Decorin-transforming growth factor- interaction regulates matrix organization and mechanical characteristics of three-dimensional collagen matrices

The small leucine-rich proteoglycan decorin has been demonstrated to be a key regulator of collagen fibrillogenesis; decorin deficiencies lead to irregularly shaped collagen fibrils and weakened material behavior in postnatal murine connective tissues. In an in vitro investigation of the contributions of decorin to tissue organization and material behavior, model tissues were engineered by seeding embryonic fibroblasts, harvested from 12.5-13.5 days gestational aged decorin null (Dcn(-/-)) or wild-type mice, within type I collagen gels. The resulting three-dimensional collagen matrices were cultured for 4 weeks under static tension. The collagen matrices seeded with Dcn(-/-) cells exhibited greater contraction, cell density, ultimate tensile strength, and elastic modulus than those seeded with wild-type cells. Ultrastructurally, the matrices seeded with Dcn(-/-) cells contained a greater density of collagen. The decorin-null tissues contained more biglycan than control tissues, suggesting that this related proteoglycan compensated for the absence of decorin. The effect of transforming growth factor-beta (TGF-beta), which is normally sequestered by decorin, was also investigated in this study. The addition of TGF-beta1 to the matrices seeded with wild-type cells improved their contraction and mechanical strength, whereas blocking TGF-beta1 in the Dcn(-/-) cell-seeded matrices significantly reduced the collagen gel contraction. These results indicate that the inhibitory interaction between decorin and TGF-beta1 significantly influenced the matrix organization and material behavior of these in vitro model tissues.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Cells Actively Stiffen Fibrin Networks by Generating Contractile Stress

During wound healing and angiogenesis, fibrin serves as a provisional extracellular matrix. We use a model system of fibroblasts embedded in fibrin gels to study how cell-mediated contraction may influence the macroscopic mechanical properties of their extracellular matrix during such processes. We demonstrate by macroscopic shear rheology that the cells increase the elastic modulus of the fibrin gels. Microscopy observations show that this stiffening sets in when the cells spread and apply traction forces on the fibrin fibers. We further show that the stiffening response mimics the effect of an external stress applied by mechanical shear. We propose that stiffening is a consequence of active myosin-driven cell contraction, which provokes a nonlinear elastic response of the fibrin matrix. Cell-induced stiffening is limited to a factor 3 even though fibrin gels can in principle stiffen much more before breaking. We discuss this observation in light of recent models of fibrin gel elasticity, and conclude that the fibroblasts pull out floppy modes, such as thermal bending undulations, from the fibrin network, but do not axially stretch the fibers. Our findings are relevant for understanding the role of matrix contraction by cells during wound healing and cancer development, and may provide design parameters for materials to guide morphogenesis in tissue engineering.     

Effects of decorin and biglycan on human airway smooth muscle cell adhesion

Growth on a decorin matrix results in decreased human airway smooth muscle cell (HASMC) number, by decreasing proliferation and increasing apoptosis. We questioned whether these effects were related to abnormal extracellular matrix (ECM)-cell adhesion. HASMCs were seeded on decorin, biglycan, collagen type I or plastic. Actin organization and focal adhesion formation were assessed by staining for filamentous (F) and globular (G) actin, and vinculin, respectively. Gene expression for focal adhesion proteins, ECM molecules and HASMC receptors was measured. Protein levels for fibronectin, α(2), α(5), α(v) and β(3) integrin subunits and, focal adhesion kinase (FAK) were assessed. F-actin filaments were prominent in cells seeded on collagen I and plastic, less apparent in cells cultured on biglycan and faint in cells on decorin. Vinculin clustering was decreased in cells seeded on decorin and biglycan, as was vinculin gene expression. Compared to cells on plastic, cells on decorin had an increase in fibronectin gene expression. Seeding on decorin caused an increase in α(2) integrin subunit and platelet-derived growth factor receptor A gene expression. There was also an increase in α(2) and α(v) integrin subunit protein. Finally, FAK protein levels in cells seeded on decorin or biglycan were decreased compared to cells seeded on plastic or collagen I. Cells grown on proteoglycan matrices demonstrate evidence of abnormalities during many of the key processes involved in normal cell adhesion. Upregulation of cell surface receptor proteins, such as α(2) integrin subunit, may represent a compensatory mechanism to overcome poor adhesion induced by growth on these matrices.     

Macrophage migration in fibrin gel matrices. II. Effects of clotting factor XIII, fibronectin, and glycosaminoglycan content on cell migration

We investigated the migration of oil-induced, guinea pig peritoneal macrophages in three-dimensional fibrin matrices, with particular attention to variables which modified fibrin gel structure and/or its adhesive properties for cells. The variables studied were fibrin concentration, gel cross-linking, and fibronectin and glycosaminoglycan content. Macrophage migration was an inverse linear function of fibrinogen concentration. Little or no fibrinolysis accompanied macrophage migration; rather, macrophages migrated through fibrin gels by an active process associated with marked distortions of cell shape and specialized plasma membrane contacts with fibrin strands. Fibrin matrices prepared from fibrinogen that had been depleted of clotting factor XIII and/or fibronectin provided a superior matrix for macrophage migration. Both the number of migrating cells and distance of migration were reduced when the gel matrix included fibronectin and was cross-linked by factor XIII. A hexapeptide containing the fibronectin cell-binding RGDS sequence reversed this migration inhibition, suggesting that fibronectin immobilized by cross-linking to fibrin may have bound macrophages and restricted cell migration. Hyaluronic acid, heparin, and heparan sulfate inhibited macrophage migration in cross-linked fibrin-fibronectin gels over a range of concentrations. These data are relevant to an understanding of macrophage migration in vivo where cross-linked fibrin-fibronectin gels containing variable amounts of glycosaminoglycans are deposited in tissues in immunologic reactions and in many other types of pathology.     

Fibronectin and fibrinolysis are not required for fibrin gel contraction by human skin fibroblasts

Human skin fibroblasts contracted fibrin gels in a time- and cell-dependent manner. Under optimal conditions, gel contraction amounted to more than 50% in 2 hr. Fibronectin did not promote contraction, and fibrinolysis was not required for contraction, although gels contracted without serum or aprotinin were lysed. Before contraction, fibrin was present in loosely packed, randomly organized fibrils. After contraction, the fibrils were more densely packed and aligned in the plane of cell spreading. Cycloheximide treatment of fibroblasts inhibited gel contraction in serum-free medium but not in serum-containing medium. Fibronectin could not substitute for serum in overcoming the cycloheximide effect. Binding sites for fibrin were distributed randomly over the cells' surfaces based on electron microscopic observations. Often small groups of fibrils were localized in indentations at the cell surface. Finally, peptides containing the arg-gly-asp-ser sequence inhibited gel contraction.     

Effects of decorin proteoglycan on fibrillogenesis,ultrastructure, and mechanics of type I collagen gels

The proteoglycan decorin is known to affect both the fibrillogenesis and the resulting ultrastructure of in vitro polymerized collagen gels. However, little is known about its effects on mechanical properties. In this study, 3D collagen gels were polymerized into tensile test specimens in the presence of decorin proteoglycan, decorin core protein, or dermatan sulfate (DS). Collagen fibrillogenesis, ultrastructure, and mechanical properties were then quantified using a turbidity assay, 2 forms of microscopy (SEM and confocal), and tensile testing. The presence of decorin proteoglycan or core protein decreased the rate and ultimate turbidity during fibrillogenesis and decreased the number of fibril aggregates (fibers) compared to control gels. The addition of decorin and core protein increased the linear modulus by a factor of 2 compared to controls, while the addition of DS reduced the linear modulus by a factor of 3. Adding decorin after fibrillogenesis had no effect, suggesting that decorin must be present during fibrillogenesis to increase the mechanical properties of the resulting gels. These results show that the inclusion of decorin proteoglycan during fibrillogenesis of type I collagen increases the modulus and tensile strength of resulting collagen gels. The increase in mechanical properties when polymerization occurs in the presence of the decorin proteoglycan is due to a reduction in the aggregation of fibrils into larger order structures such as fibers and fiber bundles.     

Neurite extension and in vitro myelination within three-dimensional modified fibrin matrices

The deposition of fibrin clots in vivo occurs after injury in the peripheral nervous system and their removal correlates with nerve regeneration. Fibrin clots provide a provisional matrix for invading cells, induce wound healing, and become proteolytically removed by regenerating tissue. Here, neurite extension and in vitro myelination were studied within three-dimensional fibrin matrices that were covalently modified with the sixth Ig-like domain of cell adhesion molecules L1 containing N-terminal transglutaminase substrate sequences (TG-L1Ig6) for covalent incorporation into fibrin matrices. TG-L1Ig6 is a specific receptor for alphavbeta3-integrin involved in neurite extension of PC12 cells and dorsal root ganglion neurons (DRGs). Neurite extension of PC12 cells depended on interactions between cell surface alphavbeta3 and RGD-sites provided by TG-L1Ig6. In addition, matrix properties such as fibrin crosslink density and matrix degradation by serine proteases were crucial. No involvement of matrix metalloproteinases was found. DRG neurite extension in native fibrin matrices was retarded as compared to neurite extension within L1Ig6-modified and laminin-1-containing matrices. Moreover, myelinated structures were almost exclusively found in TG-L1Ig6-modified and laminin-1-containing matrices. These results indicate that potential use of three-dimensional matrices in a biomaterials-based healing device to induce and/or help in vivo nerve regeneration requires specific structural and biological signals.     

Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors

We have previously shown that the tumor promoter 4 beta-phorbol 12-myristate 13-acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three-dimensional collagen gels to invade the underlying matrix and to form capillary-like tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469-477, 1985). Since angiogenesis frequently occurs within a fibrin-rich extracellular matrix, we have examined the ability of PMA-treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, epsilon-aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessel-like tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin-independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.     

Changes in secreted and cell associated proteoglycan synthesis during conversion of myoblasts to osteoblasts in response to bone morphogenetic protein-2: role of decorin in cell response to BMP-2

Proteoglycans have been identified within the extracellular matrices (ECM) of bone and are known to play a role in ECM assembly, mineralization, and bone formation. Bone morphogenetic protein-2 (BMP-2) specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells. Microarray analyses of the mouse myoblast cell line C2C12 and its differentiation into osteoblastic cells in response to BMP-2 have suggested the up-regulation of several proteoglycan species, although there is a lack of biochemical evidence for this response. In this study we have biochemically analyzed and characterized the proteoglycan populations that are induced in C2C12 cells upon osteoblastic differentiation produced by BMP-2. An important and specific increase in the synthesis of secreted decorin was observed in BMP-2-treated cells, as compared to untreated myoblasts and myoblasts induced to differentiate into myotubes. Decorin was seen to contain larger glycosaminoglycan (GAG) chains in induced than in non-induced cells. BMP-2 also produced an augment in the synthesis of different heparan sulfate proteoglycans such syndecan-2, - 3, glypican, and perlecan in detergent-soluble and non-soluble cellular fractions. We also examined whether the evident changes induced by BMP-2 in secreted decorin could have a functional role. BMP-2 signaling dependent as well as induction of alkaline phosphatase (ALP) activity was diminished in decorin null myoblasts compared to wild type myoblats although cell surface level of BPM-2 receptors was unchanged. These results are the first biochemical evidence and analysis for the effect of BMP-2 on the synthesis of proteoglycan during osteogenic conversion of myoblasts and suggest a role for decorin in cell response to BMP-2.     

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley-Liss, Inc.     

The behaviour of BHK cells and neutrophil leukocytes on collagen gels of defined mechanical strength

This study demonstrates how the mechanical strength of a series of collagen/composite gels can be measured using a penetrometer. It was found that the presence of fibrin in collagen gels resulted in increased gel strength. Similarly hyaluronic acid was found to increase the strength of collagen gels. Addition of heparin weakened collagen gels as did chondroitin-6-sulphate. Neutrophil migration into collagen gels was found to be inversely proportional to gel strength. Fibrin and hyaluronic acid containing gels inhibited neutrophil migration while the presence of heparin and chondroitin sulphate increased neutrophil migration. BHK gel contraction experiments demonstrated how the presence of fibrin prevents gel contraction. Despite increasing gel strength the presence of hyaluronic acid appeared to have no effect on BHK contraction of collagen gels. Similarly the presence of heparin or chondroitin sulphate had no effect on gel contraction by BHK cells.     

Rheological characterization and dissolution kinetics of fibrin gels crosslinked by a microbial transglutaminase

Various fibrin gels were prepared with a microbial transglutaminase under miscellaneous conditions. The gels were characterized through their rheological properties. The influence of fibronectin addition and that of covalent bonding on the viscoelastic characteristics were evaluated. Gel elasticity is proportional to fibrinogen concentration but shows a nonlinear dependence on transglutaminase concentration. Additional crosslink of fibronectin in fibrin gels has no effect on the rheological character of the matrix. Dissolution kinetics in concentrated urea solutions evidences the role of covalent bonds on gel stability. The rheological properties and gel stability are discussed in relation with the enzyme-catalyzed covalent bonding. The microbial enzyme reactions are compared to those of FXIII and tissue transglutaminases.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

Fibronectin and fibrin gel structure

Plasma fibronectin is covalently incorporated into alpha-chains of fibrin gels in the presence of Factor XIII activated by thrombin (FXIIIaT) but not by Factor XIII activated by the snake venom enzyme batroxobin (FXIIIaB). FXIIIaB catalyzes introduction of gamma-gamma cross-links in fibrin but cross-linked alpha-chains are not formed. In the presence of FXIIIaT, fibrin gels formed by batroxobin incorporated fibronectin and the alpha-chains are cross-linked indicating that FXIIIaB has a different substrate specificity from FXIIIaT. In the presence of FXIIIaT the incorporation of fibronectin approaches 1 mol/340 kDa unit weight of fibrin. Fibronectin when present in a fibrinogen thrombin mixture containing FXIII does not influence the clotting time of the system nor the release of fibrinopeptides. Incorporation of fibronectin is not appreciable before the gel point. This indicates that the polymerization and gelation of fibrinogen is essentially not perturbed by the presence of fibronectin and that fibrin in the gel matrix rather than the fibrin polymers formed prior to gel point is the preferred structure for fibronectin incorporation. Incorporation of fibronectin into fibrin gels during formation leads to an increase in turbidity and a small decrease in Ks (permeability coefficient). This suggests that the width of the strands in the gel increases as a result of fibronectin incorporation. Fibronectin is also incorporated into preformed gels having completely cross-linked gamma- and alpha-chains perhaps indicating that the sites in fibrin involved in fibronectin incorporation are different from those involved in fibrin cross-linking. FXIIIaT appeared to be adsorbed to fibrin gel matrix in the presence but not in the absence of calcium ions.     

Fibrin-fiber architecture influences cell spreading and differentiation

ABSTRACT

The mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior.     

A reproducible, high throughput method for fabricating fibrin gels

ABSTRACT: BACKGROUND: Fibrin gels are a promising biomaterial for tissue engineering. However, current fabrication methods are time intensive with inherent variation. There is a pressing need to develop new and consistent approaches for producing fibrin-based hydrogels for examination. RESULTS: We developed a high throughput method for creating fibrin gels using molds fabricated from polydimethylsiloxane (PDMS). Fibrin gels were produced by adding solutions of fibrinogen and thrombin to cylindrical defects in a PDMS sheet. Undisturbed gels were collected by removing the sheet, and fibrin gels were characterized. The characteristics of resulting gels were compared to published data by measuring compressive stiffness and osteogenic response of entrapped human mesenchymal stem cells (MSCs). Gels exhibited compressive moduli nearly identical to our previously reported fabrication method. Trends in alkaline phosphatase activity, an early marker of osteogenic differentiation in MSCs, were also consistent with previous data. CONCLUSIONS: These findings demonstrate a streamlined approach to fibrin gel production that drastically reduces the time required to make fibrin gels, while also reducing variability between gel batches. This fabrication technique provides a valuable tool for generating large numbers of gels in a cost-effective manner.     

Growth regulatory activities of endothelial extracellular matrix: mediation by transforming growth factor-beta

The potential of transforming growth factor-beta (TGF-beta) to modulate the growth of endothelial cells via alterations in the cell's extracellular matrix (ECM) was examined. Rat brain endothelial cells were cultured in the presence or absence of TGF-beta, and subsequently ECM was prepared from the cell cultures by hypotonic lysis of the cells. Untreated endothelial cells were then cultured on the various matrices. Cells grown on TGF-beta-treated ECM showed a significant decrease in cell number (41 +/- 6% mean growth inhibition at 6 days, P less than 0.005 by paired T-test) compared with cells grown on untreated ECM. The growth inhibitory activity of the ECM was depleted by 9 days of culture, and resumption of exponential cell growth was observed. A similar phenomenon was observed if anti-TGF-beta neutralizing antibodies were incubated with the ECM. When the TGF-beta-treated matrix was exposed to a brief dithiothreitol treatment in order to inactivate residual TGF-beta, an approximately equal degree of growth inhibition was observed initially, but the reversal of inhibition occurred at an earlier time point than that with unreduced TGF-beta-treated matrix. Analyses of the composition of matrices synthesized in the presence or absence of TGF-beta revealed about a twofold increase in the accumulation of various radioactive metabolic precursors in the TGF-beta-treated matrices. However, no qualitative alterations in the matrix or cellular-associated proteins or glycoproteins were observed, as analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. An increase in cell-associated heparan sulfate, however, was observed in TGF-beta-treated cells. The results suggest that certain growth regulatory effects of TGF-beta may be mediated, at least in part, by alterations in the ECM.