Continuum interpretation of mechano-adaptation in micropatterned epithelia informed by in vitro experiments.

Epithelial tissues adapt their form and function following mechanical perturbations, or mechano-adapt, and these changes often result in reactive forces that oppose the direction of the applied change. Tissues subjected to ectopic tensions, for example, employ behaviors that lower tension, such as increasing proliferation or actomyosin turnover. This oppositional behavior suggests that the tissue has a mechanical homeostasis. Whether attributed to maintenance of cellular area, cell density, or cell and tissue tensions, epithelial mechanical homeostasis has been implicated in coordinating embryonic morphogenesis, wound healing, and maintenance of adult tissues. Despite advances toward understanding the feedback between mechanical state and tissue response in epithelia, more work remains to be done to examine how tissues regulate mechanical homeostasis using epithelial sheets with defined micropatterned shapes. Here, we used cellular microbiaxial stretching (CµBS) to investigate mechano-adaptation in micropatterned tissues of different shape consisting of Madin-Darby canine kidney cells. Using the CµBS platform, tissues were subjected to a 30% stretch that was held for 24 h. We found that, following stretch, tissue stresses immediately increased then slowly evolved over time, approaching their pre-stretch values by 24 h. Organization of the actin cytoskeletal was found to play a role in this process: anisotropic ally structured tissues exhibited anisotropic stress patterns, and the cytoskeletal became more aligned following stretch and reorganized over time. Interestingly, in unstretched tissues, stresses also decreased, which was found to be driven by proliferation-induced cellular confinement and change in tissue thickness. We modeled these behaviors with a continuum-based model of epithelial growth that accounted for stress-induced actin remodeling and proliferation, and found this model to strongly capture experimental behavior. Ultimately, this combined experimental-modeling approach suggests that epithelial mechano-adaptation depends on cellular architecture and proliferation, which can be modeled with a field-averaged approach applicable to more specific contexts in which change is driven by epithelial mechanical homeostasis. Insight box Epithelial tissues adapt their form and function following mechanical perturbation, and it is thought that this 'mechano-adaptation' plays an important role in driving processes like embryonic morphogenesis, wound healing, and adult tissue maintenance. Here, we use cellular microbiaxial stretching to probe this process in vitro in small epithelial tissues whose geometries were both controlled and varied. By using a highly precise stretching device and a continuum mechanics modeling framework, we revealed that tissue mechanical state changes following stretch and over time, and that this behavior can be explained by stress-dependent changes in actin fibers and proliferation. Integration of these approaches enabled a systematic approach to empirically and precisely measure these phenomena.

The non-affine fiber network solver: A multiscale fiber network material model for finite-element analysis.

Multiscale mechanical models in biomaterials research have largely relied on simplifying the microstructure in order to make large-scale simulations tractable. The microscale simplifications often rely on approximations of the constituent distributions and assumptions on the deformation of the constituents. Of particular interest in biomechanics are fiber embedded materials, where simplified fiber distributions and assumed affinity in the fiber deformation greatly influence the mechanical behavior. The consequences of these assumptions are problematic when dealing with microscale mechanical phenomena such as cellular mechanotransduction in growth and remodeling, and fiber-level failure events during tissue failure. In this work, we propose a technique for coupling non-affine network models to finite element solvers, allowing for simulation of discrete microstructural phenomena within macroscopically complex geometries. The developed plugin is readily available as an open-source library for use with the bio-focused finite element software FEBio, and the description of the implementation allows for the adaptation to other finite element solvers.

Biology and Hemodynamics of Aneurysm Rupture.

Predicting rupture risk in intracranial aneurysms is among one of the most critical questions in vascular surgery. The processes that govern an aneurysm growth are multifaceted and complex, but may be summarized into three components: hemodynamics, biology, and mechanics. We review and connect the literature in the three disciplines, identifying considerable strides in recent history and current gaps in research. Taken together, the findings from each field elucidate how and why certain aneurysms rupture, whereas others remain stable. These parameters could eventually inform a translatable predictive model that optimizes risk evaluation and physician's decision-making in treatment options for aneurysms.

Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Cellular Microbiaxial Stretching Assay for Measurement and Characterization of the Anisotropic Mechanical Properties of Micropatterned Cells.

Characterizing the mechanical properties of single cells is important for developing descriptive models of tissue mechanics and improving the understanding of mechanically driven cell processes. Standard methods for measuring single-cell mechanical properties typically provide isotropic mechanical descriptions. However, many cells exhibit specialized geometries in vivo, with anisotropic cytoskeletal architectures reflective of their function, and are exposed to dynamic multiaxial loads, raising the need for more complete descriptions of their anisotropic mechanical properties under complex deformations. Here, we describe the cellular microbiaxial stretching (CµBS) assay in which controlled deformations are applied to micropatterned cells while simultaneously measuring cell stress. CµBS utilizes a set of linear actuators to apply tensile or compressive, short- or long-term deformations to cells micropatterned on a fluorescent bead-doped polyacrylamide gel. Using traction force microscopy principles and the known geometry of the cell and the mechanical properties of the underlying gel, we calculate the stress within the cell to formulate stress-strain curves that can be further used to create mechanical descriptions of the cells, such as strain energy density functions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assembly of CµBS stretching constructs Basic Protocol 2: Polymerization of micropatterned, bead-doped polyacrylamide gel on an elastomer membrane Support Protocol: Cell culture and seeding onto CµBS constructs Basic Protocol 3: Implementing CµBS stretching protocols and traction force microscopy Basic Protocol 4: Data analysis and cell stress measurements.

Characterizing Tissue Remodeling and Mechanical Heterogeneity in Cerebral Aneurysms.

Accurately assessing the complex tissue mechanics of cerebral aneurysms (CAs) is critical for elucidating how CAs grow and whether that growth will lead to rupture. The factors that have been implicated in CA progression - blood flow dynamics, immune infiltration, and extracellular matrix remodeling - all occur heterogeneously throughout the CA. Thus, it stands to reason that the mechanical properties of CAs are also spatially heterogeneous. Here, we present a new method for characterizing the mechanical heterogeneity of human CAs using generalized anisotropic inverse mechanics, which uses biaxial stretching experiments and inverse analyses to determine the local Kelvin moduli and principal alignments within the tissue. Using this approach, we find that there is significant mechanical heterogeneity within a single acquired human CA. These results were confirmed using second harmonic generation imaging of the CA's fiber architecture and a correlation was observed. This approach provides a single-step method for determining the complex heterogeneous mechanics of CAs, which has important implications for future identification of metrics that can improve accuracy in prediction risk of rupture.

Anisotropic Mechanics of Vascular Smooth Muscle Cells Exposed to Dynamic Loads.

Vascular smooth muscle cells (VSMCs) are the most prevalent cells in the arterial wall. In vivo, arteries are exposed to dynamic biaxial loads; thus, when characterizing VSMC mechanics, it is important to determine their anisotropic and time-dependent mechanical properties. In this work, we use cellular microbiaxial stretching to apply complex deformations to single micropatterned VSMCs and measure the resulting changes in cell stress. Previously, cellular microbiaxial stretching has been used to measure VSMC mechanical properties in response to extensional strain. Here, we measure changes in cell stress in response to both extension and compression. Additionally, we measure immediate temporal changes in stress in response to cyclically applied deformations. We find that the VSMCs display clear hysteresis when incrementally stretched and compressed and demonstrate cycle-dependent stress-relaxation when exposed to cyclic step change extension and compression. Finally, we demonstrate that a Hill-type active fiber model is capable of replicating all observed hysteresis and cycle-dependent stress-relaxation, suggesting that the temporal stress-strain behavior of the cell is regulated by acto-myosin contraction and relaxation, rather than passive viscoelasticity. This study improves upon previous studies of cellular mechanical properties by considering cellular architecture and more complex deformations when measuring the time-dependent mechanical properties of VSMCs. These findings have important implications for modeling in mechanobiology as VSMCs are mechanosensitive and actively respond to changes in their mechanical environment to maintain vascular function.

Orientation of neurites influences severity of mechanically induced tau pathology.

Chronic traumatic encephalopathy is a neurodegenerative disease associated with repeated traumatic brain injury (TBI). Chronic traumatic encephalopathy is a tauopathy, in which cognitive decline is accompanied by the accumulation of neurofibrillary tangles of the protein tau in patients' brains. We recently found that mechanical force alone can induce tau mislocalization to dendritic spines and loss of synaptic function in in vitro neuronal cultures with random cell organization. However, in the brain, neurons are highly aligned, so here we aimed to determine how neuronal organization influences early-stage tauopathy caused by mechanical injury. Using microfabricated cell culture constructs to control the growth of neurites and an in vitro simulated TBI device to apply controlled mechanical deformation, we found that neuronal orientation with respect to the direction of a uniaxial high-strain-rate stretch injury influences the degree of tau pathology in injured neurons. We found that a mechanical stretch applied parallel to the neurite alignment induces greater mislocalization of tau proteins to dendritic spines than does a stretch with the same strain applied perpendicular to the neurites. Synaptic function, characterized by the amplitude of miniature excitatory postsynaptic currents, was similarly decreased in neurons with neurites aligned parallel to stretch, whereas in neurons aligned perpendicular to stretch, it had little to no functional loss. Experimental injury parameters (strain, strain rate, direction of stretch) were combined with a standard viscoelastic solid model to show that in our in vitro model, neurite work density during stretch correlates with tau mislocalization. These findings suggest that in a TBI, the magnitude of brain deformation is not wholly predictive of neurodegenerative consequences of TBI but that deformation relative to local neuronal architecture and the neurite mechanical energy during injury are better metrics for predicting trauma-induced tauopathy.

Architecture-Dependent Mechano-Adaptation in Single Vascular Smooth Muscle Cells.

Arteries grow and remodel following mechanical perturbation. Vascular smooth muscle cells (VSMCs) within the artery undergo hyperplasia, hypertrophy, or change their contractility following sustained changes in loading. Experimental evidence in vivo and in vitro suggests that VSMCs grow and remodel to maintain a constant transmural stress, or "target" stress. This behavior is often described using a stress-dependent finite growth framework. Typically, computational models of arterial growth and remodeling account for VSMC behavior in a constrained mixture formulation that incorporates behavior of each component of the artery. However, these models do not account for differential VSMC architecture observed in situ, which may significantly influence growth and remodeling behavior. Here, we used cellular microbiaxial stretching (CµBS) to characterize how VSMCs with different cytoskeletal architectures respond to a sustained step change in strain. We find that VSMC F-actin architecture becomes more aligned following stretch and retains this alignment after 24 h. Further, we find that VSMC stress magnitude depends on cellular architecture. Qualitatively, however, stress behavior following stretch is consistent across cell architectures-stress increases following stretch and returns to prestretch magnitudes after 24 h. Finally, we formulated an architecture-dependent targeted growth law that accounts for experimentally measured cytoskeletal alignment and attributes stress evolution to individual fiber growth and find that this model robustly captures long-term stress evolution in single VSMCs. These results suggest that VSMC mechano-adaptation depends on cellular architecture, which has implications for growth and remodeling in regions of arteries with differential architecture, such as at bifurcations.

Mechanical injuries of neurons induce tau mislocalization to dendritic spines and tau-dependent synaptic dysfunction.

Chronic traumatic encephalopathy (CTE) is associated with repeated traumatic brain injuries (TBI) and is characterized by cognitive decline and the presence of neurofibrillary tangles (NFTs) of the protein tau in patients' brains. Here we provide direct evidence that cell-scale mechanical deformation can elicit tau abnormalities and synaptic deficits in neurons. Using computational modeling, we find that the early pathological loci of NFTs in CTE brains are regions of high deformation during injury. The mechanical energy associated with high-strain rate deformation alone can induce tau mislocalization to dendritic spines and synaptic deficits in cultured rat hippocampal neurons. These cellular changes are mediated by tau hyperphosphorylation and can be reversed through inhibition of GSK3ß and CDK5 or genetic deletion of tau. Together, these findings identify a mechanistic pathway that directly relates mechanical deformation of neurons to tau-mediated synaptic impairments and provide a possibly exploitable therapeutic pathway to combat CTE.

Large-deformation strain energy density function for vascular smooth muscle cells.

Vascular tissue exhibits marked mechanical nonlinearity when exposed to large strains. Vascular smooth muscle cells (VSMCs) are the most prevalent cell type in the artery wall, but it is unclear how much of the vessel nonlinearity is attributable to VSMCs. Here, we used cellular microbiaxial stretching (CµBS) to measure the large-strain mechanical properties of individual VSMCs. We find that the mechanical properties of VSMCs with native-like architectures are highly anisotropic, due to their highly aligned actomyosin cytoskeletons, and that inhibition of actomyosin contraction with rho-associated kinase inhibitor HA-1077 results in nearly isotropic material properties. We further find that when VSMCS are exposed to large strains (up to 60% stretch), the cells' stress-strain behavior is surprisingly linear. Finally, we modified a previously published Holzapfel-Gasser-Ogden type strain energy density function to characterize individual VSMCs, to account for the observed large-deformation linearity. These data have important implications in the development of models of vascular mechanics and mechanobiology.

Bimodal sensing of guidance cues in mechanically distinct microenvironments.

Contact guidance due to extracellular matrix architecture is a key regulator of carcinoma invasion and metastasis, yet our understanding of how cells sense guidance cues is limited. Here, using a platform with variable stiffness that facilitates uniaxial or biaxial matrix cues, or competing E-cadherin adhesions, we demonstrate distinct mechanoresponsive behavior. Through disruption of traction forces, we observe a profound phenotypic shift towards a mode of dendritic protrusion and identify bimodal processes that govern guidance sensing. In contractile cells, guidance sensing is strongly dependent on formins and FAK signaling and can be perturbed by disrupting microtubule dynamics, while low traction conditions initiate fluidic-like dendritic protrusions that are dependent on Arp2/3. Concomitant disruption of these bimodal mechanisms completely abrogates the contact guidance response. Thus, guidance sensing in carcinoma cells depends on both environment architecture and mechanical properties and targeting the bimodal responses may provide a rational strategy for disrupting metastatic behavior.

Architecture-Dependent Anisotropic Hysteresis in Smooth Muscle Cells.

Cells within mechanically dynamic tissues like arteries are exposed to ever-changing forces and deformations. In some pathologies, like aneurysms, complex loads may alter how cells transduce forces, driving maladaptive growth and remodeling. Here, we aimed to determine the dynamic mechanical properties of vascular smooth muscle cells (VSMCs) under biaxial load. Using cellular micro-biaxial stretching microscopy, we measured the large-strain anisotropic stress-strain hysteresis of VSMCs and found that hysteresis is strongly dependent on load orientation and actin organization. Most notably, under some cyclic loads, we found that VSMCs with elongated in-vivo-like architectures display a hysteresis loop that is reverse to what is traditionally measured in polymers, with unloading stresses greater than loading stresses. This reverse hysteresis could not be replicated using a quasilinear viscoelasticity model, but we developed a Hill-type active fiber model that can describe the experimentally observed hysteresis. These results suggest that cells in highly organized tissues, like arteries, can have strongly anisotropic responses to complex loads, which could have important implications in understanding pathological mechanotransduction.

Investigation of human iPSC-derived cardiac myocyte functional maturation by single cell traction force microscopy.

Recent advances have made it possible to readily derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). HiPSC-CMs represent a valuable new experimental model for studying human cardiac muscle physiology and disease. Many laboratories have devoted substantial effort to examining the functional properties of isolated hiPSC-CMs, but to date, force production has not been adequately characterized. Here, we utilized traction force microscopy (TFM) with micro-patterning cell printing to investigate the maximum force production of isolated single hiPSC-CMs under varied culture and assay conditions. We examined the role of length of differentiation in culture and the effects of varied extracellular calcium concentration in the culture media on the maturation of hiPSC-CMs. Results show that hiPSC-CMs developing in culture for two weeks produced significantly less force than cells cultured from one to three months, with hiPSC-CMs cultured for three months resembling the cell morphology and function of neonatal rat ventricular myocytes in terms of size, dimensions, and force production. Furthermore, hiPSC-CMs cultured long term in conditions of physiologic calcium concentrations were larger and produced more force than hiPSC-CMs cultured in standard media with sub-physiological calcium. We also examined relationships between cell morphology, substrate stiffness and force production. Results showed a significant relationship between cell area and force. Implementing directed modifications of substrate stiffness, by varying stiffness from embryonic-like to adult myocardium-like, hiPSC-CMs produced maximal forces on substrates with a lower modulus and significantly less force when assayed on increasingly stiff adult myocardium-like substrates. Calculated strain energy measurements paralleled these findings. Collectively, these findings further establish single cell TFM as a valuable approach to illuminate the quantitative physiological maturation of force in hiPSC-CMs.

Intrinsic Cell Stress is Independent of Organization in Engineered Cell Sheets.

Understanding cell contractility is of fundamental importance for cardiovascular tissue engineering, due to its major impact on the tissue's mechanical properties as well as the development of permanent dimensional changes, e.g., by contraction or dilatation of the tissue. Previous attempts to quantify contractile cellular stresses mostly used strongly aligned monolayers of cells, which might not represent the actual organization in engineered cardiovascular tissues such as heart valves. In the present study, therefore, we investigated whether differences in organization affect the magnitude of intrinsic stress generated by individual myofibroblasts, a frequently used cell source for in vitro engineered heart valves. Four different monolayer organizations were created via micro-contact printing of fibronectin lines on thin PDMS films, ranging from strongly anisotropic to isotropic. Thin film curvature, cell density, and actin stress fiber distribution were quantified, and subsequently, intrinsic stress and contractility of the monolayers were determined by incorporating these data into sample-specific finite element models. Our data indicate that the intrinsic stress exerted by the monolayers in each group correlates with cell density. Additionally, after normalizing for cell density and accounting for differences in alignment, no consistent differences in intrinsic contractility were found between the different monolayer organizations, suggesting that the intrinsic stress exerted by individual myofibroblasts is independent of the organization. Consequently, this study emphasizes the importance of choosing proper architectural properties for scaffolds in cardiovascular tissue engineering, as these directly affect the stresses in the tissue, which play a crucial role in both the functionality and remodeling of (engineered) cardiovascular tissues.

Cellular Microbiaxial Stretching to Measure a Single-Cell Strain Energy Density Function.

The stress in a cell due to extracellular mechanical stimulus is determined by its mechanical properties, and the structural organization of many adherent cells suggests that their properties are anisotropic. This anisotropy may significantly influence the cells' mechanotransductive response to complex loads, and has important implications for development of accurate models of tissue biomechanics. Standard methods for measuring cellular mechanics report linear moduli that cannot capture large-deformation anisotropic properties, which in a continuum mechanics framework are best described by a strain energy density function (SED). In tissues, the SED is most robustly measured using biaxial testing. Here, we describe a cellular microbiaxial stretching (CµBS) method that modifies this tissue-scale approach to measure the anisotropic elastic behavior of individual vascular smooth muscle cells (VSMCs) with nativelike cytoarchitecture. Using CµBS, we reveal that VSMCs are highly anisotropic under large deformations. We then characterize a Holzapfel-Gasser-Ogden type SED for individual VSMCs and find that architecture-dependent properties of the cells can be robustly described using a formulation solely based on the organization of their actin cytoskeleton. These results suggest that cellular anisotropy should be considered when developing biomechanical models, and could play an important role in cellular mechano-adaptation.

Anisotropic forces from spatially constrained focal adhesions mediate contact guidance directed cell migration.

Directed migration by contact guidance is a poorly understood yet vital phenomenon, particularly for carcinoma cell invasion on aligned collagen fibres. We demonstrate that for single cells, aligned architectures providing contact guidance cues induce constrained focal adhesion maturation and associated F-actin alignment, consequently orchestrating anisotropic traction stresses that drive cell orientation and directional migration. Consistent with this understanding, relaxing spatial constraints to adhesion maturation either through reduction in substrate alignment density or reduction in adhesion size diminishes the contact guidance response. While such interactions allow single mesenchymal-like cells to spontaneously 'sense' and follow topographic alignment, intercellular interactions within epithelial clusters temper anisotropic cell-substratum forces, resulting in substantially lower directional response. Overall, these results point to the control of contact guidance by a balance of cell-substratum and cell-cell interactions, modulated by cell phenotype-specific cytoskeletal arrangements. Thus, our findings elucidate how phenotypically diverse cells perceive ECM alignment at the molecular level.

Empirically Determined Vascular Smooth Muscle Cell Mechano-Adaptation Law.

Cardiovascular disease can alter the mechanical environment of the vascular system, leading to mechano-adaptive growth and remodeling. Predictive models of arterial mechano-adaptation could improve patient treatments and outcomes in cardiovascular disease. Vessel-scale mechano-adaptation includes remodeling of both the cells and extracellular matrix. Here, we aimed to experimentally measure and characterize a phenomenological mechano-adaptation law for vascular smooth muscle cells (VSMCs) within an artery. To do this, we developed a highly controlled and reproducible system for applying a chronic step-change in strain to individual VSMCs with in vivo like architecture and tracked the temporal cellular stress evolution. We found that a simple linear growth law was able to capture the dynamic stress evolution of VSMCs in response to this mechanical perturbation. These results provide an initial framework for development of clinically relevant models of vascular remodeling that include VSMC adaptation.

Failure of the Porcine Ascending Aorta: Multidirectional Experiments and a Unifying Microstructural Model.

The ascending thoracic aorta is poorly understood mechanically, especially its risk of dissection. To make better predictions of dissection risk, more information about the multidimensional failure behavior of the tissue is needed, and this information must be incorporated into an appropriate theoretical/computational model. Toward the creation of such a model, uniaxial, equibiaxial, peel, and shear lap tests were performed on healthy porcine ascending aorta samples. Uniaxial and equibiaxial tests showed anisotropy with greater stiffness and strength in the circumferential direction. Shear lap tests showed catastrophic failure at shear stresses (150-200 kPa) much lower than uniaxial tests (750-2500 kPa), consistent with the low peel tension (∼60 mN/mm). A novel multiscale computational model, including both prefailure and failure mechanics of the aorta, was developed. The microstructural part of the model included contributions from a collagen-reinforced elastin sheet and interlamellar connections representing fibrillin and smooth muscle. Components were represented as nonlinear fibers that failed at a critical stretch. Multiscale simulations of the different experiments were performed, and the model, appropriately specified, agreed well with all experimental data, representing a uniquely complete structure-based description of aorta mechanics. In addition, our experiments and model demonstrate the very low strength of the aorta in radial shear, suggesting an important possible mechanism for aortic dissection.