Remodeling of Fibrous Extracellular Matrices by Contractile Cells: Predictions from Discrete Fiber Network Simulations

Contractile forces exerted on the surrounding extracellular matrix (ECM) lead to the alignment and stretching of constituent fibers within the vicinity of cells. As a consequence, the matrix reorganizes to form thick bundles of aligned fibers that enable force transmission over distances larger than the size of the cells. Contractile force-mediated remodeling of ECM fibers has bearing on a number of physiologic and pathophysiologic phenomena. In this work, we present a computational model to capture cell-mediated remodeling within fibrous matrices using finite element–based discrete fiber network simulations. The model is shown to accurately capture collagen alignment, heterogeneous deformations, and long-range force transmission observed experimentally. The zone of mechanical influence surrounding a single contractile cell and the interaction between two cells are predicted from the strain-induced alignment of fibers. Through parametric studies, the effect of cell contractility and cell shape anisotropy on matrix remodeling and force transmission are quantified and summarized in a phase diagram. For highly contractile and elongated cells, we find a sensing distance that is ten times the cell size, in agreement with experimental observations.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

Elastic Anisotropy Governs the Range of Cell-Induced Displacements

The unique nonlinear mechanics of the fibrous extracellular matrix (ECM) facilitates long-range cell-cell mechanical communications that would be impossible for linear elastic substrates. Past research has described the contribution of two separated effects on the range of force transmission, including ECM elastic nonlinearity and fiber alignment. However, the relation between these different effects is unclear, and how they combine to dictate force transmission range is still elusive. Here, we combine discrete fiber simulations with continuum modeling to study the decay of displacements induced by a contractile cell in fibrous networks. We demonstrate that fiber nonlinearity and fiber reorientation both contribute to the strain-induced elastic anisotropy of the cell’s local environment. This elastic anisotropy is a “lumped” parameter that governs the slow decay of displacements, and it depends on the magnitude of applied strain, either an external tension or an internal contraction, as a model of the cell. Furthermore, we show that accounting for artificially prescribed elastic anisotropy dictates the decay of displacements induced by a contracting cell. Our findings unify previous single effects into a mechanical theory that explains force transmission in fibrous networks. This work may provide insights into biological processes that involve communication of distant cells mediated by the ECM, such as those occurring in morphogenesis, wound healing, angiogenesis, and cancer metastasis. It may also provide design parameters for biomaterials to control force transmission between cells as a way to guide morphogenesis in tissue engineering.     

The Role of Vinculin in the Regulation of the Mechanical Properties of Cells

Vinculin couples as a focal adhesion protein the extracellular matrix (ECM) through integrins to the actomyosin cytoskeleton. During the last years vinculin has become the focus of cell mechanical measurements and a key protein regulating the transmission of contractile forces. In earlier reports vinculin has been described as an inhibitor of cell migration on planar substrates, because knock-out of vinculin in F9 mouse embryonic carcinoma cells and mouse embryonic fibroblasts showed increased cell motility on 2D substrates. The role of vinculin in cell invasion through a 3D extracellular matrix is still fragmentarily investigated. This review presents vinculin in its role as a regulator of cellular mechanical functions. Contractile force generation is reduced when vinculin is absent, or enhanced when vinculin is present. Moreover, the generation of contractile forces is a prerequisite for cell invasion through a dense 3D ECM, where the pore-size is smaller than the diameter of the cell nucleus (<2 μm). Measurements of cell’s biophysical properties will be presented. In summary, vinculin’s leading role among focal adhesion proteins in regulating the mechanical properties of cells will be discussed.     

Cyclic stretch enhances reorientation and differentiation of 3-D culture model of human airway smooth muscle

Activation of airway smooth muscle (ASM) cells plays a central role in the pathophysiology of asthma. Because ASM is an important therapeutic target in asthma, it is beneficial to develop bioengineered ASM models available for assessing physiological and biophysical properties of ASM cells. In the physiological condition in vivo, ASM cells are surrounded by extracellular matrix (ECM) and exposed to mechanical stresses such as cyclic stretch. We utilized a 3-D culture model of human ASM cells embedded in type-I collagen gel. We further examined the effects of cyclic mechanical stretch, which mimics tidal breathing, on cell orientation and expression of contractile proteins of ASM cells within the 3-D gel. ASM cells in type-I collagen exhibited a tissue-like structure with actin stress fiber formation and intracellular Ca²⁺ mobilization in response to methacholine. Uniaxial cyclic stretching enhanced alignment of nuclei and actin stress fibers of ASM cells. Moreover, expression of mRNAs for contractile proteins such as α-smooth muscle actin, calponin, myosin heavy chain 11, and transgelin of stretched ASM cells was significantly higher than that under the static condition. Our findings suggest that mechanical force and interaction with ECM affects development of the ASM tissue-like construct and differentiation to the contractile phenotype in a 3-D culture model.     

Prestress in the extracellular matrix sensitizes latent TGF-β1 for activation

Integrin-mediated force application induces a conformational change in latent TGF-β1 that leads to the release of the active form of the growth factor from the extracellular matrix (ECM). Mechanical activation of TGF-β1 is currently understood as an acute process that depends on the contractile force of cells. However, we show that ECM remodeling, preceding the activation step, mechanically primes latent TGF-β1 akin to loading a mechanical spring. Cell-based assays and unique strain devices were used to produce a cell-derived ECM of controlled organization and prestrain. Mechanically conditioned ECM served as a substrate to measure the efficacy of TGF-β1 activation after cell contraction or direct force application using magnetic microbeads. The release of active TGF-β1 was always higher from prestrained ECM as compared with unorganized and/or relaxed ECM. The finding that ECM prestrain regulates the bioavailability of TGF-β1 is important to understand the context of diseases that involve excessive ECM remodeling, such as fibrosis or cancer.     

The mechanochemistry of cytoskeletal force generation

In this communication, we propose a model to study the non-equilibrium process by which actin stress fibers develop force in contractile cells. The emphasis here is on the non-equilibrium thermodynamics, which is necessary to address the mechanics as well as the chemistry of dynamic cell contractility. In this setting, we are able to develop a framework that relates (a) the dynamics of force generation within the cell and (b) the cell’s response to external stimuli to the chemical processes occurring within the cell, as well as to the mechanics of linkage between the stress fibers, focal adhesions and extracellular matrix.     

Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity

Metastatic cancers aggressively reorganize collagen in their microenvironment. For example, radially orientated collagen fibers have been observed surrounding tumor cell clusters in vivo. The degree of fiber alignment, as a consequence of this remodeling, has often been difficult to quantify. In this paper, we present an easy to implement algorithm for accurate detection of collagen fiber orientation in a rapid pixel-wise manner. This algorithm quantifies the alignment of both computer generated and actual collagen fiber networks of varying degrees of alignment within 5°°. We also present an alternative easy method to calculate the alignment index directly from the standard deviation of fiber orientation. Using this quantitative method for determining collagen alignment, we demonstrate that the number of collagen fiber intersections has a negative correlation with the degree of fiber alignment. This decrease in intersections of aligned fibers could explain why cells move more rapidly along aligned fibers than unaligned fibers, as previously reported. Overall, our paper provides an easier, more quantitative and quicker way to quantify fiber orientation and alignment, and presents a platform in studying effects of matrix and cellular properties on fiber alignment in complex 3D environments.     

Cell locomotion forces versus cell contraction forces for collagen lattice contraction: an in vitro model of wound contraction

Cultured human dermal fibroblasts suspended in a rapidly polymerizing collagen matrix produce a fibroblast-populated collagen lattice. With time, this lattice will undergo a reduction in size referred to as lattice contraction. During this process, two distinct cell populations develop. At the periphery of the lattice, highly oriented sheets of cells, morphologically identifiable as myofibroblasts, show cell-to-cell contacts and thick, actin-rich staining cytoplasmic stress fibers. It is proposed that these cells undergoing cell contraction produce a multicellular contractile unit which reorients the collagen fibrils associated with them. The cells in the central region, referred to as fibroblasts, are randomly oriented, with few cell-to-cell contacts and faintly staining actin cytoplasmic filaments. In contrast it is proposed that cells working as single units use cell locomotion forces to reorient the collagen fibrils associated with them. Using this model, we sought to determine which of these two mechanisms, cell contraction or cell locomotion, is responsible for the force that contracts collagen lattices. Our experiments showed that fibroblasts produce this contractile force, and that the mechanism for lattice contraction appears to be related to cell locomotion. This is in contrast to a myofibroblast; where the mechanism for contraction is based upon cell contractions. Fibroblasts attempting to move within the collagen matrix reorganize the surrounding collagen fibrils; when these collagen fibrils can be organized no further and cell-to-cell contacts develop, which occurs at the periphery of the lattice first, these cells can no longer participate in the dynamic aspects of lattice contraction.     

Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization

Cells generate mechanical force to organize the extracellular matrix (ECM) and drive important developmental and reparative processes. Likewise, tumor cells invading into three-dimensional (3D) matrices remodel the ECM microenvironment. Importantly, we previously reported a distinct radial reorganization of the collagen matrix surrounding tumors that facilitates local invasion. Here we describe a mechanism by which cells utilize contractility events to reorganize the ECM to provide contact guidance that facilitates 3D migration. Using novel assays to differentially organize the collagen matrix we show that alignment of collagen perpendicular to the tumor-explant boundary promotes local invasion of both human and mouse mammary epithelial cells. In contrast, organizing the collagen matrix to mimic the ECM organization associated with noninvading regions of tumors suppresses 3D migration/invasion. Moreover, we demonstrate that matrix reorganization is contractility-dependent and that the Rho/Rho kinase pathway is necessary for collagen alignment to provide contact guidance. Yet, if matrices are prealigned, inhibiting neither Rho nor Rho kinase inhibits 3D migration, which supports our conclusion that Rho-mediated matrix alignment is an early step in the invasion process, preceding and subsequently facilitating 3D migration.     

Long-Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers

Cells can sense and respond to mechanical signals over relatively long distances across fibrous extracellular matrices. Recently proposed models suggest that long-range force transmission can be attributed to the nonlinear elasticity or fibrous nature of collagen matrices, yet the mechanism whereby fibers align remains unknown. Moreover, cell shape and anisotropy of cellular contraction are not considered in existing models, although recent experiments have shown that they play crucial roles. Here, we explore all of the key factors that influence long-range force transmission in cell-populated collagen matrices: alignment of collagen fibers, responses to applied force, strain stiffening properties of the aligned fibers, aspect ratios of the cells, and the polarization of cellular contraction. A constitutive law accounting for mechanically driven collagen fiber reorientation is proposed. We systematically investigate the range of collagen-fiber alignment using both finite-element simulations and analytical calculations. Our results show that tension-driven collagen-fiber alignment plays a crucial role in force transmission. Small critical stretch for fiber alignment, large fiber stiffness and fiber strain-hardening behavior enable long-range interaction. Furthermore, the range of collagen-fiber alignment for elliptical cells with polarized contraction is much larger than that for spherical cells with diagonal contraction. A phase diagram showing the range of force transmission as a function of cell shape and polarization and matrix properties is presented. Our results are in good agreement with recent experiments, and highlight the factors that influence long-range force transmission, in particular tension-driven alignment of fibers. Our work has important relevance to biological processes including development, cancer metastasis, and wound healing, suggesting conditions whereby cells communicate over long distances.     

Exosome-mediated transduction of mechanical force regulates prostate cancer migration via microRNA

Physical cues in the extracellular microenvironment regulate cancer cell metastasis. Functional microRNA (miRNA) carried by cancer derived exosomes play a critical role in extracellular communication between cells and the extracellular microenvironment. However, little is known about the role of exosomes loaded miRNAs in the mechanical force transmission between cancer cells and extracellular microenvironment. Herein, our results suggest that stiff extracellular matrix (ECM) induced exosomes promote cancer cell migration. The ECM mechanical force regulated the exosome miRNA cargo of prostate cancer cells. Exosome miRNAs regulated by the ECM mechanical force modulated cancer cell metastasis by regulating cell motility, ECM remodeling and the interaction between cancer cells and nerves. Focal adhesion kinase mediated-ECM mechanical force regulated the intracellular miRNA expression, and F-actin mediate-ECM mechanical force regulated miRNA packaging into exosomes. The above results demonstrated that the exosome miRNA cargo promoted cancer metastasis by transmitting the ECM mechanical force. The ECM mechanical force may play multiple roles in maintaining the microenvironment of cancer metastasis through the exosome miRNA cargo.     

Quantitative assessment of local collagen matrix remodeling in 3-D culture: the role of Rho kinase

The purpose of this study was to quantitatively assess the role of Rho kinase in modulating the pattern and amount of local cell-induced collagen matrix remodeling. Human corneal fibroblasts were plated inside 100-microm thick fibrillar collagen matrices and cultured for 24 h in media with or without the Rho kinase inhibitor Y-27632. Cells were then fixed and stained with phalloidin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. Fourier transform analysis was used to assess collagen fibril alignment, and 3-D cell morphology and local collagen density were measured using MetaMorph. Culture in serum-containing media induced significant global matrix contraction, which was inhibited by blocking Rho kinase (p<0.001). Fibroblasts generally had a bipolar morphology and intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. When Rho kinase was inhibited, cells had a more cortical f-actin distribution and dendritic morphology. Both local collagen fibril density and alignment were significantly reduced (p<0.01). Overall, the data suggests that Rho kinase-dependent contractile force generation leads to co-alignment of cells and collagen fibrils along the plane of greatest resistance, and that this process contributes to global matrix contraction.     

From molecular signal activation to locomotion: an integrated, multiscale analysis of cell motility on defined matrices

The adhesion, mechanics, and motility of eukaryotic cells are highly sensitive to the ligand density and stiffness of the extracellular matrix (ECM). This relationship bears profound implications for stem cell engineering, tumor invasion and metastasis. Yet, our quantitative understanding of how ECM biophysical properties, mechanotransductive signals, and assembly of contractile and adhesive structures collude to control these cell behaviors remains extremely limited. Here we present a novel multiscale model of cell migration on ECMs of defined biophysical properties that integrates local activation of biochemical signals with adhesion and force generation at the cell-ECM interface. We capture the mechanosensitivity of individual cellular components by dynamically coupling ECM properties to the activation of Rho and Rac GTPases in specific portions of the cell with actomyosin contractility, cell-ECM adhesion bond formation and rupture, and process extension and retraction. We show that our framework is capable of recreating key experimentally-observed features of the relationship between cell migration and ECM biophysical properties. In particular, our model predicts for the first time recently reported transitions from filopodial to "stick-slip" to gliding motility on ECMs of increasing stiffness, previously observed dependences of migration speed on ECM stiffness and ligand density, and high-resolution measurements of mechanosensitive protrusion dynamics during cell motility we newly obtained for this study. It also relates the biphasic dependence of cell migration speed on ECM stiffness to the tendency of the cell to polarize. By enabling the investigation of experimentally-inaccessible microscale relationships between mechanotransductive signaling, adhesion, and motility, our model offers new insight into how these factors interact with one another to produce complex migration patterns across a variety of ECM conditions.     

A Mechanochemical Model of Cell Reorientation on Substrates under Cyclic Stretch

We report a theoretical study on the cyclic stretch-induced reorientation of spindle-shaped cells. Specifically, by taking into account the evolution of sub-cellular structures like the contractile stress fibers and adhesive receptor-ligand clusters, we develop a mechanochemical model to describe the dynamics of cell realignment in response to cyclically stretched substrates. Our main hypothesis is that cells tend to orient in the direction where the formation of stress fibers is energetically most favorable. We show that, when subjected to cyclic stretch, the final alignment of cells reflects the competition between the elevated force within stress fibers that accelerates their disassembly and the disruption of cell-substrate adhesion as well, and an effectively increased substrate rigidity that promotes more stable focal adhesions. Our model predictions are consistent with various observations like the substrate rigidity dependent formation of stable adhesions and the stretching frequency, as well as stretching amplitude, dependence of cell realignment. This theory also provides a simple explanation on the regulation of protein Rho in the formation of stretch-induced stress fibers in cells.     

The different roles of myosin IIA and myosin IIB in contraction of 3D collagen matrices by human fibroblasts

Contraction of 3D collagen matrices by fibroblasts frequently is used as an in vitro model of wound closure. Different iterations of the model – all conventionally referred to as “contraction” – involve different morphological patterns. During floating matrix contraction, cells initially are round without stress fibers and subsequently undergo spreading. During stressed matrix contraction, cells initially are spread with stress fibers and subsequently undergo shortening. In the current studies, we used siRNA silencing of myosin IIA (MyoIIA) and myosin IIB (MyoIIB) to test the roles of myosin II isoforms in fibroblast interactions with 3D collagen matrices and collagen matrix contraction. We found that MyoIIA but not MyoIIB was required for cellular global inward contractile force, formation of actin stress fibers, and morphogenic cell clustering. Stressed matrix contraction required MyoIIA but not MyoIIB. Either MyoIIA or MyoIIB was sufficient for floating matrix contraction (FMC) stimulated by platelet-derived growth factor. Neither MyoIIA or MyoIIB was necessary for FMC stimulated by serum. Our findings suggest that myosin II-dependent motor mechanisms for collagen translocation during extracellular matrix remodeling differ depending on cell tension and growth factor stimulation.     

A three-dimensional collagen-fiber network model of the extracellular matrix for the simulation of the mechanical behaviors and micro structures

The extracellular matrix (ECM) provides structural and biochemical support to cells and tissues, which is a critical factor for modulating cell dynamic behavior and intercellular communication. In order to further understand the mechanisms of the interactive relationship between cell and the ECM, we developed a three-dimensional (3D) collagen-fiber network model to simulate the micro structure and mechanical behaviors of the ECM and studied the stress–strain relationship as well as the deformation of the ECM under tension. In the model, the collagen-fiber network consists of abundant random distributed collagen fibers and some crosslinks, in which each fiber is modeled as an elastic beam and a crosslink is modeled as a linear spring with tensile limit, it means crosslinks will fail while the tensile forces exceed the limit of spring. With the given parameters of the beam and the spring, the simulated tensile stress–strain relation of the ECM highly matches the experimental results including damaged and failed behaviors. Moreover, by applying the maximal inscribed sphere method, we measured the size distribution of pores in the fiber network and learned the variation of the distribution with deformation. We also defined the alignment of the collagen-fibers to depict the orientation of fibers in the ECM quantitatively. By the study of changes of the alignment and the damaged crosslinks against the tensile strain, this paper reveals the comprehensive mechanisms of four stages of ‘toe’, ‘linear’, ‘damage’ and ‘failure’ in the tensile stress–strain relation of the ECM which can provide further insight in the study of cell-ECM interaction.     

Finite element analysis of mechanics of lateral transmission of force in single muscle fiber

Most of the myofibers in long muscles of vertebrates terminate within fascicles without reaching either end of the tendon, thus force generated in myofibers has to be transmitted laterally through the extracellular matrix (ECM) to adjacent fibers; which is defined as the lateral transmission of force in skeletal muscles. The goal of this study was to determine the mechanisms of lateral transmission of force between the myofiber and ECM. In this study, a 2D finite element model of single muscle fiber was developed to study the effects of mechanical properties of the endomysium and the tapered ends of myofiber on lateral transmission of force. Results showed that most of the force generated is transmitted near the end of the myofiber through shear to the endomysium, and the force transmitted to the end of the model increases with increased stiffness of ECM. This study also demonstrated that the tapered angle of the myofiber ends can reduce the stress concentration near the myofiber end while laterally transmitting force efficiently.     

Measurement of Skeletal Muscle Fiber Contractility with High-Speed Traction Microscopy

We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young’s modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 μN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 μN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.     

Diversity of extracellular matrix morphology in vertebrate skeletal muscle

Existing data suggest the extracellular matrix (ECM) of vertebrate skeletal muscle consists of several morphologically distinct layers: an endomysium, perimysium, and epimysium surrounding muscle fibers, fascicles, and whole muscles, respectively. These ECM layers are hypothesized to serve important functional roles within muscle, influencing passive mechanics, providing avenues for force transmission, and influencing dynamic shape changes during contraction. The morphology of the skeletal muscle ECM is well described in mammals and birds; however, ECM morphology in other vertebrate groups including amphibians, fish, and reptiles remains largely unexamined. It remains unclear whether a multilayered ECM is a common feature of vertebrate skeletal muscle, and whether functional roles attributed to the ECM should be considered in mechanical analyses of non-mammalian and non-avian muscle. To explore the prevalence of a multilayered ECM, we used a cell maceration and scanning electron microscopy technique to visualize the organization of ECM collagen in muscle from six vertebrates: bullfrogs (Lithobates catesbeianus), turkeys (Meleagris gallopavo), alligators (Alligator mississippiensis), cane toads (Rhinella marina), laboratory mice (Mus musculus), and carp (Cyprinus carpio). All muscles studied contained a collagen-reinforced ECM with multiple morphologically distinct layers. An endomysium surrounding muscle fibers was apparent in all samples. A perimysium surrounding groups of muscle fibers was apparent in all but carp epaxial muscle; a muscle anatomically, functionally, and phylogenetically distinct from the others studied. An epimysium was apparent in all samples taken at the muscle periphery. These findings show that a multilayered ECM is a common feature of vertebrate muscle and suggest that a functionally relevant ECM should be considered in mechanical models of vertebrate muscle generally. It remains unclear whether cross-species variations in ECM architecture are the result of phylogenetic, anatomical, or functional differences, but understanding the influence of such variation on muscle mechanics may prove a fruitful area for future research.