Cell-matrix entanglement and mechanical anchorage of fibroblasts in three-dimensional collagen matrices

Fibroblast-3D collagen matrix culture provides a physiologically relevant model to study cell–matrix interactions. In tissues, fibroblasts are phagocytic cells, and in culture, they have been shown to ingest both fibronectin and collagen-coated latex particles. Compared with cells on collagen-coated coverslips, phagocytosis of fibronectin-coated beads by fibroblasts in collagen matrices was found to be reduced. This decrease could not be explained by integrin reorganization, tight binding of fibronectin beads to the collagen matrix, or differences in overall bead binding to the cells. Rather, entanglement of cellular dendritic extensions with collagen fibrils seemed to interfere with the ability of the extensions to interact with the beads. Moreover, once these extensions became entangled in the matrix, cells developed an integrin-independent component of adhesion. We suggest that cell–matrix entanglement represents a novel mechanism of cell anchorage that uniquely depends on the three-dimensional character of the matrix.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.     

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.     

Dendritic fibroblasts in three-dimensional collagen matrices

Cell motility determines form and function of multicellular organisms. Most studies on fibroblast motility have been carried out using cells on the surfaces of culture dishes. In situ, however, the environment for fibroblasts is the three-dimensional extracellular matrix. In the current research, we studied the morphology and motility of human fibroblasts embedded in floating collagen matrices at a cell density below that required for global matrix remodeling (i.e., contraction). Under these conditions, cells were observed to project and retract a dendritic network of extensions. These extensions contained microtubule cores with actin concentrated at the tips resembling growth cones. Platelet-derived growth factor promoted formation of the network; lysophosphatidic acid stimulated its retraction in a Rho and Rho kinase-dependent manner. The dendritic network also supported metabolic coupling between cells. We suggest that the dendritic network provides a mechanism by which fibroblasts explore and become interconnected to each other in three-dimensional space.     

Fibroblast biology in three-dimensional collagen matrices

Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.     

Endothelial cell protrusion and migration in three-dimensional collagen matrices

Many cells display dramatically different morphologies when migrating in 3D matrices vs. on planar substrata. How these differences arise and the implications they have on cell migration are not well understood. To address these issues, we examined the locomotive structure and behavior of bovine aortic endothelial cells (ECs) either inside 3D collagen gels or on 2D surfaces. Using time-lapse imaging, immunofluorescence, and confocal microscopy, we identified key morphological differences between ECs in 3D collagen gels vs. on 2D substrata, and also demonstrated important functional similarities. In 3D matrices, ECs formed cylindrical branching pseudopodia, while on 2D substrata they formed wide flat lamellae. Three distinct cytoplasmic zones were identified in both conditions: (i) a small, F-actin-rich, rapidly moving peripheral zone, (ii) a larger, more stable, intermediate zone characterized by abundant microtubules and small organelles, and (iii) a locomotively inert central zone rich in microtubules, and containing the larger organelles. There were few differences between 2D and 3D cells in the content and behavior of their peripheral and central zones, whereas major differences were seen in the shape and types of movements displayed by the intermediate zone, which appeared critical in distributing cell-matrix adhesions and directing cytoplasmic flow. This morphological and functional delineation of cytoplasmic zones provides a conceptual framework for understanding differences in the behavior of cells in 3D and 2D environments, and indicates that cytoskeletal structure and dynamics in the relatively uncharacterized intermediate zone may be particularly important in cell motility in general.     

Biased three-dimensional cell migration and collagen matrix modification

Various tumours can be resected even for cure with complete removal. Surgical excision with a wide margin to ensure complete removal has therefore been suggested as the primary treatment for such lesions. The histological examination of the three-dimensional (3D) excision margins (3D histology) constitutes an important part of the quality control mechanisms in tumour surgery. Understanding histologically relevant properties of the constituents of the microenvironment in tumours and the circumferential portion of non-lesional tissue is therefore critical.Accompanied by the increasing availability of high performance computers in recent decades, there has been a strong movement aiming at the development of mathematical models whose implementations allow in silico simulations of biological reaction networks. Due to its relevance in numerous biological and pathological processes, there have been various attempts to model biased migration of single cells. The model introduced in this paper plays a prominent role insofar as it covers the under-represented 3D case. Moreover, it is uniformly formulated for both two and three dimensions. The velocity of each cell is characterised by a generalised Langevin equation, a stochastic differential equation, where chemotaxis as well as contact guidance are considered to simulate selected aspects of interactions between carcinoma cell groups and fibroblast-like cells.Algorithmic and numeric aspects of the developed equations are detailed in this paper and the results of the simulations are illustrated in different manners to emphasise specific features. A simple test scenario as well as a geometry based on segmentation data of a real histological slide has served for verification of the software. The resulting morphologies closely resemble that of desmoplastic stromal reaction readily identifiable in histological slides of infiltrating carcinoma, and the images can be interpreted in the context of 3D histology.     

Cell-cycle dependent tyrosine phosphorylation on mortalin regulates its interaction with fibroblast growth factor-1

We previously reported that endogenously expressed, intracellularly localized fibroblast growth factor (FGF)-1 interacts with mortalin. Here we report that FGF-1 added to the culture medium of quiescent BALB/c3T3 cells is taken up by the cells and interacts with mortalin in the cells in a regulated manner. Although both the internalized FGF-1 and mortalin were present at high levels throughout the FGF-1-initiated cell cycle, their interaction became apparent only in late G1 phase. Interestingly, mortalin was preferentially tyrosine phosphorylated at the same time, and when its normally weak phosphorylation in early G1 phase was augmented by treating the cells with vanadate, a strong interaction between mortalin and FGF-1 was established. Conversely, when phosphorylated mortalin was treated with tyrosine phosphatase, its interaction with FGF-1 was abrogated. These results indicate that FGF-1 taken up by cells preferentially interacts with mortalin in late G1 phase of the cell cycle, and that tyrosine phosphorylation of mortalin regulates this interaction.     

Protein-protein interaction regulates proteins' mechanical stability

Elastomeric proteins are molecular springs found not only in a variety of biological machines and tissues, but also in biomaterials of superb mechanical properties. Regulating the mechanical stability of elastomeric proteins is not only important for a range of biological processes, but also critical for the use of engineered elastomeric proteins as building blocks to construct nanomechanical devices and novel materials of well-defined mechanical properties. Here we demonstrate that protein-protein interactions can potentially serve as an effective means to regulate the mechanical properties of elastomeric proteins. We show that the binding of fragments of IgG antibody to a small protein, GB1, can significantly enhance the mechanical stability of GB1. The regulation of the mechanical stability of GB1 by IgG fragments is not through direct modification of the interactions in the mechanically key region of GB1; instead, it is accomplished via the long-range coupling between the IgG binding site and the mechanically key region of GB1. Although Fc and Fab bind GB1 at different regions of GB1, their binding to GB1 can increase the mechanical stability of GB1 significantly. Using alanine point mutants of GB1, we show that the amplitude of mechanical stability enhancement of GB1 by Fc does not correlate with the binding affinity, suggesting that binding affinity only affects the population of GB1/human Fc (hFc) complex at a given concentration of hFc, but does not affect the intrinsic mechanical stability of the GB1/hFc complex. Furthermore, our results indicate that the mechanical stability enhancement by IgG fragments is robust and can tolerate sequence/structural perturbation to GB1. Our results demonstrate that the protein-protein interaction is an efficient approach to regulate the mechanical stability of GB1-like proteins and we anticipate that this new methodology will help to develop novel elastomeric proteins with tunable mechanical stability and compliance.     

Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices

BackgroundThere is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.MethodsThe present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann–Whitney U, Kruskal Wallis, Student’s t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.ResultsOur findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.ConclusionsThe use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0237-z) contains supplementary material, which is available to authorized users.     

Structural and mechanical factors influencing infarct scar collagen organization

Although large collagen fibers in myocardial infarct scar are highly organized, little is known about mechanisms controlling this organization. The preexisting extracellular matrix may act as a scaffold along which fibroblasts migrate. Conversely, deformation within the ischemic area could guide fibroblasts so new collagen is oriented to counteract the stretch. To investigate these potential mechanisms, we infarcted three groups of pigs. Group 1 served as infarct controls. Group 2 had the endocardium slit longitudinally to alter local systolic deformation. Group 3 had a plug sectioned from ischemic tissue and rotated 90 degrees. The slit altered systolic deformation in the infarcted tissue, changing circumferential strain from expansion to compression and increasing radial strain and shears and the variability of collagen fiber angles but not the mean angle. In the plug pigs, when deformation, matrix orientation, and continuity are altered in the infarct area, the result is complete disarray in the organization of collagen within the infarct scar.     

PTEN regulates fibroblast elimination during collagen matrix contraction

During tissue repair, excess fibroblasts are eliminated by apoptosis. This physiologic process limits fibrosis and restores normal anatomic patterns. Replicating physiologic apoptosis associated with tissue repair, fibroblasts incorporated into type I collagen matrices undergo apoptosis in response to collagen matrix contraction. In this in vitro model of wound repair, fibroblasts first attach to collagen via alpha2beta1 integrin. This provides a survival signal via activation of the phosphatidylinositol 3-kinase/Akt signal pathway. However, during subsequent collagen matrix contraction, the level of phosphorylated Akt progressively declines, triggering apoptosis. The mechanism underlying the fall in phosphorylated Akt is incompletely understood. Here we show that PTEN phosphatase becomes activated during collagen matrix contraction and is responsible for antagonizing phosphatidylinositol 3-kinase activity and promoting a decline in phosphorylated Akt and fibroblast apoptosis in response to collagen contraction. PTEN null fibroblasts displayed enhanced levels of phosphorylated Akt and were resistant to collagen matrix contraction-induced apoptosis. Reconstitution of PTEN in PTEN null cells conferred susceptibility to apoptosis in response to contraction of collagen matrices. Consistent with this, knockdown of PTEN in PTEN(+/+) embryonic fibroblasts by small interfering RNA augmented Akt activity and suppressed apoptosis in contractile collagen matrices. Furthermore, inhibition of Akt activity restored the sensitivity of PTEN null cells to collagen contraction-induced apoptosis, indicating that the mechanism by which PTEN alters fibroblast viability is through modulation of phosphorylated Akt levels. Our work suggests that collagen matrix contraction activates PTEN by a mechanism involving cytoskeletal disassembly. Our studies indicate a key role for PTEN in regulating fibroblast viability during tissue repair.     

Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2.

Fibroblast growth factor-4 (FGF4), like other FGFs, shares a high affinity for the anionic glycosaminoglycans heparin and heparan sulfate (HS), which in turn enhance FGF-receptor (FGFR) binding and activation. Here we demonstrate using a cell free system that, at low concentrations of heparin, FGF4 binds only to FGFR-2, while much higher heparin levels are required for binding to FGFR-1. Chemical crosslinking of radiolabeled FGF4 to the soluble FGF receptors confirms the preferential formation of FGF4-FGFR-2 complexes under restricted heparin availability, with maximal ligand-receptor interactions at almost 20-fold lower heparin concentrations then those required for the affinity labeling of FGFR-1. In accordance, HS-deficient cells expressing FGFR-2 proliferate in response to FGF4 at extremely low exogenous heparin concentrations, while FGFR-1 expressing cells are completely unresponsive under the same conditions. We suggest that FGFR-2 is the preferred receptor for FGF4 under restricted HS conditions and that the bioavailability of structurally distinct HS motifs may differentially control receptor specificity of FGF4 in vivo.     

ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix

Breast epithelial cells differentiate into tubules when cultured in floating three-dimensional (3D) collagen gels, but not when the cells are cultured in the same collagen matrix that is attached to the culture dish. These observations suggest that the biophysical properties of collagenous matrices regulate epithelial differentiation, but the mechanism by which this occurs is unknown. Tubulogenesis required the contraction of floating collagen gels through Rho and ROCK-mediated contractility. ROCK-mediated contractility diminished Rho activity in a floating 3D collagen gel, and corresponded to a loss of FAK phosphorylated at Y397 localized to 3D matrix adhesions. Increasing the density of floating 3D collagen gels also disrupted tubulogenesis, promoted FAK phosphorylation, and sustained high Rho activity. These data demonstrate the novel finding that breast epithelial cells sense the rigidity or density of their environment via ROCK-mediated contractility and a subsequent down-regulation of Rho and FAK function, which is necessary for breast epithelial tubulogenesis to occur.     

Regulation of fibroblast proliferation in three-dimensional collagen gel matrix

Summary Fibroblastsin vivo reside in a three-dimensional (3-D) matrix. The 3-D culture method using collagen gels provides valuable information, but is also has some practical difficulties. In particular, the changes caused by the contraction of gels and the occasional abrupt detachment from the underlying surface have made extended culture difficult. In this study, the 3-D culture method was modified in order to observe the cells with minimal change of substrata for longer periods. The proliferation characteristics of fibroblasts cultured in gels in response to fetal calf serum (FCS), to two defined growth factors, insulin and platelet-derived growth factor (PDGF), and to a growth inhibitory factor, prostaglandin E₂ (PGE₂), were evaluated with this system in comparison with monolayer cultured fibroblasts. The DNA content of fibroblasts cultured both in gels and on dishes increased in response to FCS in a concentration-dependent manner. The proliferation of gel-cultured fibroblasts, however, was lower than that of dish-cultured cells, and higher concentrations of serum were necessary for proliferation. The response of gel-cultured cells to PDGF was also less than that of dish-cultured cells. In addition, fibroblasts cultured in gel culture did not respond to insulin, while the fibroblasts on dishes responded to insulin in a concentration-dependent manner. In contrast to the reduced response to growth stimulators, PGE₂ inhibited proliferation in gel culture and in monolayer culture similarly. The reduced responsiveness to growth stimulation but equivalent response to growth inhibition may account for reduced proliferation of fibroblasts in 3-D culture.     

Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices

Summary Here, we describe assay systems that utilize serum-free defined media to evaluate capillary morphogenesis during human endothelial cell (EC) invasion of three-dimensional collagen matrices. ECs invade these matrices over a 1–3-d period to form capillary tubes. Blocking antibodies to the α2β1 integrin interfere with invasion and morphogenesis while other integrin blocking antibodies do not. Interestingly, we observed increased invasion of ECs toward a population of underlying ECs undergoing morphogenesis. In addition, we have developed assays on microscope slides that display the invasion process horizontally, thereby enhancing our ability to image these events. Thus far, we have observed intracellular vacuoles that appear to regulate the formation of capillary lumens, and extensive cell processes that facilitate the interconnection of ECs during morphogenic events. These assays should enable further investigation of the morphologic steps and molecular events controlling human capillary tube formation in three-dimensional extracellular matrices.     

Extracellular matrices as elastic scaffold and mechanical interaction.

Development of spatial pattern and form is one of the central issues in embryology and is included under the general name of morphogenesis. Recently, many investigations have revealed how does development occurs by each embryonic stem cell or which Genes play a crucial role for morphogenesis. However, still fundamental question is unclear; such as how does each cell recognize spatial information or which kind of information guides each cell to the suitable place. Approximately, we have 6x10(13) cells in our body. If each frame of reference of each cell is included in the gene, gene must have included more than 6x10(13) of information to inform each cell where they are. We could simply suggest this kind of the idea is quite wrong because we know genes are few enough to include such informations. Recently, it has been suggested that interaction between intracellular and extracellular fiber play crucial roll for morphogenesis. The fibers inside cell are quite complicated but well organized the system, and fibers outside of the cell are comparatively very simple fiber. Each of the fibers is well studied, but quantitative investigation of their interaction is lacking although importance is suggested by many researchers. A major problem is lacking of new method or technique. In our topics, we would like to introduce how intracellular and extracellular fiber generate morphogenesis and how we could investigate them using new technique for tissue engineering, one of the promising field of applied cell biology.     

MT1-MMP promotes cell growth and ERK activation through c-Src and paxillin in three-dimensional collagen matrix

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is essential for tumor invasion and growth. We show here that MT1-MMP induces extracellular signal-regulated kinase (ERK) activation in cancer cells cultured in collagen gel, which is indispensable for their proliferation. Inhibition of MT1-MMP by MMP inhibitor or small interfering RNA suppressed activation of focal adhesion kinase (FAK) and ERK in MT1-MMP-expressing cancer cells, which resulted in up-regulation of p21^WAF1 and suppression of cell growth in collagen gel. Cell proliferation was also abrogated by the inhibitor against ERK pathway without affecting FAK phosphorylation. MT1-MMP and integrin α_vβ₃ were shown to be involved in c-Src activation, which induced FAK and ERK activation in collagen gel. These MT1-MMP-mediated signal transductions were paxillin dependent, as knockdown of paxillin reduced cell growth and ERK activation, and co-expression of MT1-MMP with paxillin induced ERK activation. The results suggest that MT1-MMP contributes to proliferation of cancer cells in the extracellular matrix by activating ERK through c-Src and paxillin.     

Measurement of mechanical tractions exerted by cells in three-dimensional matrices

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.