Breakdown of cell-collagen networks through collagen remodeling

Collagen model tissues, consisting of cells embedded in a collagen matrix at different concentrations (of cells and collagen) were analyzed. Rheological properties were measured and complementary confocal microscopy analysis carried out. An important feature, corresponding to the breakdown of the collagen network (i.e., decrease in network elasticity) was observed at high collagen concentrations, due to the presence of cells. Thanks to confocal microscopy, we showed that cells elongated within the gel and could remodel it, this being a concentration-dependent feature. A careful analysis of the remodeling process showed that cells can attract collagen in their close neighborhood, this being an irreversible process and that migrating cells create collagen-depleted regions behind them.     

Dense tissue-like collagen matrices formed in cell-free conditions.

A new protocol was developed to produce dense organized collagen matrices hierarchically ordered on a large scale. It consists of a two stage process: (1) the organization of a collagen solution and (2) the stabilization of the organizations by a sol-gel transition that leads to the formation of collagen fibrils. This new protocol relies on the continuous injection of an acid-soluble collagen solution into glass microchambers. It leads to extended concentration gradients of collagen, ranging from 5 to 1000 mg/ml. The self-organization of collagen solutions into a wide array of spatial organizations was investigated. The final matrices obtained by this procedure varied in concentration, structure and density. Changes in the liquid state of the samples were followed by polarized light microscopy, and the final stabilized gel states obtained after fibrillogenesis were analyzed by both light and electron microscopy. Typical organizations extended homogeneously by up to three centimetres in one direction and several hundreds of micrometers in other directions. Fibrillogenesis of collagen solutions of high and low concentrations led to fibrils spatially arranged as has been described in bone and derm, respectively. Moreover, a relationship was revealed between the collagen concentration and the aggregation of and rotational angles between lateral fibrils. These results constitute a strong base from which to further develop highly enriched collagen matrices that could lead to substitutes that mimic connective tissues. The matrices thus obtained may also be good candidates for the study of the three-dimensional migration of cells.     

Quantitative analysis of the effect of cancer invasiveness and collagen concentration on 3D matrix remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness.     

A critical role for the membrane-type 1 matrix metalloproteinase in collagen phagocytosis

Degradation of collagen is important for the physiological remodeling of connective tissues during growth and development as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodeling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. However, although various steps in the phagocytic pathway have been characterized, the enzyme required to initially fragment collagen fibrils for subsequent phagocytosis has not been identified. We have used laser confocal microscopy, transmission electron microscopy, and biochemical assays to show that human fibroblasts initiate degradation of collagen through the collagenase activity of the membrane-bound metalloproteinase MT1-MMP. Degradation of natural and reconstituted collagen substrates correlated with the expression of MT1-MMP, which was localized at sites of collagen cleavage at the surface of the cells and also within the cells, whereas collagen degradation was abrogated when MT1-MMP expression was blocked by small interfering RNA treatment. In contrast to MT1-MMP, the gelatinolytic activity of MMP-2 was not required for collagen phagocytosis. These studies demonstrate a pivotal role of catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation.     

In vitro study of smooth muscle cells on polycaprolactone and collagen nanofibrous matrices

Biodegradable polycaprolactone and collagen nanofibers were produced by electrospinning, with fiber diameters of around 300-700nm and features similar to the extracellular matrix of natural tissue. Human coronary artery smooth muscle cells (SMCs) seeded on nanofibrous matrices tend to maintain normal phenotypic shape and growth tends to be guided by the nanofiber orientation. The SMC and nanofibrous matrix interaction was observed by SEM, MTS assay, trypan blue exclusion method and laser scanning confocal microscopy. The results showed that the proliferation and growth rate of SMCs were not different on polycaprolactone (PCL) nanofibrous matrices coated with collagen or tissue culture plates. PCL nanofibrous matrices coated with collagen showed that the SMCs migrated towards inside the nanofibrous matrices and formed smooth muscle tissue. This approach may be useful for engineering a variety of tissues in various structures and shapes, and also to demonstrate the importance of matching both the initial mechanical properties and degradation rate of nanofibrous matrices to the specific tissue engineering.     

Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.     

Collagen concentration as a significant variable for growth and morphology of mouse mammary parenchyma in collagen matrix culture

Multicellular organoids of mouse mammary epithelium were established in culture either upon or within collagen matrices of various concentrations. Growth and tubule morphogenesis within the matrices were dependent upon the concentration of collagen, both being maximal in gels composed of 2 mg collagen/ml gel. Growth was more extensive in cultures established in gel than on gel especially at intermediate concentrations of collagen, with cell growth on gel seemingly independent of collagen concentration. Our results demonstrate that local collagen concentration can significantly affect epithelial cell growth and morphology.     

Coating electrospun collagen and gelatin fibers with perlecan domain I for increased growth factor binding

Electrospun natural polymer membranes were fabricated from collagen or gelatin coated with a bioactive recombinant fragment of perlecan, a natural heparan sulfate proteoglycan. The electrospinning process allowed the facile processing of a three-dimensional, porous fibril (2-6 microm in diameter) matrix suitable for tissue engineering. Laser scanning confocal microscopy revealed that osteoblast-like MG63 cells infiltrated the depth of the electrospun membrane evenly without visible apoptosis. Tissue engineering scaffolds ideally mimic the extracellular matrix; therefore, the electrospun membrane must contain both structural and functional matrix features. Fibers were coated, after processing, with perlecan domain I (PlnDI) to improve binding of basic fibroblast growth factor (FGF-2), which binds to native heparan sulfate chains on PlnDI. PlnDI-coated electrospun collagen fibers were ten times more effective than heparin-BSA collagen fibers at binding FGF-2. Because FGF-2 modulates cell growth, differentiation, migration and survival, the ability to effectively bind FGF-2 to an electrospun matrix is a key improvement in creating a successful tissue engineering scaffold.     

Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro

The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.Copyright 2000 John Wiley & Sons, Inc.     

Decreased level of PDGF-stimulated receptor autophosphorylation by fibroblasts in mechanically relaxed collagen matrices

The goal of our studies was to characterize the interrelationship between extracellular matrix organization and fibroblast proliferation in response to growth factors. We compared fibroblasts in monolayer culture with cells in contracted collagen matrices that were mechanically stressed or relaxed. In response to platelet-derived growth factor (PDGF), DNA synthesis by fibroblasts in mechanically relaxed collagen matrices was 80-90% lower than in monolayer culture and 50% lower than in mechanically stressed matrices. Fibroblasts in monolayer and contracted collagen matrix cultures contained similar levels of PDGF receptors, but differed in their autophosphorylation response. Cells in mechanically relaxed matrices showed lowest levels of autophosphorylation, 90% less than cells in monolayer culture. Experiments comparing receptor expression and capacity for PDGF- stimulated autophosphorylation showed that cells in mechanically relaxed collagen matrices never developed normal receptor autophosphorylation. Furthermore, when mechanically stressed collagen matrices were switched to mechanically relaxed conditions, capacity for receptor autophosphorylation decreased within 1-2 h and remained low. Based on immunomicroscopic observations and studies on down-regulation of receptors by PDGF binding, it appeared that most PDGF receptors in monolayer or contracted collagen matrix cultures were localized on the cell surface and accessible to PDGF binding. In related studies, we found that EGF receptors of fibroblasts in mechanically relaxed collagen matrices also showed low levels of autophosphorylation in response to EGF treatment. Based on these results, we suggest that mechanical interactions between cells and their surrounding matrix provide regulatory signals that modulate autophosphorylation of growth factor receptors and cell proliferation.     

Enhanced adhesion of human mononuclear cells to nonenzymatically glycosylated collagen I

Nonenzymatic glycosylation of extracellular matrix components may contribute to altered interaction of cells with the matrix. We have examined the interaction of mononuclear cells with early glycosylated collagen I. Significantly more cells attached to glycosylated collagen compared to normal collagen. Radioiodinated glycosylated collagen I specifically bound to mononuclear cells in a time and concentration dependent manner with a Kd of 2.45×10^–9 M. Maximum binding was observed in the presence of Mn⁺⁺ ions. The iodinated ligand bound to mononuclear cell membrane immobilized on nitrocellulose disks and the interaction was found to be saturable. These results suggested an alteration in the interaction of human blood mononuclear cells with collagen I, when it gets glycosylated non enzymatically and also indicate that early glycosylated collagen interacts with mononuclear cells through specific, high affinity cell surface molecules. (Mol Cell Biochem148: 115–121, 1995)     

Collagen fibril flow and tissue translocation coupled to fibroblast migration in 3D collagen matrices

In nested collagen matrices, human fibroblasts migrate from cell-containing dermal equivalents into surrounding cell-free outer matrices. Time-lapse microscopy showed that in addition to cell migration, collagen fibril flow occurred in the outer matrix toward the interface with the dermal equivalent. Features of this flow suggested that it depends on the same cell motile machinery that normally results in cell migration. Collagen fibril flow was capable of producing large-scale tissue translocation as shown by closure of a approximately 1-mm gap between paired dermal equivalents in floating, nested collagen matrices. Our findings demonstrate that when fibroblasts interact with collagen matrices, tractional force exerted by the cells can couple to matrix translocation as well as to cell migration.     

PPFIBP1 induces glioma cell migration and invasion through FAK/Src/JNK signaling pathway

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, with a 5-year survival ratio <5%. Invasive growth is a major determinant of the poor prognosis in GBM. In this study, we demonstrate that high expression of PPFIA binding protein 1 (PPFIBP1) correlates with remarkable invasion and poor prognosis of GBM patients. Using scratch and transwell assay, we find that the invasion and migration of GBM cells are promoted by overexpression of PPFIBP1, while inhibited by knockdown of PPFIBP1. Then, we illustrate that overexpression of PPFIBP1 facilitates glioma cell infiltration and reduces survival in xenograft models. Next, RNA-Seq and GO enrichment analysis reveal that PPFIBP1 regulates differentially expressed gene clusters involved in the Wnt and adhesion-related signaling pathways. Furthermore, we demonstrate that PPFIBP1 activates focal adhesion kinase (FAK), Src, c-Jun N-terminal kinase (JNK), and c-Jun, thereby enhancing Matrix metalloproteinase (MMP)-2 expression probably through interacting with SRCIN1 (p140Cap). Finally, inhibition of phosphorylation of Src and FAK significantly reversed the augmentation of invasion and migration caused by PPFIBP1 overexpression in GBM cells. In conclusion, these findings uncover a novel mechanism of glioma invasion and identify PPFIBP1 as a potential therapeutic target of glioma.Subject terms: Oncogenes, Molecular neuroscience     

Correlation of hyaluronic acid accumulation and the growth of preneoplastic mammary cells in collagen: A lonitudinal study

Summary Hyaluronic acid accumulation is characteristic of mammary tumor cells, and the amount that accumulates seems to correlate with the degree of malignancy of the producing cells. We have tested directly the relationship between hyaluronic acid accumulation and the replication rate of preneoplastic mammary cells in culture. We used nontumorigenic but immortal CL-S1 mouse mammary cells that were derived from a hyperplastic alveolar nodule. Using a collagen gel culture system, we found clear differences in the growth properties of cells before and after Passages 68 to 70. Late passage cells replicated earlier and faster than early passage cells in collagen and on plastic. The rate of cycling resembled that of tumorigenic mouse mammary cells during the first week of culture. Cells seeded at low densities cycled faster than those seeded at high densities during the second week in culture. Exogenous hyaluronic acid, at 10 to 1000μg/ml, neither enhanced nor inhibited CL-S1 cell growth significantly in collagen, regardless of passage. However, by the third day in collagen, late passage cells produced 7 times more total glycosaminoglycans and 12 times more hyaluronic acid per cell than did early passage cells. Late passage cells also deposited 12 times more labeled hyaluronic acid in the matrix than did early passage cells, on a per-cell basis. After a decline in the deposition of hyaluronic acid in the extracellular matrix, growth ceased. The late passage cells did not grow in soft agar, indicating that they had not become neoplastic spontaneously during passage. However, their accelerated growth rate, coupled with the synthesis and secretion of large amounts of hyaluronic acid into the extracellular matrix, may characterize a distinct step in tumor progression in preneoplastic CL-S1 cells.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

Characterization of 3-D collagen hydrogels for functional cell-based biosensing

To address the growing demand for functional cell-based assay technologies with accelerated drug discovery applications, we have proposed the use of human neuroblastoma cells (IMR-32) immobilized in three-dimensional (3-D) collagen hydrogel matrices. The gel protects weakly adherent cells from fluid mechanical forces while providing a more physiologically relevant 3-D environment. Hydrogels made up of collagen, between 0.5 and 1.0mg/ml, exhibited mechanical stability adequate to withstand fluid mechanical forces (<0.11 mN) typical of automated commercial fluid transfer equipment. Collagen-entrapped cells visualized with the aid of confocal microscopy and a potentiometric-sensitive dye, TMRM, exhibited round morphology in comparison to flat morphology typical of cells in two-dimensional (2-D) monolayer cultures. Morphological differentiation characterized by neurite extension and cell aggregation was observed for both 2-D and 3-D cultures. Differentiated IMR-32 cells failed to develop a resting membrane potential typical of excitable cells. Free intracellular calcium was monitored with Calcium Green-1. Depolarization-induced Ca 2+influx was only observed with differentiated 3-D cells unlike 2-D cells, where calcium flux was observed in both differentiated and undifferentiated cells. Taken together, the results revealed that collagen hydrogels (0.5 mg/ml collagen) were suitable structural supports for weakly adherent cells. However, for voltage-dependent calcium channel function applications, further investigations are needed to explain the difference between 2-D monolayer and 3-D collagen-entrapped cells.     

Retinol and extracellular collagen matrices modulate hepatic Ito cell collagen phenotype and cellular retinol binding protein levels

The hepatic vitamin A-storing Ito cell has been implicated as a causative cell in hepatic fibrogenesis. Using a modification of a recent method (Friedman, S. L., Roll, F. J., Boyles, J., and Bissell, D. M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8681-8685), rat Ito cells were isolated and passaged in vitro on collagen-coated plastic dishes through cell generation 40-50. The collagen synthetic phenotype for Ito cells grown on various extracellular matrices was demonstrated by immunofluorescence and quantitated by competition enzyme-linked immunosorbent assays. When grown on a type I collagen matrix, Ito cells produced type IV greater than type III greater than type I collagen. When grown on a type IV collagen matrix, the cells produced relatively equal amounts of types I and III collagen. The absolute amounts of type I collagen produced were greater when cells were grown on type IV versus type I matrix. When 10(-5) M retinol was added to cell cultures, there was a uniform increase in type III collagen regardless of matrix type but a decrease in type I collagen when cells were grown on a type IV matrix and a large increase in type I collagen when cells were grown on a type I collagen matrix. The levels of cellular retinol binding protein, a key cytosolic retinol transport protein, were quantitated by high performance liquid chromatography and compared for cells grown on type I versus type IV collagen matrices. It was found that cells on a type I matrix contain 4.96 +/- 2.8 times more cellular retinol binding protein than do cells grown on a type IV matrix. In conclusion, Ito cell collagen synthesis may be altered by underlying extracellular matrix and exogenous retinol. This in vitro culture system should allow the study of regulatory factors and possible therapeutic anti-fibrogenic mediators.     

Cellular remodelling of individual collagen fibrils visualized by time-lapse AFM

The extracellular matrix in tissues such as bone, tendon and cornea contains ordered, parallel arrays of collagen type I fibrils. Cells embedded in these matrices frequently co-align with the collagen fibrils, suggesting that ordered fibrils provide structural or signalling cues for cell polarization. To study mechanisms of matrix-induced cell alignment, we used nanoscopically defined two-dimensional matrices assembled of highly aligned collagen type I fibrils. On these matrices, different cell lines expressing integrin alpha(2)beta(1) polarized strongly in the fibril direction. In contrast, alpha(2)beta(1)-deficient cells adhered but polarized less well, suggesting a role of integrin alpha(2)beta(1) in the alignment process. Time-lapse atomic force microscopy (AFM) demonstrated that during alignment cells deform the matrix by reorienting individual collagen fibrils. Cells deformed the collagen matrix asymmetrically, revealing an anisotropy in matrix rigidity. When matrix rigidity was rendered uniform by chemical cross-linking or when the matrix was formed from collagen fibrils of reduced tensile strength, cell polarization was prevented. This suggested that both the high tensile strength and pliability of collagen fibrils contribute to the anisotropic rigidity of the matrix, leading to directional cellular traction and cell polarization. During alignment, cellular protrusions contacted the collagen matrix from below and above. This complex entanglement of cellular protrusions and collagen fibrils may further promote cell alignment by maximizing cellular traction.