3D observations provide striking findings in rubber elasticity.

The mechanical response of rubbers has been ubiquitously assumed to be only a function of the imposed strain. Using innovative X-ray measurements capturing the three-dimensional spatial volumetric strain fields, we demonstrate that rubbers and indeed many common engineering polymers undergo significant local volume changes. But remarkably, the overall specimen volume remains constant regardless of the imposed loading. This strange behavior which also leads to apparent negative local bulk moduli is due to the presence of a mobile phase within these materials. Combining X-ray tomographic observations with high-speed radiography to track the motion of the mobile phase, we have revised classical thermodynamic frameworks of rubber elasticity. The work opens broad avenues to understand not only the mechanical behavior of rubbers but a large class of widely used engineering polymers.

Underexcitation prevents crystallization of granular assemblies subjected to high-frequency vibration.

Crystallization of dry particle assemblies via imposed vibrations is a scalable route to assemble micro/macro crystals. It is well understood that there exists an optimal frequency to maximize crystallization with broad acceptance that this optimal frequency emerges because high-frequency vibration results in overexcitation of the assembly. Using measurements that include interrupted X-ray computed tomography and high-speed photography combined with discrete-element simulations we show that, rather counterintuitively, high-frequency vibration underexcites the assembly. The large accelerations imposed by high-frequency vibrations create a fluidized boundary layer that prevents momentum transfer into the bulk of the granular assembly. This results in particle underexcitation which inhibits the rearrangements required for crystallization. This clear understanding of the mechanisms has allowed the development of a simple concept to inhibit fluidization which thereby allows crystallization under high-frequency vibrations.

Contact guidance via heterogeneity of substrate elasticity.

Contact guidance, the widely-known phenomenon of cell alignment, is an essential step in the organization of adherent cells. This guidance is known to occur by, amongst other things, anisotropic features in the environment including elastic heterogeneity. To understand the origins of this guidance we employed a novel statistical thermodynamics framework, which recognises the non-thermal fluctuations in the cellular response, for modelling the response of the cells seeded on substrates with alternating soft and stiff stripes. Consistent with observations, the modelling framework predicts the existence of three regimes of cell guidance: (i) in regime I for stripe widths much larger than the cell size guidance is primarily entropic; (ii) for stripe widths on the order of the cell size in regime II guidance is biochemically mediated and accompanied by changes to the cell morphology while (iii) in regime III for stripe widths much less than the cell size there is no guidance as cells cannot sense the substrate heterogeneity. Guidance in regimes I and II is due to "molli-avoidance" with cells primarily residing on the stiff stripes. While the molli-avoidance tendency is not lost with decreasing density of collagen coating the substrate, the reduced focal adhesion formation with decreasing collagen density tends to inhibit contact guidance. Our results provide clear physical insights into the interplay between cell mechano-sensitivity and substrate elastic heterogeneity that ultimately leads to the contact guidance of cells in heterogeneous tissues. STATEMENT OF SIGNIFICANCE: Cellular morphology and organization play a crucial role in the micro-architecture of tissues and dictates their biological and mechanical functioning. Despite the importance of cellular organization in all facets of tissue biology, the fundamental question of how a cell organizes itself in an anisotropic environment is still poorly understood. We employ a novel statistical thermodynamics framework which recognises the non-thermal fluctuations in the cellular response to investigate cell guidance on substrates with alternating soft and stiff stripes. The propensity of cells to primarily reside on stiff stripes results in strong guidance when the period of the stripes is larger than the cell size. For smaller stripe periods, cells sense a homogeneous substrate and guidance is lost.

The influence of entropic crowding in cell monolayers.

Cell-cell interaction dictates cell morphology and organization, which play a crucial role in the micro-architecture of tissues that guides their biological and mechanical functioning. Here, we investigate the effect of cell density on the responses of cells seeded on flat substrates using a novel statistical thermodynamics framework. The framework recognizes the existence of nonthermal fluctuations in cellular response and thereby naturally captures entropic interactions between cells in monolayers. In line with observations, the model predicts that cell area and elongation decrease with increasing cell seeding density-both are a direct outcome of the fluctuating nature of the cellular response that gives rise to enhanced cell-cell interactions with increasing cell crowding. The modeling framework also predicts the increase in cell alignment with increasing cell density: this cellular ordering is also due to enhanced entropic interactions and is akin to nematic ordering in liquid crystals. Our simulations provide physical insights that suggest that entropic cell-cell interactions play a crucial role in governing the responses of cell monolayers.

Contact guidance as a consequence of coupled morphological evolution and motility of adherent cells.

Adherent cells seeded on substrates spread and evolve their morphology while simultaneously displaying motility. Phenomena such as contact guidance, viz. the alignment of cells on patterned substrates, are strongly linked to the coupling of morphological evolution with motility. Here, we employ a recently developed statistical thermodynamics framework for modelling the non-thermal fluctuating response of cells to probe this coupling. This thermodynamic framework is first extended via a Langevin style model to predict temporal responses of cells to unpatterned and patterned substrates. The Langevin model is then shown to not only predict the different experimentally observed temporal scales for morphological observables such as cell area and elongation but also the interplay of morphology with motility that ultimately leads to contact guidance.

Cell reorientation on a cyclically strained substrate.

Cyclic strain avoidance, the phenomenon of cell and cytoskeleton alignment perpendicular to the direction of cyclic strain of the underlying 2D substrate, is an important characteristic of the adherent cell organization. This alignment has typically been attributed to the stress-fiber reorganization although observations clearly show that stress-fiber reorganization under cyclic loading is closely coupled to cell morphology and reorientation of the cells. Here, we develop a statistical mechanics framework that couples the cytoskeletal stress-fiber organization with cell morphology under imposed cyclic straining and make quantitative comparisons with observations. The framework accurately predicts that cyclic strain avoidance stems primarily from cell reorientation away from the cyclic straining rather than cytoskeletal reorganization within the cell. The reorientation of the cell is a consequence of the cell lowering its free energy by largely avoiding the imposed cyclic straining. Furthermore, we investigate the kinetics of the cyclic strain avoidance mechanism and demonstrate that it emerges primarily due to the rigid body rotation of the cell rather than via a trajectory involving cell straining. Our results provide clear physical insights into the coupled dynamics of cell morphology and stress-fibers, which ultimately leads to cellular organization in cyclically strained tissues.

Nanofibril-mediated fracture resistance of bone.

Natural hard composites like human bone possess a combination of strength and toughness that exceeds that of their constituents and of many engineered composites. This augmentation is attributed to their complex hierarchical structure, spanning multiple length scales; in bone, characteristic dimensions range from nanoscale fibrils to microscale lamellae to mesoscale osteons and macroscale organs. The mechanical properties of bone have been studied, with the understanding that the isolated microstructure at micro- and nano-scales gives rise to superior strength compared to that of whole tissue, and the tissue possesses an amplified toughness relative to that of its nanoscale constituents. Nanoscale toughening mechanisms of bone are not adequately understood at sample dimensions that allow for isolating salient microstructural features, because of the challenge of performing fracture experiments on small-sized samples. We developed anin situthree-point bend experimental methodology that probes site-specific fracture behavior of micron-sized specimens of hard material. Using this, we quantify crack initiation and growth toughness of human trabecular bone with sharp fatigue pre-cracks and blunt notches. Our findings indicate that bone with fatigue cracks is two times tougher than that with blunt cracks.In situdata-correlated electron microscopy videos reveal this behavior arises from crack-bridging by nanoscale fibril structure. The results reveal a transition between fibril-bridging (∼1µm) and crack deflection/twist (∼500µm) as a function of length-scale, and quantitatively demonstrate hierarchy-induced toughening in a complex material. This versatile approach enables quantifying the relationship between toughness and microstructure in various complex material systems and provides direct insight for designing biomimetic composites.

Cellular Contact Guidance Emerges from Gap Avoidance.

In the presence of anisotropic biochemical or topographical patterns, cells tend to align in the direction of these cues-a widely reported phenomenon known as "contact guidance." To investigate the origins of contact guidance, here, we created substrates micropatterned with parallel lines of fibronectin with dimensions spanning multiple orders of magnitude. Quantitative morphometric analysis of our experimental data reveals two regimes of contact guidance governed by the length scale of the cues that cannot be explained by enforced alignment of focal adhesions. Adopting computational simulations of cell remodeling on inhomogeneous substrates based on a statistical mechanics framework for living cells, we show that contact guidance emerges from anisotropic cell shape fluctuation and "gap avoidance," i.e., the energetic penalty of cell adhesions on non-adhesive gaps. Our findings therefore point to general biophysical mechanisms underlying cellular contact guidance, without the necessity of invoking specific molecular pathways.

Entropic Forces Drive Cellular Contact Guidance.

Contact guidance-the widely known phenomenon of cell alignment induced by anisotropic environmental features-is an essential step in the organization of adherent cells, but the mechanisms by which cells achieve this orientational ordering remain unclear. Here, we seeded myofibroblasts on substrates micropatterned with stripes of fibronectin and observed that contact guidance emerges at stripe widths much greater than the cell size. To understand the origins of this surprising observation, we combined morphometric analysis of cells and their subcellular components with a, to our knowledge, novel statistical framework for modeling nonthermal fluctuations of living cells. This modeling framework is shown to predict not only the trends but also the statistical variability of a wide range of biological observables, including cell (and nucleus) shapes, sizes, and orientations, as well as stress-fiber arrangements within the cells with remarkable fidelity with a single set of cell parameters. By comparing observations and theory, we identified two regimes of contact guidance: 1) guidance on stripe widths smaller than the cell size (w ≤ 160 µm), which is accompanied by biochemical changes within the cells, including increasing stress-fiber polarization and cell elongation; and 2) entropic guidance on larger stripe widths, which is governed by fluctuations in the cell morphology. Overall, our findings suggest an entropy-mediated mechanism for contact guidance associated with the tendency of cells to maximize their morphological entropy through shape fluctuations.

A microscopically motivated model for the remodeling of cardiomyocytes.

We present a thermodynamically based model that captures the remodeling effects in cardiac muscle cells. This work begins with the formulation of the kinematics of a cardiomyocyte resulting from a prescribed macroscopic deformation and the reorganization of the internal structure. Specifically, relations between the macroscopic deformation and the number of sarcomeres, the sarcomere stretch, and the number of myofibrils in the cell are determined. The remodeling process is split into two separate phases-(1) elongation/shortening of the existing myofibrils by addition/detachment of sarcomeres and (2) formation of new myofibrils. The remodeling associated with each phase is modeled through a dissipation postulate. We show that remodeling is based on a competition between the internal energy, the entropy, the energy supplied to the system by ATP and other sources to drive the remodeling process, and dissipation mechanisms. While the variations in entropy associated with phase (1) are neglected, the substantial entropy loss associated with the formation of new myofibrils is determined. To illustrate the merit of the proposed framework, we compute the response of cardiomyocytes subjected to isometric axial stretch that are either free to deform or fixed in the transverse direction. We also examine the predictions of this model for cardiomyocytes subjected to various cyclic loadings. The proposed framework is capable of capturing a wide range of remodeling effects and agrees with experimental observations.

Transient active force generation and stress fibre remodelling in cells under cyclic loading.

The active cytoskeleton is known to play an important mechanistic role in cellular structure, spreading, and contractility. Contractility is actively generated by stress fibres (SF), which continuously remodel in response to physiological dynamic loading conditions. The influence of actin-myosin cross-bridge cycling on SF remodelling under dynamic loading conditions has not previously been uncovered. In this study, a novel SF cross-bridge cycling model is developed to predict transient active force generation in cells subjected to dynamic loading. Rates of formation of cross-bridges within SFs are governed by the chemical potentials of attached and unattached myosin heads. This transient cross-bridge cycling model is coupled with a thermodynamically motivated framework for SF remodelling to analyse the influence of transient force generation on cytoskeletal evolution. A 1D implementation of the model is shown to correctly predict complex patterns of active cell force generation under a range of dynamic loading conditions, as reported in previous experimental studies.

High strength metallic wood from nanostructured nickel inverse opal materials.

This paper describes a nickel-based cellular material, which has the strength of titanium and the density of water. The material's strength arises from size-dependent strengthening of load-bearing nickel struts whose diameter is as small as 17 nm and whose 8 GPa yield strength exceeds that of bulk nickel by up to 4X. The mechanical properties of this material can be controlled by varying the nanometer-scale geometry, with strength varying over the range 90-880 MPa, modulus varying over the range 14-116 GPa, and density varying over the range 880-14500 kg/m3. We refer to this material as a "metallic wood," because it has the high mechanical strength and chemical stability of metal, as well as a density close to that of natural materials such as wood.

Thermodynamic Modeling of the Statistics of Cell Spreading on Ligand-Coated Elastic Substrates.

Biological spread cells exist in a perpetually fluctuating state and therefore cannot be described in terms of a unique deterministic system. For modeling approaches to provide novel insight and uncover new mechanisms that drive cell behavior, a framework is required that progresses from traditional deterministic methods (whereby simulation of an experiment predicts a single outcome). In this study, we implement a new, to our knowledge, modeling approach for the analysis of cell spreading on ligand-coated substrates, extending the framework for nonequilibrium thermodynamics of cells developed by Shishvan et al. to include active focal adhesion assembly. We demonstrate that the model correctly predicts the coupled influence of surface collagen density and substrate stiffness on cell spreading, as reported experimentally by Engler et al. Low surface collagen densities are shown to result in a high probability that cells will be restricted to low spread areas. Furthermore, elastic free energy induced by substrate deformation lowers the probability of observing a highly spread cell, and, consequentially, lower cell tractions affect the assembly of focal adhesions. Experimentally measurable observables such as cell spread area and aspect ratio can be directly postprocessed from the computed homeostatic ensemble of (several million) spread states. This allows for the prediction of mean and SDs of such experimental observables. This class of cell mechanics modeling presents a significant advance on conventional deterministic approaches.

Role of boundary conditions in determining cell alignment in response to stretch.

The ability of cells to orient in response to mechanical stimuli is essential to embryonic development, cell migration, mechanotransduction, and other critical physiologic functions in a range of organs. Endothelial cells, fibroblasts, mesenchymal stem cells, and osteoblasts all orient perpendicular to an applied cyclic stretch when plated on stretchable elastic substrates, suggesting a common underlying mechanism. However, many of these same cells orient parallel to stretch in vivo and in 3D culture, and a compelling explanation for the different orientation responses in 2D and 3D has remained elusive. Here, we conducted a series of experiments designed specifically to test the hypothesis that differences in strains transverse to the primary loading direction give rise to the different alignment patterns observed in 2D and 3D cyclic stretch experiments ("strain avoidance"). We found that, in static or low-frequency stretch conditions, cell alignment in fibroblast-populated collagen gels correlated with the presence or absence of a restraining boundary condition rather than with compaction strains. Cyclic stretch could induce perpendicular alignment in 3D culture but only at frequencies an order of magnitude greater than reported to induce perpendicular alignment in 2D. We modified a published model of stress fiber dynamics and were able to reproduce our experimental findings across all conditions tested as well as published data from 2D cyclic stretch experiments. These experimental and model results suggest an explanation for the apparently contradictory alignment responses of cells subjected to cyclic stretch on 2D membranes and in 3D gels.

Notch sensitivity of orthotropic solids: interaction of tensile and shear damage zones.

The macroscopic tensile strength of a panel containing a centre-crack or a centre-hole is predicted, assuming the simultaneous activation of multiple cohesive zones. The panel is made from an orthotropic elastic solid, and the stress raiser has both a tensile cohesive zone ahead of its tip, and shear cohesive zones in an orthogonal direction in order to represent two simultaneous damage mechanisms. These cohesive zones allow for two modes of fracture: (i) crack extension by penetration, and (ii) splitting in an orthogonal direction. The sensitivity of macroscopic tensile strength and failure mode to the degree of orthotropy is explored. The role of notch acuity and notch size are assessed by comparing the response of the pre-crack to that of the circular hole. This study reveals the role of the relative strength and relative toughness of competing damage modes in dictating the macroscopic strength of a notched panel made from an orthotropic elastic solid. Universal failure mechanism maps are constructed for the pre-crack and hole for a wide range of material orthotropies. The maps are useful for predicting whether failure is by penetration or kinking. Case studies are developed to compare the predictions with observations taken from the literature for selected orthotropic solids. It is found that synergistic strengthening occurs: when failure is by crack penetration ahead of the stress raiser, the presence of shear plastic zones leads to an enhancement of macroscopic strength. In contrast, when failure is by crack kinking, the presence of a tensile plastic zone ahead of the stress raiser has only a mild effect upon the macroscopic strength.

Energetics: An emerging frontier in cellular mechanosensing: Reply to comments on "Cellular mechanosensing of the biophysical microenvironment: A review of mathematical models of biophysical regulation of cell responses".

How do cells can sense the substrate stiffness? Our recent review highlighted a range of theoretical models and simulations that have been proposed to answer this important question. In response to this review, three leading groups in the field noted some important omissions not only from our review itself but also from the field. These groups noted, correctly, that much of our understanding of cellular mechanosensing arises from models that take advantage of equilibrium thermodynamics, and that this is inappropriate because living cells are never in thermodynamic equilibrium. In this response, we highlight some promising research aimed at resolving this conundrum.

Cellular mechanosensing of the biophysical microenvironment: A review of mathematical models of biophysical regulation of cell responses.

Cells in vivo reside within complex microenvironments composed of both biochemical and biophysical cues. The dynamic feedback between cells and their microenvironments hinges upon biophysical cues that regulate critical cellular behaviors. Understanding this regulation from sensing to reaction to feedback is therefore critical, and a large effort is afoot to identify and mathematically model the fundamental mechanobiological mechanisms underlying this regulation. This review provides a critical perspective on recent progress in mathematical models for the responses of cells to the biophysical cues in their microenvironments, including dynamic strain, osmotic shock, fluid shear stress, mechanical force, matrix rigidity, porosity, and matrix shape. The review highlights key successes and failings of existing models, and discusses future opportunities and challenges in the field.

Free energy analysis of cell spreading.

In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to "V-shaped" and "Y-shaped" ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing cytoskeletal free energy and increasing passive elastic free energy.

Crack kinking at the tip of a mode I crack in an orthotropic solid.

The competition between crack penetration and crack kinking is addressed for a mode I macroscopic crack in an orthotropic elastic solid. Cohesive zones of finite peak strength and finite toughness are placed directly ahead of and orthogonal to the plane of the parent crack. The cohesive zone ahead of the crack tip is tensile in nature and leads to crack penetration, whereas the inclined zones slide without opening under a combined shear and normal traction, and give crack kinking. Thereby, the competition between continued crack growth by penetration ahead of the crack tip versus kinking is determined as a function of the relative strength and relative toughness of the cohesive zones. This competition is plotted in the form of a failure mechanism map, with the role of material orthotropy emphasized. Synergistic toughening is observed, whereby the parent crack tip is shielded by the activation of both the tensile and shear (kinking) cohesive zones, and the macroscopic toughness is elevated. The study is used to assess the degree to which various classes of composite have the tendency to undergo kinking.