Musculoskeletal mechanobiology: Interpretation by external force and engineered substratum

Mechanobiology aims to discover how the mechanical environment affects the biological activity of cells and how cells’ ability to sense these mechanical cues is converted into elicited cellular responses. Musculoskeletal mechanobiology is of particular interest given the high mechanical loads that musculoskeletal tissues experience on a daily basis. How do cells within these mechanically active tissues interpret external loads imposed on their extracellular environment, and, how are cell–substrate interactions converted into biochemical signals? This review outlines many of the main mechanotransduction mechanisms known to date, and describes recent literature examining effects of both external forces and cell–substrate interactions on musculoskeletal cells. Whether via application of external forces and/or cell–substrate interactions, our understanding and regulation of musculoskeletal mechanobiology can benefit by expanding upon traditional models, and shedding new light through novel investigative approaches. Current and future work in this field is focused on identifying specific forces, stresses, and strains at the cellular and tissue level through both experimental and computational approaches, and analyzing the role of specific proteins through fluorescence-based investigations and knockdown models.     

Actin cytoskeleton dynamics and the cell division cycle

The network of actin filaments is one of the crucial cytoskeletal structures contributing to the morphological framework of a cell and which participates in the dynamic regulation of cellular functions. In adherent cell types, cells adhere to the substratum during interphase and spread to assume their characteristic shape supported by the actin cytoskeleton. This actin cytoskeleton is reorganized during mitosis to form rounded cells with increased cortical rigidity. The actin cytoskeleton is re-established after mitosis, allowing cells to regain their extended shape and attachment to the substratum. The modulation of such drastic changes in cell shape in coordination with cell cycle progression suggests a tight regulatory interaction between cytoskeleton signalling, cell–cell/cell–matrix adhesions and mitotic events. Here, we review the contribution of the actin cytoskeleton to cell cycle progression with an emphasis on the effectors responsible for the regulation of the actin cytoskeleton and integration of their activities with the cell cycle machinery.     

An inverse method for predicting tissue-level mechanics from cellular mechanical input

Extracellular matrix (ECM) provides a dynamic three-dimensional structure which translates mechanical stimuli to cells. This local mechanical stimulation may direct biological function including tissue development. Theories describing the role of mechanical regulators hypothesize the cellular response to variations in the external mechanical forces on the ECM. The exact ECM mechanical stimulation required to generate a specific pattern of localized cellular displacement is still unknown. The cell to tissue inverse problem offers an alternative approach to clarify this relationship. Developed for structural dynamics, the inverse dynamics problem translates measurements of local state variables (at the cell level) into an unknown or desired forcing function (at the tissue or ECM level). This paper describes the use of eigenvalues (resonant frequencies), eigenvectors (mode shapes), and dynamic programming to reduce the mathematical order of a simplified cell–tissue system and estimate the ECM mechanical stimulation required for a specified cellular mechanical environment. Finite element and inverse numerical analyses were performed on a simple two-dimensional model to ascertain the effects of weighting parameters and a reduction of analytical modes leading toward a solution. Simulation results indicate that the reduced number of mechanical modes (from 30 to 14 to 7) can adequately reproduce an unknown force time history on an ECM boundary. A representative comparison between cell to tissue (inverse) and tissue to cell (boundary value) modeling illustrates the multiscale applicability of the inverse model.     

Force Measurement Tools to Explore Cadherin Mechanotransduction

Abstract

Cell–cell adhesions serve to mechanically couple cells, allowing for long-range transmission of forces across cells in development, disease, and homeostasis. Recent work has shown that such contacts also play a role in transducing mechanical cues into a wide variety of cellular behaviors important to tissue function. As such, understanding the mechanical regulation of cells through their adhesion molecules has become a point of intense focus. This review will highlight the existing and emerging technologies and models that allow for exploration of cadherin-based adhesions as sites of mechanotransduction.     

Mechanical Cell–Cell Communication in Fibrous Networks: The Importance of Network Geometry

Cells contracting in extracellular matrix (ECM) can transmit stress over long distances, communicating their position and orientation to cells many tens of micrometres away. Such phenomena are not observed when cells are seeded on substrates with linear elastic properties, such as polyacrylamide (PA) gel. The ability for fibrous substrates to support far reaching stress and strain fields has implications for many physiological processes, while the mechanical properties of ECM are central to several pathological processes, including tumour invasion and fibrosis. Theoretical models have investigated the properties of ECM in a variety of network geometries. However, the effects of network architecture on mechanical cell–cell communication have received little attention. This work investigates the effects of geometry on network mechanics, and thus the ability for cells to communicate mechanically through different networks. Cell-derived displacement fields are quantified for various network geometries while controlling for network topology, cross-link density and micromechanical properties. We find that the heterogeneity of response, fibre alignment, and substrate displacement fields are sensitive to network choice. Further, we show that certain geometries support mechanical communication over longer distances than others. As such, we predict that the choice of network geometry is important in fundamental modelling of cell–cell interactions in fibrous substrates, as well as in experimental settings, where mechanical signalling at the cellular scale plays an important role. This work thus informs the construction of theoretical models for substrate mechanics and experimental explorations of mechanical cell–cell communication.     

Slow stress propagation in adherent cells

Mechanical cues influence a wide range of cellular behaviors including motility, differentiation, and tumorigenesis. Although previous studies elucidated the role of specific players such as ion channels and focal adhesions as local mechanosensors, the investigation of how mechanical perturbations propagate across the cell is necessary to understand the spatial coordination of cellular processes. Here we quantify the magnitude and timing of intracellular stress propagation, using atomic force microscopy and particle tracking by defocused fluorescence microscopy. The apical cell surface is locally perturbed by atomic force microscopy cantilever indentation, and distal displacements are measured in three dimensions by tracking integrin-bound fluorescent particles. We observe an immediate response and slower equilibration, occurring over times that increase with distance from perturbation. This distance-dependent equilibration occurs over several seconds and can be eliminated by disruption of the actin cytoskeleton. Our experimental results are not explained by traditional viscoelastic models of cell mechanics, but they are consistent with predictions from poroelastic models that include both cytoskeletal deformation and flow of the cytoplasm. Our combined atomic force microscopy-particle tracking measurements provide direct evidence of slow, distance-dependent dissipative stress propagation in response to external mechanical cues and offer new insights into mechanical models and physiological behaviors of adherent cells.     

Myosin II Activity Softens Cells in Suspension

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.     

Probing cell structure by controlling the mechanical environment with cell–substrate interactions

Recent results demonstrate the exquisite sensitivity of cell morphology and structure to mechanical stimulation. Mechanical stimulation is often coupled with cell–substrate interactions that can, in turn, influence molecular response and determine cellular fates including apoptosis, proliferation, and differentiation. To understand these effects as they specifically relate to compressive mechanical stimulation and topographic control, we developed a microfabricated system to grow cells on polydimethylsiloxane (PDMS) microchannel surfaces where we maintained compression stimulation. We also probed cellular response following compressive mechanical stimulation to PDMS substrates of varying stiffness. In these instances, we examined cytoskeletal and morphologic changes in living cells attached to our substrate following the application of localized compressive stimulation. We found that the overall morphology and cell structure, including the actin cytoskeleton, oriented in the direction of the compressive strain applied and along the topographic microchannels. Furthermore by comparing topographic response to material stiffness, we found a 40% increase in cell area for cells cultured on the microchannels versus softer PDMS as well as a decreased cell area of 30% when using softer PDMS over unmodified PDMS. These findings have implications for research in a diversity of fields including cell–material interactions, mechanotransduction, and tissue engineering.     

Effects of blocking integrin β1 and N-cadherin cellular interactions on mechanical properties of vascular smooth muscle cells

Experimental measurements of cellular mechanical properties have shown large variability in whole-cell mechanical properties between cells from a single population. This heterogeneity has been observed in many cell populations and with several measurement techniques but the sources are not yet fully understood. Cell mechanical properties are directly related to the composition and organization of the cytoskeleton, which is physically coupled to neighboring cells through adherens junctions and to underlying matrix through focal adhesion complexes. This high level of heterogeneity may be attributed to varying cellular interactions throughout the sample. We tested the effect of cell-cell and cell-matrix interactions on the mechanical properties of vascular smooth muscle cells (VSMCs) in culture by using antibodies to block N-cadherin and integrin β1 interactions. VSMCs were cultured on substrates of varying stiffness with and without tension. Under each of these conditions, cellular mechanical properties were characterized by performing atomic force microscopy (AFM) and cellular structure was analyzed through immunofluorescence imaging. As expected, VSMC mechanical properties were greatly affected by the underlying culture substrate and applied tension. Interestingly, the cell-to-cell variation in mechanical properties within each sample decreased significantly in the antibody conditions. Thus, the cells grown with blocking antibodies were more homogeneous in their mechanical properties on both glass and soft substrates. This suggests that diversified adhesion binding between cells and the ECM is responsible for a significant amount of mechanical heterogeneity that is observed in 2D cell culture studies.     

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration.     

Actin and microtubules drive differential aspects of planar cell polarity in multiciliated cells

Planar cell polarization represents the ability of cells to orient within the plane of a tissue orthogonal to the apical basal axis. The proper polarized function of multiciliated cells requires the coordination of cilia spacing and cilia polarity as well as the timing of cilia beating during metachronal synchrony. The planar cell polarity pathway and hydrodynamic forces have been shown to instruct cilia polarity. In this paper, we show how intracellular effectors interpret polarity to organize cellular morphology in accordance with asymmetric cellular function. We observe that both cellular actin and microtubule networks undergo drastic reorganization, providing differential roles during the polarized organization of cilia. Using computational angular correlation analysis of cilia orientation, we report a graded cellular organization downstream of cell polarity cues. Actin dynamics are required for proper cilia spacing, global coordination of cilia polarity, and coordination of metachronic cilia beating, whereas cytoplasmic microtubule dynamics are required for local coordination of polarity between neighboring cilia.     

Tissue‐disruption‐induced cellular stochasticity and epigenetic drift: Common origins of aging and cancer?

Age‐related and cancer‐related epigenomic modifications have been associated with enhanced cell‐to‐cell gene expression variability that characterizes increased cellular stochasticity. Since gene expression variability appears to be highly reduced by—and epigenetic and phenotypic stability acquired through—direct or long‐range cellular interactions during cell differentiation, we propose a common origin for aging and cancer in the failure to control cellular stochasticity by cell–cell interactions. Tissue‐disruption‐induced cellular stochasticity associated with epigenetic drift would be at the origin of organ dysfunction because of an increase in phenotypic variation among cells, ultimately leading to cell death and organ failure through a loss of coordination in cellular functions, and eventually to cancerization. We propose mechanistic research perspectives to corroborate this hypothesis and explore its evolutionary consequences, highlighting a positive correlation between the median age of mass loss onset (a proxy for the onset of organ aging) and the median age at cancer diagnosis.     

Design principles of tissue organisation: How single cells coordinate across scales

Cells act as building blocks of multicellular organisms, forming higher-order structures at different biological scales. Niches, tissues and, ultimately, entire organisms consist of single cells that remain in constant communication. Emergence of developmental patterns and tissue architecture thus relies on single cells acting as a collective, coordinating growth, migration, cell fate transitions and cell type sorting. For this, information has to be transmitted forward from cells to tissues and fed back to the individual cell to allow dynamic and robust coordination. Here, we define the design principles of tissue organisation integrating chemical, genetic and mechanical cues. We also review the state-of-the-art technologies used for dissecting collective cellular behaviours at single cell– and tissue-level resolution. We finally outline future challenges that lie in a comprehensive understanding of how single cells coordinate across biological scales to insure robust development, homoeostasis and regeneration of tissues.     

A hybrid computational model for collective cell durotaxis

Collective cell migration is regulated by a complex set of mechanical interactions and cellular mechanisms. Collective migration emerges from mechanisms occurring at single cell level, involving processes like contraction, polymerization and depolymerization, of cell–cell interactions and of cell–substrate adhesion. Here, we present a computational framework which simulates the dynamics of this emergent behavior conditioned by substrates with stiffness gradients. The computational model reproduces the cell’s ability to move toward the stiffer part of the substrate, process known as durotaxis. It combines the continuous formulation of truss elements and a particle-based approach to simulate the dynamics of cell–matrix adhesions and cell–cell interactions. Using this hybrid approach, researchers can quickly create a quantitative model to understand the regulatory role of different mechanical conditions on the dynamics of collective cell migration. Our model shows that durotaxis occurs due to the ability of cells to deform the substrate more in the part of lower stiffness than in the stiffer part. This effect explains why cell collective movement is more effective than single cell movement in stiffness gradient conditions. In addition, we numerically evaluate how gradient stiffness properties, cell monolayer size and force transmission between cells and extracellular matrix are crucial in regulating durotaxis.     

Cell Surface Deformation during an Action Potential

The excitation of many cells and tissues is associated with cell mechanical changes. The evidence presented herein corroborates that single cells deform during an action potential. It is demonstrated that excitation of plant cells (Chara braunii internodes) is accompanied by out-of-plane displacements of the cell surface in the micrometer range (～1–10 μm). The onset of cellular deformation coincides with the depolarization phase of the action potential. The mechanical pulse: 1) propagates with the same velocity as the electrical pulse (within experimental accuracy, ～10 mm s^?1), 2) is reversible, 3) in most cases is of biphasic nature (109 out of 152 experiments), and 4) is presumably independent of actin-myosin-motility. The existence of transient mechanical changes in the cell cortex is confirmed by micropipette aspiration experiments. A theoretical analysis demonstrates that this observation can be explained by a reversible change in the mechanical properties of the cell surface (transmembrane pressure, surface tension, and bending rigidity). Taken together, these findings contribute to the ongoing debate about the physical nature of cellular excitability.     

Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation

Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas‐exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell–cell, cell–extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle‐actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load‐bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas‐exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization. Birth Defects Research (Part A) 100:227–239, 2014. © 2014 Wiley Periodicals, Inc.     

Invited review: engineering approaches to cytoskeletal mechanics.

An outstanding problem in cell biology is how cells sense mechanical forces and how those forces affect cellular functions. Various biophysical and biochemical mechanisms have been invoked to answer this question. A growing body of evidence indicates that the deformable cytoskeleton (CSK), an intracellular network of interconnected filamentous biopolymers, provides a physical basis for transducing mechanical signals into biochemical signals. Therefore, to understand how mechanical forces regulate cellular functions, it is important to know how cells respond to changes in the CSK force balance and to identify the underlying mechanisms that control transmission of mechanical forces throughout the CSK and bring it to equilibrium. Recent developments of new experimental techniques for measuring cell mechanical properties and novel theoretical models of cellular mechanics make it now possible to identify and quantitate the contributions of various CSK structures to the overall balance of mechanical forces in the cell. This review focuses on engineering approaches that have been used in the past two decades in studies of the mechanics of the CSK.     

Polar growth in pollen tubes is associated with spatially confined dynamic changes in cell mechanical properties

Cellular morphogenesis involves changes to cellular size and shape which in the case of walled cells implies the mechanical deformation of the extracellular matrix. So far, technical challenges have made quantitative mechanical measurements of this process at subcellular scale impossible. We used micro-indentation to investigate the dynamic changes in the cellular mechanical properties during the onset of spatially confined growth activities in plant cells. Pollen tubes are cellular protuberances that have a strictly unidirectional growth pattern. Micro-indentation of these cells revealed that the initial formation of a cylindrical protuberance is preceded by a local reduction in cellular stiffness. Similar cellular softening was observed before the onset of a rapid growth phase in cells with oscillating growth pattern. These findings provide the first quantitative cytomechanical data that confirm the important role of the mechanical properties of the cell wall for local cellular growth processes. They are consistent with a conceptual model that explains pollen tube oscillatory growth based on the relationship between turgor pressure and tensile resistance in the apical cell wall. To further confirm the significance of cell mechanics, we artificially manipulated the mechanical cell wall properties as well as the turgor pressure. We observed that these changes affected the oscillation profile and were able to induce oscillatory behavior in steadily growing tubes.     

Metabolic responses induced by compression of chondrocytes in variable-stiffness microenvironments

Cells sense and respond to mechanical loads in a process called mechanotransduction. These processes are disrupted in the chondrocytes of cartilage during joint disease. A key driver of cellular mechanotransduction is the stiffness of the surrounding matrix. Many cells are surrounded by extracellular matrix that allows for tissue mechanical function. Although prior studies demonstrate that extracellular stiffness is important in cell differentiation, morphology and phenotype, it remains largely unknown how a cell’s biological response to cyclical loading varies with changes in surrounding substrate stiffness. Understanding these processes is important for understanding cells that are cyclically loaded during daily in vivo activities (e.g. chondrocytes and walking). This study uses high-performance liquid chromatography – mass spectrometry to identify metabolomic changes in primary chondrocytes under cyclical compression for 0–30 minutes in low- and high-stiffness environments. Metabolomic analysis reveals metabolites and pathways that are sensitive to substrate stiffness, duration of cyclical compression, and a combination of both suggesting changes in extracellular stiffness in vivo alter mechanosensitive signaling. Our results further suggest that cyclical loading minimizes matrix deterioration and increases matrix production in chondrocytes. This study shows the importance of modeling in vivo stiffness with in vitro models to understand cellular mechanotransduction.