Development of a three-dimensional extracellular matrix synthesized by human diploid fibroblasts in vitro

1986 ◽  
Vol 84 (1) ◽  
pp. 183-200
Author(s):  
E.E. Qwarnstrom ◽  
R.C. Page
Keyword(s):  
Extracellular Matrix ◽  
Light And Electron Microscopy ◽  
Root Surface ◽  
Human Tooth ◽  
Immunocytochemical Staining ◽  
Cell Contact ◽  
Collagen Fibres ◽  
Intense Staining ◽  
Fibrillar Material ◽  
The Matrix

Development and maturation of an extracellular matrix, synthesized by human gingival fibroblasts, have been studied microscopically. Pairs of demineralized, fibronectin-coated slices of human tooth root, 300 micron thick, were placed on confluent cell layers, defining a 0.5 mm wide space. The cultures were grown under standard conditions with ascorbic acid (50 micrograms ml-1) added daily. At various times up to 13 weeks, the cultures were fixed and the samples prepared for light and electron microscopy. Cells from the monolayer became attached to, and migrated up, the vertical root surface and, during the time studied, completely filled the space between the root slices with an extracellular matrix. A close association was seen between the cell membrane and collagen fibres in the demineralized surface initially. A thin layer of fibrillar material was deposited between the cell and the vertical surface, and eventually an extracellular matrix surrounding the cells and attaching to the root surface was present. Samples fixed in the presence of Ruthenium Red showed intense staining of the fibrillar material, indicating the presence of anionic molecules. Additional cells migrated onto the newly synthesized matrix and up the root surface. Growth of the fibrillar networks on either side, horizontally and vertically, continued and, eventually, an extracellular matrix attaching to the vertical surfaces completely filled the previously empty space. Immunocytochemical staining showed that the matrix contained hyaluronic acid, chondroitin sulphate, dermatan sulphate and fibronectin at this time. Collagen fibres were observed at 6 weeks, and at later times collagen types I, III and V were the primary matrix components. The fibroblasts attaching to the root slice and those present at the edge of the matrix had an elongated, polar form. The cells within the matrix frequently showed a stellate appearance with numerous extended processes, in contact with fibrillar material or collagen fibres. Fibroblast processes were at later times seen to enclose bundles of collagen fibres and to mediate cell-to-cell contact, occasionally via desmosome-like structures. The structure and composition of the matrix and the appearance and apparent behaviour of the cells were similar to that observed in the healing wound. This system thus could provide a model for studying various aspects of regeneration of extracellular matrix.

scholarly journals The role of intermolecular disulfide bonding in deposition of GP140 in the extracellular matrix.

1984 ◽  
Vol 99 (1) ◽  
pp. 105-114 ◽  
Author(s):  
W G Carter
Keyword(s):  
Extracellular Matrix ◽  
Culture Media ◽  
Molecular Forms ◽  
Cell Protein ◽  
Cell Contact ◽  
Relative Molecular Mass ◽  
Disulfide Bonding ◽  
Sequence Of Events ◽  
The Matrix ◽  
Matrix Precipitation

Human WI-38 fibroblasts in cultures synthesized at least three molecular forms of the major, extracellular matrix glycoprotein (GP), GP140: (a) cytoplasmic GP140 (1.2 ng of GP140/micrograms of cell protein) was detergent-soluble, underglycosylated, and possessed detectable levels of intermolecular disulfide bonding; (b) matrix GP140 (3.6 ng of GP140/micrograms of cell protein) was detergent-insoluble, more highly glycosylated and polymerized by intermolecular disulfide bonding, and co-distributed in the extracellular matrix with fibronectin; and (c) released GP140 (2 ng of GP140/micrograms of cell protein per 24 h) was recovered in the conditioned culture media and lacked intermolecular disulfide bonding. Cytoplasmic GP140 was the immediate biosynthetic precursor of the matrix form of GP140. In addition, various human adult and fetal tissues contained a form of GP140 that resembled the fibroblast matrix GP140 in the degree of intermolecular disulfide bonding, relative molecular mass, and immunological reactivity. Analysis of the sequence of events in assembly of GP140 and fibronectin in the extracellular matrix detected the following: (a) fibronectin was first to appear in the extracellular matrix; (b) GP140 accumulated in the cytoplasm, then deposited in the extracellular matrix and co-aligned with the established fibronectin; and (c) maturation of the extracellular matrix proceeded by continued intermolecular disulfide bonding. To evaluate possible roles for intermolecular disulfide bonding in cell interactions, a unique assay system was utilized based on the ability of labeled cells to incorporate radioactive matrix components into a biotinylated exogenous matrix. Precipitation of the biotinylated matrix from extracts of the cultures using avidin indicated: (a) disulfide bonding of radioactive GP140 and fibronectin into the exogenous biotinylated matrix required cell contact with the matrix. The newly deposited GP140 and fibronectin derived from the cells and not from GP140 and fibronectin present in the conditioned culture media. (b) Pro-alpha 1 and Pro-alpha 2 procollagens, present in the culture media, bound to the exogenous matrix in a noncovalent manner and were independent of cell contact. (c) SV40 transformed cells (WI-38 VA13) synthesized released form GP140 but did not deposit GP140 into the biotinylated matrix.

Effects of EDTA, Hyaluronidase and Collagenase on Epiphyseal Cartilage Matrix

1968 ◽  
Vol 26 ◽  
pp. 56-57
Author(s):  
H. Clarke Anderson ◽  
Priscilla R. Coulter
Keyword(s):  
Light And Electron Microscopy ◽  
Matrix Vesicles ◽  
Cartilage Matrix ◽  
Collagen Fibrils ◽  
Epiphyseal Cartilage ◽  
Dense Matrix ◽  
Type Iv ◽  
Bovine Testicular Hyaluronidase ◽  
The Matrix ◽  
Testicular Hyaluronidase

Epiphyseal cartilage matrix contains fibrils and particles of at least 5 different types: 1. Banded collagen fibrils, present throughout the matrix, but not seen in the lacunae. 2. Non-periodic fine fibrils <100Å in diameter (Fig. 1), which are most notable in the lacunae, and may represent immature collagen. 3. Electron dense matrix granules (Fig. 1) which are often attached to fine fibrils and collagen fibrils, and probably contain protein-polysaccharide although the possibility of a mineral content has not been excluded. 4. Matrix vesicles (Fig. 2) which show a selective distribution throughout the epiphysis, and may play a role in calcification. 5. Needle-like apatite crystals (Fig. 2).Blocks of formalin-fixed epiphysis from weanling mice were digested with the following agents in 0.1M phosphate buffer: a) 5% ethylenediaminetetraacetate (EDTA) at pH 8.3, b) 0.015% bovine testicular hyaluronidase (Sigma, type IV, 750 units/mg) at pH 5.5, and c) 0.1% collagenase (Worthington, chromatograhically pure, 200 units/mg) at pH 7.4. All digestions were carried out at 37°C overnight. Following digestion tissues were examined by light and electron microscopy to determine changes in the various fibrils and particles of the matrix.

Extracellular matrix: the gatekeeper of tumor angiogenesis

2019 ◽  
Vol 47 (5) ◽  
pp. 1543-1555 ◽  
Author(s):  
Maurizio Mongiat ◽  
Simone Buraschi ◽  
Eva Andreuzzi ◽  
Thomas Neill ◽  
Renato V. Iozzo
Keyword(s):  
Extracellular Matrix ◽  
Tumor Angiogenesis ◽  
Signaling Cascades ◽  
Cancer Growth ◽  
Cell Behavior ◽  
Metastatic Progression ◽  
Surface Receptors ◽  
The Matrix ◽  
Multiple Signaling

Abstract The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action.

Development of an in vitro Model for the Study of the Response of Equine Tendon Fibroblasts to Injury and Medication

1997 ◽  
Vol 10 (01) ◽  
pp. 6-11 ◽  
Author(s):  
R. F. Rosenbusch ◽  
L. C. Booth ◽  
L. A. Dahlgren
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Light Microscopy ◽  
Light And Electron Microscopy ◽  
Tendon Healing ◽  
Matrix Proteins ◽  
Isolated Cells ◽  
Superficial Digital Flexor Tendon ◽  
Vitro Model

SummaryEquine tendon fibroblasts were isolated from explants of superficial digital flexor tendon, subcultured and maintained in monolayers. The cells were characterized by light microscopy, electron microscopy and radiolabel studies for proteoglycan production. Two predominant cell morphologies were identified. The cells dedifferentiated toward a more spindle shape with repeated subcultures. Equine tendon fibroblasts were successfully cryopreserved and subsequently subcultured. The ability to produce proteoglycan was preserved.The isolated cells were identified as fibroblasts, based on their characteristic shape by light microscopy and ultrastructure and the active production of extracellular matrix proteins. Abundant rough endoplasmic reticulum and the production of extracellular matrix products demonstrated active protein production and export. Proteoglycans were measurable via liquid scintillation counting in both the cell-associated fraction and free in the supernatant. This model is currently being utilized to study the effects of polysulfated glycosaminoglycan on tendon healing. Future uses include studying the effects of other pharmaceuticals, such as hyaluronic acid, on tendon healing.A model was developed for in vitro investigations into tendon healing. Fibroblasts were isolated from equine superficial digital flexor tendons and maintained in monolayer culture. The tenocytes were characterized via light and electron microscopy. Proteoglycan production was measured, using radio-label techniques. The fibroblasts were cryopreserved and subsequently subcultured. The cells maintained their capacity for proteoglycan production, following repeated subculturing and cryopreservation.

Cell-to-cell contact and extracellular matrix

1994 ◽  
Vol 6 (5) ◽  
pp. 645-647 ◽  
Author(s):  
Alan F. Horwitz ◽  
Jean Paul Thiery
Keyword(s):  
Extracellular Matrix ◽  
Cell Contact

scholarly journals von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1531-1534 ◽  
Author(s):  
LA Sporn ◽  
VJ Marder ◽  
DD Wagner
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Immunofluorescent Staining ◽  
Cultured Endothelial Cells ◽  
Human Foreskin Fibroblasts ◽  
Platelet Binding ◽  
The Matrix ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.

Cell-to-cell contact and extracellular matrix

1999 ◽  
Vol 11 (5) ◽  
pp. 535-536
Author(s):  
Jonathon Pines ◽  
Luca Toldo ◽  
Frank Lafont
Keyword(s):  
Extracellular Matrix ◽  
Cell Contact

Web alert: Cell-to-cell contact and extracellular matrix

2001 ◽  
Vol 13 (5) ◽  
pp. 523-524 ◽  
Author(s):  
Jonathon Pines ◽  
Frank Lafont
Keyword(s):  
Extracellular Matrix ◽  
Cell Contact

Cell-to-cell contact and extracellular matrix

2007 ◽  
Vol 19 (5) ◽  
pp. 493-494
Author(s):  
Lawrence Shapiro ◽  
Barry Honig
Keyword(s):  
Extracellular Matrix ◽  
Cell Contact

scholarly journals Proteolytic Events of Wound-Healing — Coordinated Interactions Among Matrix Metalloproteinases (MMPs), Integrins, and Extracellular Matrix Molecules

2001 ◽  
Vol 12 (5) ◽  
pp. 373-398 ◽  
Author(s):  
Bjorn Steffensen ◽  
Lari Häkkinen ◽  
Hannu Larjava
Keyword(s):  
Extracellular Matrix ◽  
Wound Healing ◽  
Matrix Metalloproteinases ◽  
Wound Repair ◽  
Enzyme Activation ◽  
Processing Enzymes ◽  
The Matrix ◽  
Signaling Receptors ◽  
State Of Rest ◽  
And Function

During wound-healing, cells are required to migrate rapidly into the wound site via a proteolytically generated pathway in the provisional matrix, to produce new extracellular matrix, and, subsequently, to remodel the newly formed tissue matrix during the maturation phase. Two classes of molecules cooperate closely to achieve this goal, namely, the matrix adhesion and signaling receptors, the integrins, and matrix-degrading and -processing enzymes, the matrix metalloproteinases (MMPs). There is now substantial experimental evidence that blocking key molecules of either group will prevent or seriously delay wound-healing. It has been known for some time now that cell adhesion by means of the integrins regulates the expression of MMPs. In addition, certain MMPs can bind to integrins or other receptors on the cell surface involved in enzyme activation, thereby providing a mechanism for localized matrix degradation. By proteolytically modifying the existing matrix molecules, the MMPs can then induce changes in cell behavior and function from a state of rest to migration. During wound repair, the expression of integrins and MMPs is simultaneously up-regulated. This review will focus on those aspects of the extensive knowledge of fibroblast and keratinocyte MMPs and integrins in biological processes that relate to wound-healing.

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