of cell-matrix adhesion sites correlate with force generation by corneal fibroblasts on a fibrillar collagen extracellular matrix. Primary cultures of rabbit corneal fibroblasts were transfected using a vector encoding GFP-zyxin to allow visualization of adhesion sites. Cells were plated at low density on top of 100 micro m thick fibrillar collagen lattices embedded with 2 micro m diameter red fluorescent beads. Time-lapse imaging was performed at one minute intervals for up to 3 hours. At each time interval, GFP-zyxin, bead and DIC images were acquired in rapid succession using filter wheels. Cells were treated with cytochalasin D and/or Triton X-100 at the end of each experiment. The movements of adhesions and nearby matrix landmarks were measured and correlated from the time-lapse digital images, and the size, intensity and orientation of the adhesions were quantified. GFP-zyxin was detected in adhesions of transfected corneal fibroblasts as confirmed using vinculin counterstaining. Time-lapse imaging revealed extensions and retractions of cell processes and displacements of the fiduciary beads that were similar to control cells. Extending processes exhibited the most complex behavior, with new adhesions continuously forming at the leading edge while existing adhesions moved backward in a retrograde fashion. This process generated tractional forces as indicated by pulling in of the extracellular matrix in front of the cell. Interestingly, during extension, adhesions along the ventral surface of the cell body generally moved toward those at the tip, resulting in contractile-like shortening and matrix compression at the base of lamellipodia. Overall, a high correlation was found between both the magnitude (R=0.87, P<0.001) and direction (R=0.98, P<0.001) of the adhesions and nearby matrix displacements. Cytochalasin D induced rapid and reversible disassembly of adhesions, cell elongation and matrix relaxation, including decompression at the base of the lamellipodia. This new experimental model allows direct, dynamic assessment of cell-matrix interactions on a fibrillar collagen matrix. Our results are consistent with the previously described 'frontal towing' model of cell motility and demonstrate for the first time that this mechanism is employed by cells interacting with a fibrillar extracellular matrix.

Actins↗
PubMed2001

Experimental observations of a nuclear matrix.

Nuclei are intricately structured, and nuclear metabolism has an elaborate spatial organization. The architecture of the nucleus includes two overlapping and nucleic-acid-containing structures - chromatin and a nuclear matrix. The nuclear matrix is observed by microscopy in live, fixed and extracted cells. Its ultrastructure and composition show it to be, in large part, the ribonucleoprotein (RNP) network first seen in unfractionated cells more than 30 years ago. At that time, the discovery of this RNP structure explained surprising observations that RNA, packaged in proteins, is attached to an intranuclear, non-chromatin structure. Periodic and specific attachments of chromatin fibers to the nuclear matrix create the chromatin loop domains that can be directly observed by microscopy or inferred from biochemical experiments. The ultrastructure of the nuclear matrix is well characterized and consists of a nuclear lamina and an internal nuclear network of subassemblies linked together by highly structured fibers. These complex fibers are built on an underlying scaffolding of branched 10-nm filaments that connect to the nuclear lamina. The structural proteins of the nuclear lamina have been well characterized, but the structural biochemistry of the internal nuclear matrix has received less attention. Many internal matrix proteins have been identified, but far less is known about how these proteins assemble to make the fibers, filaments and other assemblies of the internal nuclear matrix. Correcting this imbalance will require the combined application of biochemistry and electron microscopy. The central problem in trying to define nuclear matrix structure is to identify the proteins that assemble into the 10-nm filaments upon which the interior architecture of the nucleus is constructed. Only by achieving a biochemical characterization of the nuclear matrix will we advance beyond simple microscopic observations of structure to a better understanding of nuclear matrix function, regulation and post-mitotic assembly.

Animals↗
PubMed2007

Mechanical regulation of matrix metalloproteinases.

Matrix metalloproteinases can degrade and modify almost all components of the extracellular matrix hence their enzymatic activity is tightly regulated under physiological conditions. Primary modes of enzyme regulation include transcriptional control, zymogen activation and dynamic inhibition by tissue inhibitors of matrix metalloproteinases. Recent studies have demonstrated that mechanical regulation of matrix metalloproteinases largely operate through these regulatory pathways. Over the last decade a large cohort of studies have been conducted on many tissue/cell types using diverse loading parameters in vivo and in vitro suggesting that mechanical load is essential in maintaining normal tissue function via the matrix metalloproteinases. However there may be a mechanically-regulated homeostasis, with cells responding to and interpreting growth factors and other biochemical signals within the context of mechanical forces to provide a suitable cellular matrix metalloproteinase response. On the contrary, mechanical overload can result in unrestrained matrix metalloproteinase activities eventually leading to matrix degradation, mechanical dysfunction and failure of the tissue. In this chapter, the effect of mechanical load on matrix metalloproteinase expression will be reviewed, and the signal transduction pathways involved in modulating the metabolic homeostasis of various tissues including blood vessels, intervertebral disc and components of the synovial joint with emphasis on articular cartilage discussed. Both mechanically-induced stimulation and inhibition of matrix metalloproteinases will be discussed and placed into context with their potential relevance to disease.

Animals↗
PubMed2004

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration.

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast--mineralized matrix complex. Histologically, at this time point a tissue structure with a "connective tissue"-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of immunodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

Animals↗
PubMed2006

Comparative effects of interferon-gamma and all- trans retinoic acid on secreted and surface-associated matrix metalloproteinase-9 expression of human monocytes.

Matrix metalloproteinase-9 is involved in inflammation and tumor progression. We previously demonstrated that interferon type I (alpha/beta) and II (gamma) inhibit matrix metalloproteinase-9 (92kDa) gene expression on lymphocytes from patients with B chronic lymphocytic leukemia and human monocytes. Since all-trans retinoic acid (ATRA) can regulate some interferon -responsive genes, we studied here the effects of all-trans retinoic acid onto matrix metalloproteinase-9 levels in these cells. By using RT-PCR, ELISA and zymography experiments, we showed that all-trans retinoic acid down-regulated matrix metalloproteinase-9 synthesis (mRNA,protein) and secretion. The inhibitory action of all-trans retinoic acid toward matrix metalloproteinase-9 was however not associated with the STAT1/IRF-1 pathway involved in interferon-mediated matrix metalloproteinase-9 inhibition indicating that all-trans retinoic acid did not bypass IFN receptor signaling. Using flow cytometry, we detected on the surface of monocytes low expression of matrix metalloproteinase-9 and Fc-gammaRI, and high expression of HLA-DR, beta1 and beta2 integrins. Enhancement of Fc-gammaRI and HLA-DR on monocytes by interferon-gamma, but not by all-trans retinoic acid, was accompanied by up-regulation of surface matrix metalloproteinase-9. Furthermore, we showed that all-trans retinoic acid down-regulated matrix metalloproteinase-9 expression in lymphocytes of untreated patients with early stage B chronic lymphocytic leukemia. Together, our data suggest the potential relevance of all-trans retinoic acid as a pharmacological tool to attenuate matrix metalloproteinase-9 secretion in pathological situations.

Antigens, Surface↗
PubMed1988

Septal distribution and the relationship of matrix vesicle size to cartilage mineralization.

To estimate matrix vesicle distribution between longitudinal and transversal septal matrix in the proliferative, hypertrophic and calcifying zones of normal epiphyseal cartilage, the volume density of matrix vesicles in the longitudinal septal matrix was compared to that of total extralacunar matrix. The results confirm the qualitative observation by Anderson that matrix vesicles are located mainly in the longitudinal septa. To elucidate whether cartilage mineralization can be related to the disappearance of matrix vesicles of particular size classes, epiphyseal growth cartilage from three groups of animals were studied: normal rats, rats with florid rickets and rats with early healing rickets. The study was focused on the proliferative, hypertrophic and calcifying zones and in each zone the matrix vesicles were classified into four size classes: 1, less than or equal to 50 nm; 2, 51-67 nm; 3, 68-84 nm; 4, greater than or equal to 85 nm. The results show that the decrease in volume density previously demonstrated in normal rats to a large extent is due to a decreased number of larger vesicles. In florid rickets the decrease in this size group is much smaller while the values for healing rachitic animals fall between those of florid rachitic rats and those of controls. The data indicate that the decrease in the number of larger vesicles, which represents a considerable vesicle volume, is of particular importance. The heterogenous change in the concentration of matrix vesicles of different size classes during cartilage mineralization as well as under conditions of arrested calcification, is compatible with the existence of a matrix vesicle subpopulation of larger size.

Animals↗
PubMed1986

Mesangial cell hillocks. Nodular foci of exaggerated growth of cells and matrix in prolonged culture.

To examine the capability of glomerular mesangial cells (MCs) to produce extracellular matrix, the authors studied MCs in culture by light and electron microscopy as well as immunocytochemistry. MCs were obtained from isolated rat glomeruli and maintained up to 12 weeks in medium containing 20% fetal calf serum. MC outgrowth of primary culture and of up to three subcultures showed characteristic organization consisting of bands of elongated or stellate intertwined cells. After confluency at 10-16 days, MCs continued to grow in irregular multilayers. MCs produced extracellular matrix material within 2-4 days after plating, and large amounts of matrix accumulated with time. By 2-3 weeks, foci of exaggerated MC proliferation, matrix secretion, and necrotic cell debris formed nodular protrusions, which gradually produced large hillocks. Immunocytochemical studies of MC outgrowths were performed on culture plates or on sectioned material with the use of specific rabbit polyclonal antibodies to isolated matrix proteins and FITC-conjugated, affinity-purified second antibodies. Within 3 days of culture, MCs elaborated fibronectin and collagen Types I, III, IV, and V. With time, strands of matrix, notably in the central mass of hillocks, stained extensively for these constituents. Staining for laminin was less pronounced. Smooth muscle cell myosin was regularly found on distinct intracellular fibrils and in the extracellular material of hillocks. Electron microscopy revealed the hillocks to be composed of elongated cells on the surface and stellate cells intermingled with matrix and necrotic cell debris in the core. The results show that proliferating MCs can be maintained in homogeneous culture for a prolonged time period. MCs produce large amounts of the extracellular matrix proteins (Type IV and V collagen, fibronectin, laminin), which are found in normal glomeruli. Cultured MCs also produce interstitial collagen Types I and III. MC hillocks show the nodular accumulation of matrix similar to that seen in the mesangium of diseased glomeruli. It is concluded that the in vitro model of prolonged MC outgrowth may facilitate the investigation of factors that govern mesangial matrix production. Such a model could be used in examining the response of the mesangium to defined inflammatory or metabolic stimuli.

Animals↗
PubMed1987

Inhibition of growth and induction of differentiation in a malignant human glioma cell line by normal leptomeningeal extracellular matrix proteins.

We devised a model system to study the effects of extracellular matrix proteins on the malignant phenotype of an anaplastic glioma cell line, U 343 MG-A. Well-characterized cultures derived from normal human leptomeninges were grown to confluence and maintained for 2 weeks. The leptomeningeal cells were then removed with base and detergent, leaving behind an extracellular matrix enriched in laminin, fibronectin, type I and IV collagen, and procollagen III. U 343 MG-A tumor cells planted on top of this normal extracellular matrix were profoundly growth inhibited compared with glioma cells grown on plastic alone. Glioma cells grown on the extracellular matrix developed multiple, slender processes and assumed a more differentiated astrocytic phenotype; immunostains for glial fibrillary acidic protein revealed a more extensive intracytoplasmic network of intensely staining filaments than in control glioma cells. When glioma cells grown on the extracellular matrix were analyzed by an enzyme-linked immunosorbent assay for glial fibrillary acidic protein, the amount of this intermediate filament per cell was increased 20-fold compared with glioma cells growing on plastic. The growth and differentiation of U 343 MG-A glioma cells in flasks coated with purified fibronectin or laminin was not significantly perturbed; however, glioma cell cultures grown in flasks coated with purified type I or IV collagen showed decreased cellular proliferation, stellate cell formation, and increased levels of glial fibrillary acidic protein per cell compared with glioma cells growing on plastic. Gelatin gel analysis showed that U 343 MG-A glioma cells growing on plastic secreted a 65,000-D metalloproteinase that was not secreted by glioma cells grown on the leptomeningeal extracellular matrix. We conclude that in this system, the extracellular matrix of a normal human leptomeningeal culture substantially inhibited the proliferation of and induced differentiation in an anaplastic glioma cell line. Our analysis of single components of the extracellular matrix suggests that these effects may be mediated in part by type I and IV collagen. The mechanism by which the leptomeningeal extracellular matrix inhibits glioma cell proliferation may be by diminishing tumor-associated protease secretion so that the degradation of extracellular matrix macromolecules in the tumor cell microenvironment is prevented and tumor cell migration becomes less likely.

Cell Differentiation↗
PubMed1986

Invasion of reconstituted basement membrane matrix by metastatic human tumor cells.

A gel-like reconstituted basement membrane matrix containing type IV collagen, laminin, entactin, nidogen, and heparan sulfate proteoglycan was used to examine the invasive properties of human HT1080 fibrosarcoma cells. Within several hours after seeding, the tumor cells initiated a random migration, leaving behind channels etched in the surface of the matrix. Eventually the channels became interconnected into a complex network. As the tumor cells proliferated, the channels became filled until the surrounding matrix was gradually dissolved. Cells then migrated outward, forming the typical disorganized cell monolayer normally observed when fibrosarcoma cells are cultured on plastic surfaces. In contrast to the fibrosarcoma cells, normal skin fibroblasts, while able to attach to the matrix, exhibited minimal migration, tracking, and invasion during the same time period. When tumor cells were seeded onto thick layers of matrix, the cells ultimately invaded downward into the matrix, leaving behind open tunnels. At the front of the invading cells, long irregular pseudopodia projected in the direction of movement. Electron microscopy demonstrated these filopodial and lamellopodial projections to directly extend into the surrounding matrix, with focal clearing of the matrix in the immediate vicinity of these invading pseudopodia. Thus, tumor cell invasion of extracellular matrices, including basal lamina, may proceed by the formation of specialized pseudopodia that not only form adhesion contacts with the matrix but also provide an efficient mechanism for the focal hydrolysis of the matrix at the site of directed cell movement.

Basement Membrane↗
PubMed1983

Role of plasminogen in matrix breakdown by neoplastic cells.

Destruction of the extracellular matrix is often observed during tumor invasion, and proteolytic enzymes may participate actively in the degradation of matrix proteins. The present report elucidates the role of plasminogen in the degradation by tumor cells of an in vitro elaborated extracellular matrix. Matrices produced by rat smooth muscle cells in the presence of [3H]proline or [3H]fucose were used as substrates for human fibrosarcoma cells (HT-1080), mouse melanoma cells (B16F1), or human rhabdomyosarcoma cells (RD). All three cell lines degraded part of the glycoprotein compartment of the matrix. HT-1080 cells digested the matrices in a density-dependent manner, and while matrix glycoprotein degradation was plasminogen-dependent at the beginning of the experiment and at low cell densities, the zymogen was not essential for further glycoprotein digestion at high cell densities. Depletion of plasminogen from the growth medium resulted in a threefold reduction of matrix degradation by B16F1 cells showing a distinct plasminogen dependency at low cell numbers. RD cells digested only matrix glycoproteins, and this degradation was completely dependent on the presence of plasminogen at all cell densities. These results suggested that plasmin generated from plasminogen by a tumor cell-associated plasminogen activator may be most important for matrix hydrolysis at low cell densities, and while certain tumor cell lines showed a definite plasminogen-independent matrix degradation with increased cell numbers, other neoplastic cells hydrolyzed the matrix only in the presence of the zymogen at all cell densities.

Animals↗
PubMed1995

Cultured endothelial cells regulate platelet adhesion to their extracellular matrix by regulating its von Willebrand factor content.

Endothelial cells and their extracellular matrix formed in vitro are often used as a model for subendothelium in studies on platelet-vessel wall interaction. We have characterized the influence of culture conditions of endothelial cells on the formation of extracellular matrix and on the interaction of the matrix with platelets. Passage number, time of confluence, serum concentration and the addition of heparin, growth factors and antibiotics to the culture medium were varied and the extracellular matrices were isolated. The amount of fibronectin and von Willebrand factor present in the matrix were measured and the number of platelets adhering to these matrices after perfusion with citrated whole blood at a shear rate of 1000 s-1 was determined. A three times increase of the amount of von Willebrand factor in the matrix was found when the serum concentration was increased from 2.5% to 30%. When the passage number of the cells was increased or the period during which the cells were at confluence was extended, the amount of von Willebrand factor in the matrix was decreased up to 50%. Addition of heparin or ECGS (endothelial cell growth supplement) decreased the von Willebrand factor content in the matrix. Addition of penicillin or streptomycin to the culture medium had no influence on the amount of von Willebrand factor deposited in the matrix or secreted into the medium, however, other antibiotics such as gentamycin and neomycin decrease the amount of von Willebrand factor in the matrix. No influence on the amount of fibronectin in the matrix was found under all conditions tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Platelets↗
PubMed1995

Modulation of opioid binding associated with nuclear matrix and nuclear membranes of NG108-15 cells.

Opioid binding sites were found in nuclear matrix preparations from NG108-15 neurohybrid cells. Binding parameters of delta-specific radioligands indicated that high-affinity binding sites discovered in purified nuclei were present in nuclear membranes and nuclear matrix fractions. Agonists bind with low affinity, if at all, to nuclear matrix preparations. Neither sensitivity of agonist binding to the GTP analog 5-guanylylimidodiphosphate nor adenylyl cyclase activity were detected in this fraction, suggesting the presence of guanine nucleotide binding regulatory protein/effector uncoupled sites. Opioid inhibition of basal and forskolin-stimulated adenylyl cyclase activity was found in nuclear membrane preparations. Cycloheximide treatment of cells inhibited opioid binding to nuclear membrane fractions to a greater extent than that associated with membranes sedimenting at 20,000 x g (P20) or nuclear matrix. Colchicine, a microtubule disrupter and inhibitor of receptor internalization, caused up-regulation of nuclear membrane and P20 opioid receptors and a loss in nuclear matrix associated sites. Taxol, a microtubule stabilizing agent, prevented the effect of colchicine. Etorphine-elicited down-regulation increased nuclear matrix associated binding while diminishing that in nuclear membranes and P20 fractions. Agonist-induced desensitization completely abolished nuclear matrix binding. In vitro preincubation of nuclear matrix preparations with protein kinase A catalytic subunit mimicked the desensitization effect. Forskolin treatment of cells potentiated nuclear matrix and P20 binding. These data suggest that nuclear membrane opioid receptors represent newly synthesized molecules en route to the cell surface, whereas nuclear matrix contains internalized delta sites.

Analgesics↗
PubMed1994

Aggrecan synthesized by mature bovine chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools.

Proteoglycans synthesized by chondrocytes in alginate beads are found in two compartments: the cell-associated matrix and the further removed matrix (Häuselmann, H. J., Aydelotte M. B., Schumacher B. L., Kuettner K. E., Gitelis, S. H., and Thonar, E. J.-M. A. (1992) Matrix 12, 116-129). To study the metabolism of aggrecan in these two compartments, mature bovine articular chondrocytes in alginate beads were pulsed with [35S]sulfate for 30 min or 16 h on day 7 of culture and then chased in isotope-free medium for up to 21 days. At different times, the two matrix pools were separately isolated, and the 35S-proteoglycans quantified, purified, and characterized. Radiolabeled aggrecan molecules exhibited a very long average half-life in the beads (t1/2 = 95 days). In contrast, small non-aggregating proteoglycans, which made up approximately 4% of the 35S-proteoglycans synthesized, were rapidly lost from the beads (t1/2 = < 24 h). Approximately half the 35S-aggrecan subunits, representing mostly molecules which showed a delay in ability to form aggregates in the presence of exogenous hyaluronan and link protein, spent only a short time (t1/2 = 4 h) in the cell-associated matrix before moving into the further removed matrix. They exhibited a much longer average half-life in the beads than 35S-aggrecan molecules which became resident of the cell-associated matrix (t1/2 = > 95 days versus 15 days). Radiolabeled aggrecan subunits in the two matrix compartments had a similar average hydrodynamic size and polydispersity; importantly, the size of these molecules did not change during the chase period. Catabolism of 35S-aggrecan in the cell-associated matrix was the only significant contributor to the appearance in the medium of partially degraded 35S-aggrecan which had lost the ability to bind to hyaluronan. These results strongly suggest aggrecan molecules which reside in the pericellular and territorial matrix compartments in close proximity to the chondrocytes have a much faster rate of turnover than their counterpart in the interterritorial areas further removed from the cells.

Aggrecans↗
PubMed1994

alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood.

Previous studies have shown that thrombin binds to the extracellular endothelial matrix and remains biologically active. In the present study, the role of matrix-bound alpha-thrombin in thrombus formation was investigated by utilizing a model system of thrombogenesis. Plastic cover-slips coated with either matrix-bound alpha-thrombin or matrix-bound active site inactivated thrombin (DIP-alpha-thrombin) were positioned in parallel-plate perfusion chambers and subsequently exposed to non-anticoagulated human blood at a venous wall shear rate of 100/s. The blood was drawn directly from an antecubital vein by a roller pump placed distally to the perfusion chamber. The thrombotic deposits on the matrix, fibrin deposition and platelet thrombus volume, were morphologically evaluated. Matrix-bound alpha-thrombin enhanced the fibrin deposition and thrombus volume on matrices of non-stimulated endothelium with 91% (P < 0.001) and 94% (P < 0.05), respectively. In contrast, binding of DIP-alpha-thrombin to matrices of stimulated endothelium reduced the fibrin deposition by 33% (P < 0.05), but had no effect on the platelet thrombus volume. Translocation of thrombin molecules from upstream matrix areas to binding sites farther downstream on the matrix was indicated in experiments with matrices of stimulated endothelium, which showed enhanced fibrin deposition on downstream areas. Our findings are compatible with a prominent role for matrix-bound alpha-thrombin in thrombogenesis, and in particular on endothelial matrices without tissue factor. The role of matrix-bound alpha-thrombin on tissue factor containing matrices appears less prominent, although it is significant.

Blood Coagulation↗
PubMed1994

Assembly of amino-terminal fibronectin dimers into the extracellular matrix.

Fibronectin is a dimeric adhesion molecule that consists of three types of repeating modules. Adherent cells bind soluble fibronectin and incorporate it into insoluble fibrils in the extracellular matrix. The amino-terminal 70-kDa portion of fibronectin mediates binding to the cell surface, but amino-terminal fragments do not accumulate in the extracellular matrix. The ninth type I and first type III modules, the cell adhesion region, and the cysteines that form the interchain disulfide bonds have also been implicated in matrix assembly. To further define which regions of fibronectin are essential for matrix assembly, we generated a dimeric protein (d70 kDa) in which the 70-kDa amino terminus is directly linked to the last 51 amino acids of fibronectin, which contain the cysteines involved in interchain disulfide bonding. d70 kDa bound to cells and accumulated in the extracellular matrix. Incorporation of d70 kDa into the extracellular matrix was dependent upon protein synthesis; in cycloheximide-treated cultures that lacked a pre-existing matrix, d70 kDa accumulated in the extracellular matrix only in the presence of intact fibronectin. Monomeric 70-kDa protein was not incorporated into the matrix in the presence or absence of cycloheximide. These data indicate that fibronectin molecules containing only the amino-terminal 70-kDa region and the carboxyl-terminal 51 amino acids can become assembled into the extracellular matrix.

Animals↗
PubMed1993

Type X collagen does not bind to matrix vesicles.

Cultured chick embryo tibial hypertrophic chondrocytes released matrix vesicles and Type X collagen into the culture medium. When the culture medium was filtered through a 0.1 micron nitrocellulose filter, both the matrix vesicles, measured as alkaline phosphatase, and the Type X collagen were retained quantitatively. None of the other collagen types in the culture medium was retained on the filter. Dissolution of the matrix vesicles on the filter in detergent solutions resulted in quantitative solubilization of the Type X collagen also. These results suggested that the Type X collagen was intimately associated with the matrix vesicles. However, when membrane filters that were composed of materials other than nitrocellulose, and that had a range of pore sizes, were used to filter the culture medium, the ratios of total matrix vesicles to total Type X collagen retained on the filters ranged from 53 (polysulfone membranes) to 0.3 (nitrocellulose-cellulose acetate membranes). Thus it is concluded that the quantitative retention of matrix vesicles and Type X collagen on 0.1 micron nitrocellulose filters was due to true filtration of the matrix vesicles and to selective adsorption of the Type X collagen. Removal of the noncollagenous extensions from the Type X collagen by brief pepsin or trypsin treatment converted the Type X collagen to its 45 kDa collagenous domain, which was no longer retained on nitrocellulose filters, suggesting that the adsorption of the Type X collagen on the filters was through one or both of its noncollagenous extensions. When the culture medium was subjected to ultracentrifugation to pellet the matrix vesicles, 98% of the membrane-associated alkaline phosphatase (matrix vesicles) was pelleted but only 15-20% of the Type X collagen was recovered in the pellet. These results indicate that matrix vesicles and Type X collagen are not the associated products of hypertrophic chondrocyte.

Alkaline Phosphatase↗
PubMed1995

Comparison between allograft plus demineralized bone matrix versus autograft in anterior cervical fusion. A prospective multicenter study.

STUDY DESIGN: This study analyzed the fusion results of an allograft-demineralized bone matrix composite versus autograft in a prospective series of patients undergoing surgery for cervical disc disease. OBJECTIVES: To determine the fusion rates of allograft-demineralized bone matrix composite in anterior cervical fusion as compared with the gold standard autograft. SUMMARY OF BACKGROUND DATA: For the anterior cervical fusion, the use of freeze-dried allograft is well documented in the literature, citing its effectiveness and inferior fusion rates. The use of demineralized bone matrix in conjunction with freeze-dried allograft in anterior cervical fusion has not been reported. METHODS: This study was done in a prospective fashion in two medical centers. One group received autograft from the anterior iliac crest, whereas others received freeze-dried allograft augmented with demineralized bone matrix (Grafton, Osteotech, Inc., Shrewsbury, New Jersey). For the autograft group, the standard Smith-Robinson grafting technique was used. For the allograft composite group, demineralized bone matrix was pasted onto the freeze-dried allograft and into the disc space before graft insertion. The autograft group consisted of 38 patients with age ranging 26-71 years (mean, 46.1 years) and follow-up periods of 12-33 months (mean, 18.4 months). There were 19 one-level, 17 two-level, and two three-level fusions. Similarly, the allograft group consisted of 39 patients with age ranging 28-80 years (mean, 48.0 years) with follow-up period of 12-31 months (mean, 17.5 months). There were 19 one-level, 16 two-level, and four three-level fusions. Clinical and radiographic follow-up evaluations were completed at 3-month intervals. Radiographs taken 12 months after surgery were analyzed blindly. RESULTS: Pseudarthrosis developed in 46.2% of patients (33.3% of levels) in the allograft-demineralized bone matrix group compared with 26.3% (22% of levels) in the autograft group (P = 0.11 for patients, P = 0.23 for levels). For patients undergoing two-level fusions, 37.5% of allograft-demineralized bone matrix failed compared with 23.5% of autografts. For single-level fusions, 47.4% of allograft patients developed a pseudarthrosis compared with 26.3% in the autograft group. Graft collapse of > or = 3 mm was noted in 11% of the autograft group versus 19% in the allograft-demineralized bone matrix group (P = 0.32). Graft collapse of > or = 2 mm occurred in 24.4% of autograft patients compared with 39.7% of the allograft-demineralized bone matrix group (P = 0.09). Smokers had an increased rate of pseudarthrosis (47.1%) compared with nonsmokers (27.9%, P = 0.13). CONCLUSIONS: The study revealed that the allograft-demineralized bone matrix construct gives a higher rate of graft collapse and pseudarthrosis when compared with autograft in a prospective series, although the differences were not statistically significant. The pseudarthrosis rate in the series may be high because of the large percentage of smokers and radiographic evaluation techniques. For the purpose of solid radiographic fusion, the use of autograft is recommended in anterior cervical surgery until other acceptable osteoinductive materials are developed.

Adult↗