Fiber alignment in 3D collagen networks as a biophysical marker for cell contractility.

Cells cultured in 3D fibrous biopolymer matrices exert traction forces on their environment that induce deformations and remodeling of the fiber network. By measuring these deformations, the traction forces can be reconstructed if the mechanical properties of the matrix and the force-free matrix configuration are known. These requirements limit the applicability of traction force reconstruction in practice. In this study, we test whether force-induced matrix remodeling can instead be used as a proxy for cellular traction forces. We measure the traction forces of hepatic stellate cells and different glioblastoma cell lines and quantify matrix remodeling by measuring the fiber orientation and fiber density around these cells. In agreement with simulated fiber networks, we demonstrate that changes in local fiber orientation and density are directly related to cell forces. By resolving Rho-kinase (ROCK) inhibitor-induced changes of traction forces, fiber alignment, and fiber density in hepatic stellate cells, we show that the method is suitable for drug screening assays. We conclude that differences in local fiber orientation and density, which are easily measurable, can be used as a qualitative proxy for changes in traction forces. The method is available as an open-source Python package with a graphical user interface.

Emergent mechanical control of vascular morphogenesis.

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.

Endothelium and Subendothelial Matrix Mechanics Modulate Cancer Cell Transendothelial Migration.

Cancer cell extravasation, a key step in the metastatic cascade, involves cancer cell arrest on the endothelium, transendothelial migration (TEM), followed by the invasion into the subendothelial extracellular matrix (ECM) of distant tissues. While cancer research has mostly focused on the biomechanical interactions between tumor cells (TCs) and ECM, particularly at the primary tumor site, very little is known about the mechanical properties of endothelial cells and the subendothelial ECM and how they contribute to the extravasation process. Here, an integrated experimental and theoretical framework is developed to investigate the mechanical crosstalk between TCs, endothelium and subendothelial ECM during in vitro cancer cell extravasation. It is found that cancer cell actin-rich protrusions generate complex push-pull forces to initiate and drive TEM, while transmigration success also relies on the forces generated by the endothelium. Consequently, mechanical properties of the subendothelial ECM and endothelial actomyosin contractility that mediate the endothelial forces also impact the endothelium's resistance to cancer cell transmigration. These results indicate that mechanical features of distant tissues, including force interactions between the endothelium and the subendothelial ECM, are key determinants of metastatic organotropism.

Poroelastic osmoregulation of living cell volume.

Cells maintain their volume through fine intracellular osmolarity regulation. Osmotic challenges drive fluid into or out of cells causing swelling or shrinkage, respectively. The dynamics of cell volume changes depending on the rheology of the cellular constituents and on how fast the fluid permeates through the membrane and cytoplasm. We investigated whether and how poroelasticity can describe volume dynamics in response to osmotic shocks. We exposed cells to osmotic perturbations and used defocusing epifluorescence microscopy on membrane-attached fluorescent nanospheres to track volume dynamics with high spatiotemporal resolution. We found that a poroelastic model that considers both geometrical and pressurization rates captures fluid-cytoskeleton interactions, which are rate-limiting factors in controlling volume changes at short timescales. Linking cellular responses to osmotic shocks and cell mechanics through poroelasticity can predict the cell state in health, disease, or in response to novel therapeutics.

Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension.

Pulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

Dynamic filopodial forces induce accumulation, damage, and plastic remodeling of 3D extracellular matrices.

The mechanical properties of the extracellular matrix (ECM)-a complex, 3D, fibrillar scaffold of cells in physiological environments-modulate cell behavior and can drive tissue morphogenesis, regeneration, and disease progression. For simplicity, it is often convenient to assume these properties to be time-invariant. In living systems, however, cells dynamically remodel the ECM and create time-dependent local microenvironments. Here, we show how cell-generated contractile forces produce substantial irreversible changes to the density and architecture of physiologically relevant ECMs-collagen I and fibrin-in a matter of minutes. We measure the 3D deformation profiles of the ECM surrounding cancer and endothelial cells during stages when force generation is active or inactive. We further correlate these ECM measurements to both discrete fiber simulations that incorporate fiber crosslink unbinding kinetics and continuum-scale simulations that account for viscoplastic and damage features. Our findings further confirm that plasticity, as a mechanical law to capture remodeling in these networks, is fundamentally tied to material damage via force-driven unbinding of fiber crosslinks. These results characterize in a multiscale manner the dynamic nature of the mechanical environment of physiologically mimicking cell-in-gel systems.

Complex mechanics of the heterogeneous extracellular matrix in cancer.

The extracellular matrix (ECM) performs many critical functions, one of which is to provide structural and mechanical integrity, and many of the constituent proteins have clear mechanical roles. The composition and structural characteristics of the ECM are widely variable among different tissues, suiting diverse functional needs. In diseased tissues, particularly solid tumors, the ECM is complex and influences disease progression. Cancer and stromal cells can significantly influence the matrix composition and structure and thus the mechanical properties of the tumor microenvironment (TME). In this review, we describe the interactions that give rise to the structural heterogeneity of the ECM and present the techniques that are widely employed to measure ECM properties and remodeling dynamics. Furthermore, we review the tools for measuring the distinct nature of cell-ECM interactions within the TME.

Cell contraction induces long-ranged stress stiffening in the extracellular matrix.

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.

In Vitro Modeling of Mechanics in Cancer Metastasis.

In addition to a multitude of genetic and biochemical alterations, abnormal morphological, structural, and mechanical changes in cells and their extracellular environment are key features of tumor invasion and metastasis. Furthermore, it is now evident that mechanical cues alongside biochemical signals contribute to critical steps of cancer initiation, progression, and spread. Despite its importance, it is very challenging to study mechanics of different steps of metastasis in the clinic or even in animal models. While considerable progress has been made in developing advanced in vitro models for studying genetic and biological aspects of cancer, less attention has been paid to models that can capture both biological and mechanical factors realistically. This is mainly due to lack of appropriate models and measurement tools. After introducing the central role of mechanics in cancer metastasis, we provide an outlook on the emergence of novel in vitro assays and their combination with advanced measurement technologies to probe and recapitulate mechanics in conditions more relevant to the metastatic disease.

Patient-specific fracture risk assessment of vertebrae: A multiscale approach coupling X-ray physics and continuum micromechanics.

While in clinical settings, bone mineral density measured by computed tomography (CT) remains the key indicator for bone fracture risk, there is an ongoing quest for more engineering mechanics-based approaches for safety analyses of the skeleton. This calls for determination of suitable material properties from respective CT data, where the traditional approach consists of regression analyses between attenuation-related grey values and mechanical properties. We here present a physics-oriented approach, considering that elasticity and strength of bone tissue originate from the material microstructure and the mechanical properties of its elementary components. Firstly, we reconstruct the linear relation between the clinically accessible grey values making up a CT, and the X-ray attenuation coefficients quantifying the intensity losses from which the image is actually reconstructed. Therefore, we combine X-ray attenuation averaging at different length scales and over different tissues, with recently identified 'universal' composition characteristics of the latter. This gives access to both the normally non-disclosed X-ray energy employed in the CT-device and to in vivo patient-specific and location-specific bone composition variables, such as voxel-specific mass density, as well as collagen and mineral contents. The latter feed an experimentally validated multiscale elastoplastic model based on the hierarchical organization of bone. Corresponding elasticity maps across the organ enter a finite element simulation of a typical load case, and the resulting stress states are increased in a proportional fashion, so as to check the safety against ultimate material failure. In the young patient investigated, even normal physiological loading is probable to already imply plastic events associated with the hydrated mineral crystals in the bone ultrastructure, while the safety factor against failure is still as high as five. Copyright © 2016 John Wiley & Sons, Ltd.

On the relative relevance of subject-specific geometries and degeneration-specific mechanical properties for the study of cell death in human intervertebral disk models.

Capturing patient- or condition-specific intervertebral disk (IVD) properties in finite element models is outmost important in order to explore how biomechanical and biophysical processes may interact in spine diseases. However, disk degenerative changes are often modeled through equations similar to those employed for healthy organs, which might not be valid. As for the simulated effects of degenerative changes, they likely depend on specific disk geometries. Accordingly, we explored the ability of continuum tissue models to simulate disk degenerative changes. We further used the results in order to assess the interplay between these simulated changes and particular IVD morphologies, in relation to disk cell nutrition, a potentially important factor in disk tissue regulation. A protocol to derive patient-specific computational models from clinical images was applied to different spine specimens. In vitro, IVD creep tests were used to optimize poro-hyperelastic input material parameters in these models, in function of the IVD degeneration grade. The use of condition-specific tissue model parameters in the specimen-specific geometrical models was validated against independent kinematic measurements in vitro. Then, models were coupled to a transport-cell viability model in order to assess the respective effects of tissue degeneration and disk geometry on cell viability. While classic disk poro-mechanical models failed in representing known degenerative changes, additional simulation of tissue damage allowed model validation and gave degeneration-dependent material properties related to osmotic pressure and water loss, and to increased fibrosis. Surprisingly, nutrition-induced cell death was independent of the grade-dependent material properties, but was favored by increased diffusion distances in large IVDs. Our results suggest that in situ geometrical screening of IVD morphology might help to anticipate particular mechanisms of disk degeneration.

Numerical exploration of the combined effect of nutrient supply, tissue condition and deformation in the intervertebral disc.

Novel strategies to heal discogenic low back pain could highly benefit from comprehensive biophysical studies that consider both mechanical and biological factors involved in intervertebral disc degeneration. A decrease in nutrient availability at the bone-disc interface has been indicated as a relevant risk factor and as a possible initiator of cell death processes. Mechanical behaviour of both healthy and degenerated discs could highly interact with cell death in these compromised situations. In the present study, a mechano-transport finite element model was used to investigate the nature of mechanical effects on cell death processes via load-induced metabolic transport variations. Cycles of static sustained compression were chosen to simulate daily human activity. Healthy and degenerated cases were simulated as well as a reduced supply of solutes and an increase in solute exchange area at the bone-disc interface. Results showed that a reduction in metabolite concentrations at the bone-disc boundaries induced cell death, even when the increased exchange area was simulated. Slight local mechanical enhancements of glucose in the disc centre were capable of decelerating cell death but occurred only with healthy mechanical properties. However, mechanical deformations were responsible for a worsening in terms of cell death in the inner annulus, a disadvantaged zone far from the boundary supply with both an increased cell demand and a strain-dependent decrease of diffusivity. Such adverse mechanical effects were more accentuated when degenerative properties were simulated. Overall, this study paves the way for the use of biophysical models for a more integrated understanding of intervertebral disc pathophysiology.

Regional annulus fibre orientations used as a tool for the calibration of lumbar intervertebral disc finite element models.

The collagen network of the annulus fibrosus largely controls the functional biomechanics of the lumbar intervertebral discs (IVDs). Quantitative anatomical examinations have shown bundle orientation patterns, possibly coming from regional adaptations of the annulus mechanics. This study aimed to show that the regional differences in annulus mechanical behaviour could be reproduced by considering only fibre orientation changes. Using the finite element method, a lumbar annulus was modelled as a poro-hyperelastic material in which fibres were represented by a direction-dependent strain energy density term. Fibre orientations were calibrated to reproduce the annulus tensile behaviours measured for four different regions: posterior outer, anterior outer, posterior inner and anterior inner. The back-calculated fibre angles and regional patterns as well as the global disc behaviour were comparable with anatomical descriptions reported in the literature. It was concluded that annulus fibre variations might be an effective tool to calibrate lumbar spine IVD and segment models.

The effect of sustained compression on oxygen metabolic transport in the intervertebral disc decreases with degenerative changes.

Intervertebral disc metabolic transport is essential to the functional spine and provides the cells with the nutrients necessary to tissue maintenance. Disc degenerative changes alter the tissue mechanics, but interactions between mechanical loading and disc transport are still an open issue. A poromechanical finite element model of the human disc was coupled with oxygen and lactate transport models. Deformations and fluid flow were linked to transport predictions by including strain-dependent diffusion and advection. The two solute transport models were also coupled to account for cell metabolism. With this approach, the relevance of metabolic and mechano-transport couplings were assessed in the healthy disc under loading-recovery daily compression. Disc height, cell density and material degenerative changes were parametrically simulated to study their influence on the calculated solute concentrations. The effects of load frequency and amplitude were also studied in the healthy disc by considering short periods of cyclic compression. Results indicate that external loads influence the oxygen and lactate regional distributions within the disc when large volume changes modify diffusion distances and diffusivities, especially when healthy disc properties are simulated. Advection was negligible under both sustained and cyclic compression. Simulating degeneration, mechanical changes inhibited the mechanical effect on transport while disc height, fluid content, nucleus pressure and overall cell density reductions affected significantly transport predictions. For the healthy disc, nutrient concentration patterns depended mostly on the time of sustained compression and recovery. The relevant effect of cell density on the metabolic transport indicates the disturbance of cell number as a possible onset for disc degeneration via alteration of the metabolic balance. Results also suggest that healthy disc properties have a positive effect of loading on metabolic transport. Such relation, relevant to the maintenance of the tissue functional composition, would therefore link disc function with disc nutrition.

Comparison of four methods to simulate swelling in poroelastic finite element models of intervertebral discs.

Osmotic phenomena influence the intervertebral disc biomechanics. Their simulation is challenging and can be undertaken at different levels of complexity. Four distinct approaches to simulate the osmotic behaviour of the intervertebral disc (a fixed boundary pore pressure model, a fixed osmotic pressure gradient model in the whole disc or only in the nucleus pulposus, and a swelling model with strain-dependent osmotic pressure) were analysed. Predictions were compared using a 3D poroelastic finite element model of a L4-L5 spinal unit under three different loading conditions: free swelling for 8 h and two daily loading cycles: (i) 200 N compression for 8 h followed by 500 N compression for 16 h; (ii) 500 N for 8 h followed by 1000 N for 16 h. Overall, all swelling models calculated comparable results, with differences decreasing under greater loads. Results predicted with the fixed boundary pore pressure and the fixed osmotic pressure in the whole disc models were nearly identical. The boundary pore pressure model, however, cannot simulate differential osmotic pressures in disc regions. The swelling model offered the best potential to provide more accurate results, conditional upon availability of reliable values for the required coefficients and material properties. Possible fields of application include mechanobiology investigations and crack opening and propagation. However, the other approaches are a good compromise between the ease of implementation and the reliability of results, especially when considering higher loads or when the focus is on global results such as spinal kinematics.

Statistical factorial analysis on the poroelastic material properties sensitivity of the lumbar intervertebral disc under compression, flexion and axial rotation.

A statistical factorial analysis approach was conducted on a poroelastic finite element model of a lumbar intervertebral disc to analyse the influence of six material parameters (permeabilities of annulus, nucleus, trabecular vertebral bone, cartilage endplate and Young's moduli of annulus and nucleus) on the displacement, fluid pore pressure and velocity fields. Three different loading modes were investigated: compression, flexion and axial rotation. Parameters were varied considering low and high levels in agreement with values found in the literature for both healthy and degenerated lumbar discs. Results indicated that annulus stiffness and cartilage endplate permeability have a strong effect on the overall fluid- and solid-phase responses in all loading conditions studied. Nucleus stiffness showed its main relevance in compression while annulus permeability influenced mainly the annular pressure field. This study confirms the permeability's central role in biphasic modelling and highlights for the lumbar disc which experiments of material property characterization should be performed. Moreover, such sensitivity study gives important guidelines in poroelastic material modelling and finite element disc validation.

An accurate estimation of bone density improves the accuracy of subject-specific finite element models.

An experimental-numerical study was performed to investigate the relationships between computed tomography (CT)-density and ash density, and between ash density and apparent density for bone tissue, to evaluate their influence on the accuracy of subject-specific FE models of human bones. Sixty cylindrical bone specimens were examined. CT-densities were computed from CT images while apparent and ash densities were measured experimentally. The CT/ash-density and ash/apparent-density relationships were calculated. Finite element models of eight human femurs were generated considering these relationships to assess their effect on strain prediction accuracy. CT and ash density were linearly correlated (R(2)=0.997) over the whole density range but not equivalent (intercep t <0, slope >1). A constant ash/apparent-density ratio (0.598+/-0.004) was found for cortical bone. A lower ratio, with a larger dispersion, was found for trabecular bone (0.459+/-0.100), but it became less dispersed, and equal to that of cortical tissue, when testing smaller trabecular specimens (0.598+/-0.036). This suggests that an experimental error occurred in apparent-density measurements for large trabecular specimens and a constant ratio can be assumed valid for the whole density range. Introducing the obtained relationships in the FE modelling procedure improved strain prediction accuracy (R(2)=0.95, RMSE=7%). The results suggest that: (i) a correction of the densitometric calibration should be used when evaluating bone ash-density from clinical CT scans, to avoid ash-density underestimation and overestimation for low- and high-density bone tissue, respectively; (ii) the ash/apparent-density ratio can be assumed constant in human femurs and (iii) the correction improves significantly the model accuracy and should be considered in subject-specific bone modelling.

Subject-specific finite element models can accurately predict strain levels in long bones.

The prediction of the stress-state and fracture risk induced in bones by various loading conditions in individual patients using subject-specific finite element models still represents a challenge in orthopaedic biomechanics. The accuracy of the strain predictions reported in the literature is variable and generally not satisfactory. The aim of the present study was to evaluate if a proper choice of the density-elasticity relationship can lead to accurate strain predictions in the frame of an automatic subject-specific model generation strategy. To this aim, a combined numerical-experimental study was performed comparing finite element predicted strains with strain-gauges measurements obtained on eight cadaver proximal femurs, each instrumented with 15 rosettes mostly concentrated in the bone metaphyses, tested non-destructively in vitro under six different loading scenarios. Three different density-elasticity power relationships were selected from the literature and implemented in the finite element models derived from computed tomography data. The results of the present study confirm the great influence of the density-elasticity relationship used on the accuracy of numerical predictions. One of the tested constitutive laws provided a very good agreement (R(2)=0.91, RMSE lower than 10% of the maximum measured value) between numerical calculations and experimental measurements. The presented results show, in addition, that the adoption of a single density-elasticity relationship over the whole bone density range is adequate to obtain an accuracy that is already suitable for many applications.