Loss of Vimentin Enhances Cell Motility through Small Confining Spaces

The migration of cells through constricting spaces or along fibrous tracks in tissues is important for many biological processes and depends on the mechanical properties of a cytoskeleton made up of three different filaments: F‐actin, microtubules, and intermediate filaments. The signaling pathways and cytoskeletal structures that control cell motility on 2D are often very different from those that control motility in 3D. Previous studies have shown that intermediate filaments can promote actin‐driven protrusions at the cell edge, but have little effect on overall motility of cells on flat surfaces. They are however important for cells to maintain resistance to repeated compressive stresses that are expected to occur in vivo. Using mouse embryonic fibroblasts derived from wild‐type and vimentin‐null mice, it is found that loss of vimentin increases motility in 3D microchannels even though on flat surfaces it has the opposite effect. Atomic force microscopy and traction force microscopy experiments reveal that vimentin enhances perinuclear cell stiffness while maintaining the same level of acto‐myosin contractility in cells. A minimal model in which a perinuclear vimentin cage constricts along with the nucleus during motility through confining spaces, providing mechanical resistance against large strains that could damage the structural integrity of cells, is proposed.     

Evolution of PVA gels prepared without crosslinking agents as a cell adhesive surface

Physical parameters (such as crosslinking density, crystallinity and mechanical properties) have been found to largely affect cellular behavior on polymer scaffolds. This study demonstrated that transparent pure Poly (vinyl alcohol) hydrogels prepared via a freeze–thaw method can be made to support cell adhesion by controlling physical parameters such as concentration and the number of freeze–thaw cycles. For a given number of freeze–thaw cycles, (specifically 45), polymer concentration dependent structural and mechanical properties (such as tensile strength and stiffness) were correlated with cell adhesion. The maximum cell attachment occurred on the hydrogels with the greatest mechanical properties, crystallinity and crosslinking density. The hydrogel surfaces were more favorable to human dermal fibroblasts than human lens epithelial cells and retained their transparency as well as dimensional stability with only a small degree of swelling. Fibroblast laden hydrogels showed extensive alkaline phosphatase activity which confirmed their healthy proliferation and function. In this manner, this study suggests that transparent Poly (vinyl alcohol) hydrogels prepared by the freeze thaw method described here should be further studied for numerous tissue engineering applications.     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

Abstract

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy

The nanomechanical properties of living cells, such as their surface elastic response and adhesion, have important roles in cellular processes such as morphogenesis, mechano-transduction, focal adhesion, motility, metastasis and drug delivery. Techniques based on quasi-static atomic force microscopy techniques can map these properties, but they lack the spatial and temporal resolution that is needed to observe many of the relevant details. Here, we present a dynamic atomic force microscopy method to map quantitatively the nanomechanical properties of live cells with a throughput (measured in pixels/minute) that is ～10-1,000 times higher than that achieved with quasi-static atomic force microscopy techniques. The local properties of a cell are derived from the 0th, 1st and 2nd harmonic components of the Fourier spectrum of the AFM cantilevers interacting with the cell surface. Local stiffness, stiffness gradient and the viscoelastic dissipation of live Escherichia coli bacteria, rat fibroblasts and human red blood cells were all mapped in buffer solutions. Our method is compatible with commercial atomic force microscopes and could be used to analyse mechanical changes in tumours, cells and biofilm formation with sub-10?nm detail.     

圆管状立体机织复合材料的多尺度分析

采用多尺度耦合的数值模型研究了圆管状立体机织复合材料的力学性能。建立了反映纤维束中纤维/基体二相材料的微观尺度单胞和反映周期性编织结构的细观尺度扇形单胞,并重点讨论了扇形单胞的周期性边界条件。通过逐级计算微观单胞、细观单胞的平均弹性常数,得到了圆管状立体机织复合材料的刚度参数,实现了由组分材料性能及编织参数预测圆管的宏观弹性性能,模型预测刚度与试验结果吻合。另一方面,研究了从大到小各尺度耦合的应力分析,对于圆管环向应力非周期分布的情况,建立了嵌入细观单胞的环状模型,进行了复杂荷载下从宏观圆管结构、到细观纤维束尺度、再到微观纤维尺度之间的逐级应力分析。     

Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell–matrix interaction, using poly-ε-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.     

Shear thickening of F-actin networks crosslinked with non-muscle myosin IIB

The material properties of cytoskeletal F-actin networks facilitate a broad range of cellular behaviors, whereby in some situations cell shape is preserved in the presence of force and, at other times, force results in irreversible shape change. These behaviors strongly suggest that F-actin networks can variably deform elastically or viscously. While a significant amount is known about the regulation of the elastic stiffness of F-actin networks, our understanding of the regulation of viscous behaviors of F-actin networks is largely lacking. Here, we study the rheological behavior of F-actin networks formed with heavy meromyosin non-muscle IIB (NMMIIB). We show that NMMIIB quenched with ADP crosslinks F-actin into networks that, for sufficient densities, display stress stiffening behavior. By performing a series of creep tests, we show that densely crosslinked actin/NMMIIB-ADP networks undergo viscous deformation over a wide range of stresses, ranging from 0.001 to 10 Pa. At high stresses, networks that stress stiffen are also observed to shear thicken, whereby the effective viscosity increases as a function of stress. Shear thickening results in a reduction in the extent of irreversible, viscous deformation in actin/NMMIIB-ADP networks at high stresses compared to that expected for a linear viscoelastic material. Thus, viscous deformation contributes less to the overall mechanical response at high levels of applied force. Our results indicate mechanisms by which the fluid-like nature of the actomyosin cytoskeleton can be reduced under high load.     

Characterization of edge effects of composite lattice structures

To analyze the mechanical behaviors of composite lattice structures with finite sizes and strengthened edges, periodic expansion method was developed to get the corresponding equivalent infinite periodic structures. Finite element method was also adopted to testify the theoretical predictions. The strengthened edges greatly influence the properties of composite lattice structures made up of only several layers of unit cells. Reductions of the specific stiffness and strength of the lattice composites in most cases stem from the mass enhancement. Made up of more than 10–20 unit cell layers, the lattices possess nearly identical properties of the intact composite lattice structures and the edge effect is small enough. Compared with the weak edges, the major advantage of strong edges is that they distribute loads more extensively and evenly and greatly decrease the localization of strains and stresses on the boundaries under concentrated loads.     

Three-dimensional traction forces of Schwann cells on compliant substrates

The mechanical interaction between Schwann cells (SCs) and their microenvironment is crucial for the development, maintenance and repair of the peripheral nervous system. In this paper, we present a detailed investigation on the mechanosensitivity of SCs across a physiologically relevant substrate stiffness range. Contrary to many other cell types, we find that the SC spreading area and cytoskeletal actin architecture were relatively insensitive to substrate stiffness with pronounced stress fibre formation across all moduli tested (0.24–4.80 kPa). Consistent with the presence of stress fibres, we found that SCs generated large surface tractions on stiff substrates and large, finite material deformations on soft substrates. When quantifying the three-dimensional characteristics of the SC traction profiles, we observed a significant contribution from the out-of-plane traction component, locally giving rise to rotational moments similar to those observed in mesenchymal embryonic fibroblasts. Taken together, these measurements provide the first set of quantitative biophysical metrics of how SCs interact with their physical microenvironment, which are anticipated to aid in the development of tissue engineering scaffolds designed to promote functional integration of SCs into post-injury in vivo environments.     

Homogenization of helical beam-like structures: application to single-walled carbon nanotubes

This work is devoted to the computation of axial stiffness of helical beam-like structures. Starting from the homogenization theory of periodic slender domains and taking benefit of the property of helical symmetry, the overall elastic behavior can be obtained from the solution of three-dimensional problems posed on a reduced basic cell. The mechanical analysis of this reduced basic cell performed using a concise FE model allows therefore to compute easily the anisotropic beam homogenized stiffness coefficients. The accuracy and usefulness of this approach is demonstrated by comparisons with reference solutions and large FE model results for two numerical volume structure examples: a wire spring and a stranded “6 + 1” rope. The homogenization procedure is then applied to single-walled carbon nanotubes and it is shown from the two helical symmetries that their basic cell can be reduced to three beam elements.     

Actin-binding proteins sensitively mediate F-actin bundle stiffness

Bundles of filamentous actin (F-actin) form primary structural components of a broad range of cytoskeletal processes including filopodia, sensory hair cell bristles and microvilli. Actin-binding proteins (ABPs) allow the cell to tailor the dimensions and mechanical properties of the bundles to suit specific biological functions. Therefore, it is important to obtain quantitative knowledge on the effect of ABPs on the mechanical properties of F-actin bundles. Here we measure the bending stiffness of F-actin bundles crosslinked by three ABPs that are ubiquitous in eukaryotes. We observe distinct regimes of bundle bending stiffness that differ by orders of magnitude depending on ABP type, concentration and bundle size. The behaviour observed experimentally is reproduced quantitatively by a molecular-based mechanical model in which ABP shearing competes with F-actin extension/compression. Our results shed new light on the biomechanical function of ABPs and demonstrate how single-molecule properties determine mesoscopic behaviour. The bending mechanics of F-actin fibre bundles are general and have implications for cytoskeletal mechanics and for the rational design of functional materials.     

Effects of hypergravity on the cell shape and on the organization of cytoskeleton and extracelluar matrix molecules of in vitro human dermal fibroblasts

In vitro human dermal fibroblasts were submitted to normal gravity (1 g) or to chronic hypergravity ranging from 2 to 20 g for 8 days. Changes only appeared above 15 g. The majority of 20 g-subjected cells showed fine filipods in the shape of a star whereas most control cells had rounded shapes and spread by forming lamellipodia. Indirect immunofluorescence staining of vinculin, alpha-actinin and actin stress fibers showed changes of the arrangement anchoring points of stress fibers under hypergravity. Tubulin staining showed that the centrosomal material generally located above the nucleus in control cells had migrated to the nucleus side in 20 g-exposed cells. After 8 d of culture under 20 g hypergravity the thickness of fibronectin network seemed to be increased and bundles of fibrils appeared linking ordered arrays of fibers. The fibrils of collagen I formed better delimited and thicker bundles of fibers. We may assume that 20 g hypergravity can induce changes in fibroblast cell shape, migration way, and anchorage leading to a reorganization of extracellular matrix without concomitant change of cell proliferation.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

Abstract

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

Substrate stiffness affects skeletal myoblast differentiation in vitro

To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ε-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.     

The Fibrillar Matrix: Novel Avenues for Breast Cancer Detection and Treatment

Breast cancer is marked by large increases in the protein fibers around tumor cells. These fibers increase the mechanical stiffness of the tissue, which has long been used for tumor diagnosis by manual palpation. Recent research in bioengineering has led to the development of novel biomaterials that model the mechanical and architectural properties of the tumor microenvironment and can be used to understand how these cues regulate the growth and spread of breast cancer. Herein, we provide an overview of how the mechanical properties of breast tumor tissues differ from those of normal breast tissue and non-cancerous lesions. We also describe how biomaterial models make it possible to understand how the stiffness and viscosity of the extracellular environment regulate cell migration and breast cancer metastasis. We highlight the need for biomaterial models that allow independent analysis of the individual and different mechanical properties of the tumor microenvironment and that use cells derived from different regions within tumors. These models will guide the development of novel mechano-based therapies against breast cancer metastasis.     

Contributions of talin-1 to glioma cell�Cmatrix tensional homeostasis

The ability of cells to adapt their mechanical properties to those of the surrounding microenvironment (tensional homeostasis) has been implicated in the progression of a variety of solid tumours, including the brain tumour glioblastoma multiforme (GBM). GBM tumour cells are highly sensitive to extracellular matrix (ECM) stiffness and overexpress a variety of focal adhesion proteins, such as talin. While talin has been shown to play critical early roles in integrin-based force-sensing in non-tumour cells, it remains unclear whether this protein contributes to tensional homeostasis in GBM cells. Here, we investigate the role of the talin isoform talin-1 in enabling human GBM cells to adapt to ECM stiffness. We show that human GBM cells express talin-1, and we use RNA interference to suppress talin-1 expression without affecting levels of talin-2, vinculin or phosphorylated focal adhesion kinase. Knockdown of talin-1 strongly reduces both cell spreading area and random migration speed but does not significantly affect overall focal adhesion size distributions. Most strikingly, atomic force microscopy indentation reveals that talin-1 suppression compromises adaptation of cell stiffness to changes in ECM stiffness. Together, these data support a role for talin-1 in the maintenance of tensional homeostasis in GBM and suggest a functional role for enriched talin expression in this tumour.     

Synthesis, characterization, and in vitro evaluation of a hydrogel-based corneal onlay

Blindness due to opacity of the cornea is treated by corneal transplantation with donor tissue. Due to the limited supply of suitable donor corneas, the need for synthetic corneal equivalents is clear. Herein we report the design and in vitro characterization of a hydrogel-based implant; this implant will serve as a permanent, transparent, space-filling onlay with a two-layer design that mimics the native corneal stratification to support surface epithelialization and foster integration with the surrounding tissue. The top layer of the implant was composed of a 2-hydroxyethylmethacrylate hydrogel containing methacrylic acid as the co-monomer (HEMA-co-MAA) with tunable dimensions and compressive modulus ranging from 700-1000 kPa. The bottom layer, which constitutes the bulk of the implant and is designed to provide integration with the corneal stroma, is a dendrimer hydrogel with high water content and compressive modulus ranging from 500-1200 kPa. Both hydrogels were found to possess optical and diffusion properties similar to those of the human cornea. In addition, composite implants with uniform and structurally sound interfaces were formed when the gels were sequentially injected and cross-linked in the same mold. HEMA-co-MAA hydrogels were covalently modified with type I collagen to enable corneal epithelial cell adhesion and spreading that was dependent upon the collagen coating density but independent of hydrogel stiffness. Similarly, dendrimer hydrogels supported the adhesion and spreading of corneal fibroblasts upon modification with the adhesion ligand arginine-glycine-aspartic acid (RGD). Fibroblast adhesion was not dependent upon dendrimer hydrogel stiffness for the formulations studied and, after in vitro culture for 4 weeks, fibroblasts remained able to adhere to and conformally coat the hydrogel surface. In conclusion, the tunable physical properties and structural integrity of the laminated interface suggests that this design is suitable for further study. The judicious tuning of material properties and inclusion of bioactive moieties is a promising strategy for promotion of implant epithelialization and tissue integration.     

面向灰库清理的超大伸缩比机械臂结构设计与刚度优化

火电厂灰库内壁粉尘粘壁、板结、搭桥等现象严重影响火电机组的安全运营,因此须对灰库内壁积灰进行定期全域清理。针对现有灰库清理机器人作业区间有限及因机械臂伸缩比不足、刚度不大导致的清理效果不佳等问题,通过设计引入往复绳排驱动机构和多级嵌套箱型臂体,设计了大刚度、伸缩比为1∶7的机械臂整体结构,研制出一款面向灰库内壁全方位清理的高效作业机器人。基于机械臂级联臂体本征结构和内外耦合作用力系深度分析,结合Castigliano第二定理和线弹性原理,建立了在复杂交变载荷作用下变长度机械臂整体刚度模型。综合考虑在极端工况下机械臂应力分布特征,通过周期性拓扑优化低应力区各离散子域孔洞形状,提出了在严苛质量约束下机械臂全域刚度优化方法。机械臂性能仿真和测试实验表明：优化后机械臂质量大幅减小,且具备理想的伸缩区间和刚度特性;机械臂末端偏移量的测试值与仿真值存在一定偏差,系机械臂加工和装配误差所致。研究结果为灰库清理机器人的结构设计和刚度优化提供了重要参考。     

A micromechanical characterization of angular bidirectional fibrous composites

A micromechanical numerical algorithm to efficiently determine the homogenized elastic properties of bidirectional fibrous composites is presented. A repeating unit cell (RUC) based on a pre-determined bidirectional fiber packing is assumed to represent the microstructure of the composite. For angular bidirectional fiber distribution, the symmetry lines define a parallelepiped unit cell, representing the periodic microstructure of an angular bidirectional fiber composite. The lines of symmetry extrude a volume to capture a three dimensional unit cell. Finite element analysis of this unit cell under six possible independent loading conditions is carried out to study and quantify the homogenized mechanical property of the cell. A volume averaging scheme is implemented to determine the average response as a function of loading in terms of stresses and strains. The individual elastic properties of the constituents’ materials, as well as, the composite can be assumed to be completely isotropic to completely anisotropic. The output of the analysis can determine this degree. The logic behind the selection of the unit cell and the implementation of the periodic boundary conditions as well as the constraints are presented. To verify this micromechanics algorithm, the results for four composites are presented. The results in this paper are mainly focused on the impact of the fiber cross angles on the stiffness properties of the composites chosen. The accuracy of the results from this micromechanics modeling procedure has been compared with the stiffness/compliance solutions from lamination theory. The methodology is to be accurate and efficient to the extent that periodicity of the composite material is maintained. In addition, the results will show the impact of fiber volume fraction on the material properties of the composite. This micromechanics tool could make a powerful viable algorithm for determination of many linear as well as nonlinear properties in continuum mechanics material characterization and analysis.     

Actin flow and talin dynamics govern rigidity sensing in actin–integrin linkage through talin extension

At cell–substrate adhesion sites, the linkage between actin filaments and integrin is regulated by mechanical stiffness of the substrate. Of potential molecular regulators, the linker proteins talin and vinculin are of particular interest because mechanical extension of talin induces vinculin binding with talin, which reinforces the actin–integrin linkage. For understanding the molecular and biophysical mechanism of rigidity sensing at cell–substrate adhesion sites, we constructed a simple physical model to examine a role of talin extension in the stiffness-dependent regulation of actin–integrin linkage. We show that talin molecules linking between retrograding actin filaments and substrate-bound integrin are extended in a manner dependent on substrate stiffness. The model predicts that, in adhesion complexes containing ≈30 talin links, talin is extended enough for vinculin binding when the substrate is stiffer than 1 kPa. The lifetime of talin links needs to be 2–5 s to achieve an appropriate response of talin extension against substrate stiffness. Furthermore, changes in actin velocity drastically shift the range of substrate stiffness that induces talin–vinculin binding. Our results suggest that talin extension is a key step in sensing and responding to substrate stiffness at cell adhesion sites.