Heterogeneous identity, stiffness and growth characterise the shoot apex of Arabidopsis stem cell mutants.

Stem cell homeostasis in the shoot apical meristem involves a core regulatory feedback loop between the signalling peptide CLAVATA3, produced in stem cells, and the transcription factor WUSCHEL, expressed in the underlying organising centre. clavata mutant meristems display massive overgrowth, which is thought to be caused by stem cell overproliferation, although it is unknown how uncontrolled stem cell divisions lead to this altered morphology. Here we reveal local buckling defects in mutant meristems, and use analytical models to show how mechanical properties and growth rates may contribute to the phenotype. Indeed, clavata meristems are mechanically more heterogeneous than the wild type, and also display regional growth heterogeneities. Furthermore, stereotypical wild-type meristem organisation is lost in mutants, in which cells simultaneously express distinct fate markers. Finally, cells in mutant meristems are auxin responsive, suggesting that they are functionally distinguishable from wild-type stem cells. Thus all benchmarks show that clavata meristem cells are different from wild-type stem cells, suggesting that overgrowth is caused by the disruption of a more complex regulatory framework that maintains distinct genetic and functional domains in the meristem.

Physical biology of cell-substrate interactions under cyclic stretch.

Mechanosensitive focal adhesion (FA) complexes mediate dynamic interactions between cells and substrates and regulate cellular function. Integrins in FA complexes link substrate ligands to stress fibers (SFs) and aid load transfer and traction generation. We developed a one-dimensional, multi-scale, stochastic finite element model of a fibroblast on a substrate that includes calcium signaling, SF remodeling, and FA dynamics. We linked stochastic dynamics, describing the formation and clustering of integrins to substrate ligands via motor-clutches, to a continuum level SF contractility model at various locations along the cell length. We quantified changes in cellular responses with substrate stiffness, ligand density, and cyclic stretch. Results show that tractions and integrin recruitments varied along the cell length; tractions were maximum at lamellar regions and reduced to zero at the cell center. Optimal substrate stiffness, based on maximum tractions exerted by the cell, shifted toward stiffer substrates at high ligand densities. Mean tractions varied biphasically with substrate stiffness and peaked at the optimal substrate stiffness. Cytosolic calcium increased monotonically with substrate stiffness and accumulated near lamellipodial regions. Cyclic stretch increased the cytosolic calcium, integrin concentrations, and tractions at lamellipodial and intermediate regions on compliant substrates. The optimal substrate stiffness under stretch shifted toward compliant substrates for a given ligand density. Stretch also caused cell deadhesions beyond a critical substrate stiffness. FA's destabilized on stiff substrates under cyclic stretch. An increase in substrate stiffness and cyclic stretch resulted in higher fibroblast contractility. These results show that chemomechanical coupling is essential in mechanosensing responses underlying cell-substrate interactions.

Substrate Stiffness Modulates TGF-ß Activation and ECM-Associated Gene Expression in Fibroblasts.

Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that regulates the expression of ECM-associated genes during early injury. Tissue fibrosis development is driven by synergistic cues between the evolving biochemical and mechanical milieu. Few studies have addressed the role of substrate stiffness on TGF-ß activity and extracellular matrix (ECM)-associated genes. We used a commercial formulation of polydimethylsiloxane (PDMS) to fabricate substrates of 40 kPa, 300 kPa, and 1.5 MPa stiffness, and cultured the HMF3S fibroblasts on substrates. We quantified TGF-ß protein secreted by HMF3S cells on different substrates using a TGF-ß responsive promoter reporter assay. We also tested for variations in gene expression levels on the substrates using RT-PCR and Western blotting and determined the MMP-2 and MMP-9 activities with gelatin zymography. The results showed that TGF-ß protein activation was significantly compromised at lower stiffnesses. The expression of integrin α5 decreased on lower stiffness substrates and correlated with inefficient TGF-ß protein activation. Collagen I, collagen III, and MMP-2 expression levels were lower on softer substrates; there was little MMP-9 activity on all substrates. Cell and nuclear morphologies were more rounded on compliant substrates, correlating with increased tubulin expression. Proliferations were higher on stiffer substrates, whereas cells on softer substrates showed cell cycle arrest. These results demonstrated critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts, relevant in fibrosis.

Crack propagation and arrests in gelatin hydrogels are linked to tip curvatures.

Gelatin hydrogels are attractive scaffold materials for tissue engineering applications as they provide motifs for cell attachment, undergo large deformations, and are tunable. Low toughness and brittle fractures however limit their use in load bearing applications. An investigation of crack tip processes and mechanisms of crack propagation is warranted to link fracture properties with material microstructure. We cross-linked gelatin using glutaraldehyde to obtain low cross-linked control hydrogels and used an additional cross-linker, methylglyoxal, to fabricate MGO hydrogels with higher cross-links. We quantified fractures in the gelatin hydrogels from both groups using pure shear notch tests and characterized strain fields near the crack tip using 2-D digital image correlation. We used a numerical method based on Taylor's series expansion to measure the crack tip curvatures in the hydrogels. This method captures tip curvatures better than the parabolic method routinely used in studies. Results from our study show that cracks in gelatin hydrogels underwent frequent arrests during propagation through the specimen width in both groups. MGO hydrogels had 85% enhanced fracture toughness and a significantly higher number of stalls compared to the control group. Crack initiations following stalls in the sample correlated with low tip curvatures in both hydrogel groups. We also show that mechanical stretching blunts the crack tip before crack propagation; the degree of blunting was independent of the cross-link density and elastic modulus of the gelatin hydrogels. These results show a link between crack growth and the tip curvature in cross-linked gelatin hydrogels, and offer potential insights for the development of tougher hydrogels.

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness.

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free way to characterize cell-substrate interactions and heterogeneities in cell populations. We quantified the critical adhesion strengths of MCF-7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower critical adhesion strengths (τ50) than their non-invasive counterparts, HPL1D and MCF-7. Detachment dynamics inversely correlated with cell invasion potentials. A theoretical model, based on τ50 values and the distribution of cell areas on substrates, provided good fits to results from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions; non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. Fluorescence intensity of actin was significantly lower in invasive cells as compared to non invasive cells. These correlations in adhesion strengths and traction stresses with cell invasiveness may be useful in cancer diagnostics and other pathologies featuring mis-regulation in adhesion.

Stress fiber growth and remodeling determines cellular morphomechanics under uniaxial cyclic stretch.

Stress fibers in the cytoskeleton are essential in maintaining cellular shape and influence cellular adhesion and migration. Cyclic uniaxial stretching results in cellular reorientation orthogonal to the applied stretch direction. The mechanistic cues underlying changes to cellular form and function to stretch stimuli are currently underexplored. We show stretch-induced stress fiber lengthening, their realignment, and increased cortical actin in NIH 3T3 fibroblasts stretched over varied amplitudes and durations. Higher amounts of actin and stress fiber alignment were accompanied with an increase in the effective elastic modulus of cells. Microtubules did not contribute to the measured stiffness or reorientation response but were essential to the nuclear reorientation. We used a phenomenological growth and remodeling law, based on the experimental data, to model stress fiber elongation and reorientation dynamics based on a nonlinear, orthotropic, fiber-reinforced continuum representation of the cell. The model predicts the changes observed fibroblast morphology and increased cellular stiffness under uniaxial cyclic stretch which agrees with experimental results. Such studies are important in exploring the differences underlying mechanotransduction and cellular contractility under stretch.

Role of Fiber Orientations in the Mechanics of Bioinspired Fiber-Reinforced Elastomers.

Fiber reinforcement is a crucial attribute of soft-bodied muscular hydrostats that have the ability to undergo large deformations and maintain their posture. Helically wound fibers around the cylindrical worm body help control the tube diameter and length. Geometric considerations show that a fiber winding angle of 54.7°, called the magic angle, results in a maximum enclosed volume. Few studies have combined both experimental and theoretical techniques to explore the effects of fiber winding at varied angles on the large deformation mechanics of fiber-reinforced elastomers (FRE). We fabricated FRE materials in transversely isotropic layouts varying from 0° to 90° using a custom filament winding technique and characterized the nonlinear stress-strain relationships using uniaxial and equibiaxial experiments. We used these data within a continuum mechanical framework to propose a novel constitutive model for incompressible FRE materials with embedded extensible fibers. The model includes individual contributions from the matrix and fibers in addition to coupled terms in strain invariants, I1 and I4. The deviatoric stress components show inversion at fiber orientation angles near the magic angle in the FRE composites. These results are useful in soft robotic applications and in the biomechanics of fiber-reinforced tissues such as the myocardium, arteries, and skin.

Making the cut: mechanics of cutting and steering of insect probes.

Many insects forage, oviposit or inject venom in their prey by penetrating or cutting through substrates. From a physical perspective, cutting involves creation of new free surfaces. The cutting parts of insects, such as their mandibles or ovipositor tips, are often zinc-enriched and hardened as compared to the other cuticular regions. Whereas tip hardening is key to their ability to penetrate surfaces, it is often also important for probes to be maneuverable through substrates. How do insect probes negotiate the trade-off between cutting and steering through substrates of diverse stiffness? To address this question, we review the morphology, mechanics, and adaptations in the cutting parts of various insects. Understanding these mechanisms will allow us to develop biomimetic tools, including agricultural and surgical tools, that can both cut and steer through diverse substrates.

Cutting mechanics of wood by beetle larval mandibles.

Wood boring is a feature of several insect species and is a major cause of severe and irreparable damage to trees. Adult females typically deposit their eggs on the stem surface under bark scales. The emerging hatchlings live within wood during their larval phase, avoiding possible predation, whilst continually boring and tunneling through wood until pupation. A study of wood boring by insects offers unique insights into the bioengineering principles that drive evolutionary adaptations. We show that larval mandibles of the coffee wood stem borer beetle (Xylotrechus quadripes: Cerambycidae) have a highly sharp cusp edge to initiate fractures in Arabica wood and a suitable shape to generate small wood chips that are suitable for digestion. Cuticle hardness at the tip is significantly enhanced through zinc-enrichment. A hollow architecture significantly reduces bending stresses at the mandibular base without compromising the structural integrity. Finite element model of the mandible showed highest stresses in the tip region; these decreased to significantly lower values at the start of the hollow section. A scaling model based on a fracture mechanics framework shows the importance of the mandible shape in generating optimal chip sizes. These findings contain general principles in tool design and put in focus interactions of insects and their woody hosts.

Shear stress rosettes capture the complex flow physics in diseased arteries.

Wall shear stress (WSS) is an important parameter in arterial mechanobiology. Various flow metrics, such as time averaged WSS (TAWSS), oscillatory shear index (OSI), and transWSS, have been used to characterize and relate possible WSS variations in arterial diseases like aneurysms and atherosclerosis. We use a graphical representation of WSS using shear rosettes to map temporal changes in the flow dynamics during a cardiac cycle at any spatial location on the vessel surface. The presence of secondary flows and flow reversals can be interpreted directly from the shape of the shear rosette. The mean WSS is given by the rosette centroid, the OSI by the splay around the rosette origin, and the transWSS by its width. We define a new metric, anisotropy ratio (AR), based on the ratio of the length to width of the shear rosette, to capture flow bi-directionality. We characterized the flow physics in controls and patient specific geometries of the ascending aorta (AA) and internal carotid artery (ICA) that have fundamentally different flow dynamics due to differences in the Reynolds and Womersley numbers. The differences in the flow dynamics are well reflected in the shapes of the WSS rosettes and the corresponding flow metrics.

Biophysics of Cell-Substrate Interactions Under Shear.

Cells adhere to substrates through mechanosensitive focal adhesion complexes. Measurements that probe how cells detach from substrates when they experience an applied force connect molecular-scale aspects of cell adhesion with the biophysical properties of adherent cells. Such forces can be applied through shear devices that flow fluid in a controlled manner across cells. The signaling pathways associated with focal adhesions, in particular those that involve integrins and receptor tyrosine kinases, are complex, receiving mechano-chemical feedback from the sensing of substrate stiffness as well as of external forces. This article reviews the signaling processes involved in mechanosensing and mechanotransduction during cell-substrate interactions, describing the role such signaling plays in cancer metastasis. We examine some recent progress in quantifying the strength of these interactions, describing a novel fluid shear device that allows for the visualization of the cell and its sub-cellular structures under a shear flow. We also summarize related results from a biophysical model for cellular de-adhesion induced by applied forces. Quantifying cell-substrate adhesions under shear should aid in the development of mechano-diagnostic techniques for diseases in which cell-adhesion is mis-regulated, such as cancers.

Correction to: The unexplained success of stentplasty vasospasm treatment : Insights using Mechanistic Mathematical Modeling.

Correction to: Clin Neuroradiol 2019 https://doi.org/10.1007/s00062-019-00776-2 The original version of this article unfortunately contained a mistake. The Acknowledgements were missing. The correct information is given ….

The unexplained success of stentplasty vasospasm treatment : Insights using Mechanistic Mathematical Modeling.

BACKGROUND: Cerebral vasospasm (CVS) following subarachnoid hemorrhage occurs in up to 70% of patients. Recently, stents have been used to successfully treat CVS. This implies that the force required to expand spastic vessels and resolve vasospasm is lower than previously thought. OBJECTIVE: We develop a mechanistic model of the spastic arterial wall to provide insight into CVS and predict the forces required to treat it. MATERIAL AND METHODS: The arterial wall is modelled as a cylindrical membrane using a constrained mixture theory that accounts for the mechanical roles of elastin, collagen and vascular smooth muscle cells (VSMC). We model the pressure diameter curve prior to CVS and predict how it changes following CVS. We propose a stretch-based damage criterion for VSMC and evaluate if several commercially available stents are able to resolve vasospasm. RESULTS: The model predicts that dilatation of VSMCs beyond a threshold of mechanical failure is sufficient to resolve CVS without damage to the underlying extracellular matrix. Consistent with recent clinical observations, our model predicts that existing stents have the potential to provide sufficient outward force to successfully treat CVS and that success will be dependent on an appropriate match between stent and vessel. CONCLUSION: Mathematical models of CVS can provide insights into biological mechanisms and explore treatment approaches. Improved understanding of the underlying mechanistic processes governing CVS and its mechanical treatment may assist in the development of dedicated stents.

Traction cytometry: regularization in the Fourier approach and comparisons with finite element method.

Traction forces exerted by adherent cells are quantified using displacements of embedded markers on polyacrylamide substrates due to cell contractility. Fourier Transform Traction Cytometry (FTTC) is widely used to calculate tractions but has inherent limitations due to errors in the displacement fields; these are mitigated through a regularization parameter (γ) in the Reg-FTTC method. An alternate finite element (FE) approach computes tractions on a domain using known boundary conditions. Robust verification and recovery studies are lacking but essential in assessing the accuracy and noise sensitivity of the traction solutions from the different methods. We implemented the L2 regularization method and defined a maximum curvature point in the traction with γ plot as the optimal regularization parameter (γ*) in the Reg-FTTC approach. Traction reconstructions using γ* yield accurate values of low and maximum tractions (Tmax) in the presence of up to 5% noise. Reg-FTTC is hence a clear improvement over the FTTC method but is inadequate to reconstruct low stresses such as those at nascent focal adhesions. FE, implemented using a node-by-node comparison, showed an intermediate reconstruction compared to Reg-FTTC. We performed experiments using mouse embryonic fibroblast (MEF) and compared results between these approaches. Tractions from FTTC and FE showed differences of ∼92% and 22% as compared to Reg-FTTC. Selection of an optimum value of γ for each cell reduced variability in the computed tractions as compared to using a single value of γ for all the MEF cells in this study.

TGF-ß induces changes in breast cancer cell deformability.

Mechanical properties of cells are shown to regulate cell behaviors leading to phenotypic changes that may aid in the development and progression of disease. In this study, we used atomic force microscopy (AFM) indentation with a spherical probe to characterize the elastic and viscoelastic properties of invasive (MDA-MB-231) and noninvasive (MCF-7) breast cancer cells treated with transforming growth factor-ß (TGF-ß). We also used confocal fluorescence imaging to investigate the sub-membrane cytoskeletal structure of the cells. Results showed significant alterations in moduli of both cell types after 24 h TGF-ß treatment which had a context dependent response; moduli for MDA-MB-231 decreased whereas MCF-7 demonstrated stiffening response. Stress relaxation tests showed increased fluid-like nature of MDA-MB-231 following TGF-ß treatment and lower fluidity for MCF-7 cells. We also observed significant alterations in the expression and orientation of actin stress fibers with TGF-ß treatment which correlated with the changes in cell mechanics. Less invasive MCF-7 cells had a delayed overall increase in cell deformability after 48 h exposure to TGF-ß; a similar trend was observed for MDA-MB cells. These changes may be important to facilitate migration, for instance, during metastasis of cancer cells through submicron sized spaces.

Mechanics of the thorax in flies.

Insects represent more than 60% of all multicellular life forms, and are easily among the most diverse and abundant organisms on earth. They evolved functional wings and the ability to fly, which enabled them to occupy diverse niches. Insects of the hyper-diverse orders show extreme miniaturization of their body size. The reduced body size, however, imposes steep constraints on flight ability, as their wings must flap faster to generate sufficient forces to stay aloft. Here, we discuss the various physiological and biomechanical adaptations of the thorax in flies which enabled them to overcome the myriad constraints of small body size, while ensuring very precise control of their wing motion. One such adaptation is the evolution of specialized myogenic or asynchronous muscles that power the high-frequency wing motion, in combination with neurogenic or synchronous steering muscles that control higher-order wing kinematic patterns. Additionally, passive cuticular linkages within the thorax coordinate fast and yet precise bilateral wing movement, in combination with an actively controlled clutch and gear system that enables flexible flight patterns. Thus, the study of thoracic biomechanics, along with the underlying sensory-motor processing, is central in understanding how the insect body form is adapted for flight.

Fabrication and Mechanical Characterization of Hydrogel Infused Network Silk Scaffolds.

Development and characterization of porous scaffolds for tissue engineering and regenerative medicine is of great importance. In recent times, silk scaffolds were developed and successfully tested in tissue engineering and drug release applications. We developed a novel composite scaffold by mechanical infusion of silk hydrogel matrix into a highly porous network silk scaffold. The mechanical behaviour of these scaffolds was thoroughly examined for their possible use in load bearing applications. Firstly, unconfined compression experiments show that the denser composite scaffolds displayed significant enhancement in the elastic modulus as compared to either of the components. This effect was examined and further explained with the help of foam mechanics principles. Secondly, results from confined compression experiments that resemble loading of cartilage in confinement, showed nonlinear material responses for all scaffolds. Finally, the confined creep experiments were performed to calculate the hydraulic permeability of the scaffolds using soil mechanics principles. Our results show that composite scaffolds with some modifications can be a potential candidate for use of cartilage like applications. We hope such approaches help in developing novel scaffolds for tissue engineering by providing an understanding of the mechanics and can further be used to develop graded scaffolds by targeted infusion in specific regions.

Gelatin Methacrylate Hydrogels as Biomimetic Three-Dimensional Matrixes for Modeling Breast Cancer Invasion and Chemoresponse in Vitro.

Recent studies have shown that three-dimensional (3D) culture environments allow the study of cellular responses in a setting that more closely resembles the in vivo milieu. In this context, hydrogels have become popular scaffold options for the 3D cell culture. Because the mechanical and biochemical properties of culture matrixes influence crucial cell behavior, selecting a suitable matrix for replicating in vivo cellular phenotype in vitro is essential for understanding disease progression. Gelatin methacrylate (GelMA) hydrogels have been the focus of much attention because of their inherent bioactivity, favorable hydration and diffusion properties, and ease-of-tailoring of their physicochemical characteristics. Therefore, in this study we examined the efficacy of GelMA hydrogels as a suitable platform to model specific attributes of breast cancer. We observed increased invasiveness in vitro and increased tumorigenic ability in vivo in breast cancer cells cultured on GelMA hydrogels. Further, cells cultured on GelMA matrixes were more resistant to paclitaxel treatment, as shown by the results of cell-cycle analysis and gene expression. This study, therefore, validates GelMA hydrogels as inexpensive, cell-responsive 3D platforms for modeling key characteristics associated with breast cancer metastasis, in vitro.

Advancing Edge Speeds of Epithelial Monolayers Depend on Their Initial Confining Geometry.

Collective cell migrations are essential in several physiological processes and are driven by both chemical and mechanical cues. The roles of substrate stiffness and confinement on collective migrations have been investigated in recent years, however few studies have addressed how geometric shapes influence collective cell migrations. Here, we address the hypothesis that the relative position of a cell within the confinement influences its motility. Monolayers of two types of epithelial cells--MCF7, a breast epithelial cancer cell line, and MDCK, a control epithelial cell line--were confined within circular, square, and cross-shaped stencils and their migration velocities were quantified upon release of the constraint using particle image velocimetry. The choice of stencil geometry allowed us to investigate individual cell motility within convex, straight and concave boundaries. Cells located in sharp, convex boundaries migrated at slower rates than those in concave or straight edges in both cell types. The overall cluster migration occurred in three phases: an initial linear increase with time, followed by a plateau region and a subsequent decrease in cluster speeds. An acto-myosin contractile ring, present in the MDCK but absent in MCF7 monolayer, was a prominent feature in the emergence of leader cells from the MDCK clusters which occurred every ~125 µm from the vertex of the cross. Further, coordinated cell movements displayed vorticity patterns in MDCK which were absent in MCF7 clusters. We also used cytoskeletal inhibitors to show the importance of acto-myosin bounding cables in collective migrations through translation of local movements to create long range coordinated movements and the creation of leader cells within ensembles. To our knowledge, this is the first demonstration of how bounding shapes influence long-term migratory behaviours of epithelial cell monolayers. These results are important for tissue engineering and may also enhance our understanding of cell movements during developmental patterning and cancer metastasis.

Biomechanical Properties of Human Ascending Thoracic Aortic Dissections.

Thoracic aortic dissections are associated with a significant risk of morbidity and mortality, and currently challenge our understanding of the biomechanical factors leading to their initiation and propagation. We quantified the biaxial mechanical properties of human type A dissections (n = 16) and modeled the stress-strain data using a microstructurally motivated form of strain energy function. Our results show significantly higher stiffness for dissected tissues as compared to control aorta without arterial disease. Higher stiffness of dissected tissues did not, however, correlate with greater aortic diameter measured prior to surgery nor were there any age dependent differences in the tissue properties.