Uniaxial Cyclic Strain Research Articles

Ti3C2Tx MXene-Reduced Graphene Oxide Composite Electrodes for Stretchable Supercapacitors.

The development of stretchable electronics requires the invention of compatible high-performance power sources, such as stretchable supercapacitors and batteries. In this work, two-dimensional (2D) titanium carbide (Ti3C2Tx) MXene is being explored for flexible and printed energy storage devices by fabrication of a robust, stretchable high-performance supercapacitor with reduced graphene oxide (RGO) to create a composite electrode. The Ti3C2Tx/RGO composite electrode combines the superior electrochemical and mechanical properties of Ti3C2Tx and the mechanical robustness of RGO resulting from strong nanosheet interactions, larger nanoflake size, and mechanical flexibility. It is found that the Ti3C2Tx/RGO composite electrodes with 50 wt % RGO incorporated prove to mitigate cracks generated under large strains. The composite electrodes exhibit a large capacitance of 49 mF/cm2 (∼490 F/cm3 and ∼140 F/g) and good electrochemical and mechanical stability when subjected to cyclic uniaxial (300%) or biaxial (200% × 200%) strains. The as-assembled symmetric supercapacitor demonstrates a specific capacitance of 18.6 mF/cm2 (∼90 F/cm3 and ∼29 F/g) and a stretchability of up to 300%. The developed approach offers an alternative strategy to fabricate stretchable MXene-based energy storage devices and can be extended to other members of the large MXene family.

Resident multipotent vascular stem cells exhibit amplitude dependent strain avoidance similar to that of vascular smooth muscle cells

Atherosclerosis is one of the leading causes of mortality worldwide, and presents as a narrowing or occlusion of the arterial lumen. Interventions to re-open the arterial lumen can result in re-occlusion through intimal hyperplasia. Historically only de-differentiated vascular smooth muscle cells were thought to contribute to intimal hyperplasia. However recent significant evidence suggests that resident medial multipotent vascular stem cells (MVSC) may also play a role. We therefore investigated the strain response of MVSC since these resident cells are also subjected to strain within their native environment. Accordingly, we applied uniaxial 1 Hz cyclic uniaxial tensile strain at three amplitudes around a mean strain of 5%, (4–6%, 2–8% and 0–10%) to either rat MVSC or rat VSMC before their strain response was evaluated. While both cell types strain avoid, the strain avoidant response was greater for MVSC after 24 h, while VSMC strain avoid to a greater degree after 72 h. Additionally, both cell types increase strain avoidance as strain amplitude is increased. Moreover, MVSC and VSMC both demonstrate a strain-induced decrease in cell number, an effect more pronounced for MVSC. These experiments demonstrate for the first time the mechano-sensitivity of MVSC that may influence intimal thickening, and emphasizes the importance of strain amplitude in controlling the response of vascular cells in tissue engineering applications.

Myoblast maturity on aligned microfiber bundles at the onset of strain application impacts myogenic outcomes

Engineered skeletal muscle grafts may be employed in various applications including the treatment of volumetric muscle loss (VML) and pharmacological drug screening. To recapitulate the well-defined structure of native muscle, tensile strains have been applied to the grafts. In this study, we cultured C2C12 murine myoblasts on electrospun fibrin microfiber bundles for 7 days in custom-built bioreactor units and investigated the impact of strain regimen and delayed onset of tensile straining on myogenic outcomes. The substrate topography induced uniaxial alignment of cells in all (strained and unstrained) groups. The engineered grafts in strained groups were subjected to 10% strain amplitude for 6 h per day. We found that both static and cyclic uniaxial strains resulted in similar morphological and gene expression outcomes. However, relative to 0% strain groups, there were stark increases in myotube diameter, myosin heavy chain (MHC) coverage, and expression of key myogenic genes (Pax 7, Troponin, MHC I, MHC IIb, MHC IIx) only if strain was applied at Days 5–7 rather than Days 3–7. This finding suggests that a critical indicator of myogenic improvement under strain in our system is the phenotype of the cells at the onset of strain and suggests that this is a key parameter that should be considered in studies where myoblasts are subjected to biophysical stimulation to promote tissue formation. Statement of SignificanceThis is the first report on the impact of the timing of the initial application of mechanical strain for improving the myogenic outcomes of 3D engineered skeletal muscle grafts. In this work, immature skeletal myoblasts were grown on topographically aligned, electrospun fibrin microfiber bundles and we applied 10% uniaxial static or cyclic strain. We concluded that the maturity of myoblasts prior to strain application, rather than strain waveform, was the primary predictor of improved myogenic outcomes, including myogenic gene expression and myotube morphology. Elucidating the optimal conditions for strain application is a vital step in recapitulating physiological myogenic properties in tissue engineered skeletal muscle constructs, with applications for treating volumetric muscle loss, disease modeling, and drug testing.

A microscale biomimetic platform for generation and electro-mechanical stimulation of 3D cardiac microtissues.

Organs-on-chip technology has recently emerged as a promising tool to generate advanced cardiac tissue in vitro models, by recapitulating key physiological cues of the native myocardium. Biochemical, mechanical, and electrical stimuli have been investigated and demonstrated to enhance the maturation of cardiac constructs. However, the combined application of such stimulations on 3D organized constructs within a microfluidic platform was not yet achieved. For this purpose, we developed an innovative microbioreactor designed to provide a uniform electric field and cyclic uniaxial strains to 3D cardiac microtissues, recapitulating the complex electro-mechanical environment of the heart. The platform encompasses a compartment to confine and culture cell-laden hydrogels, a pressure-actuated chamber to apply a cyclic uniaxial stretch to microtissues, and stainless-steel electrodes to accurately regulate the electric field. The platform was exploited to investigate the effect of two different electrical stimulation patterns on cardiac microtissues from neonatal rat cardiomyocytes: a controlled electric field [5 V/cm, or low voltage (LV)] and a controlled current density [74.4 mA/cm2, or high voltage (HV)]. Our results demonstrated that LV stimulation enhanced the beating properties of the microtissues. By fully exploiting the platform, we combined the LV electrical stimulation with a physiologic mechanical stretch (10% strain) to recapitulate the key cues of the native cardiac microenvironment. The proposed microbioreactor represents an innovative tool to culture improved miniaturized cardiac tissue models for basic research studies on heart physiopathology and for drug screening.

Directing Neuronal Outgrowth and Network Formation of Rat Cortical Neurons by Cyclic Substrate Stretch.

Neuronal mechanobiology plays a vital function in brain development and homeostasis with an essential role in neuronal maturation, pathfinding, and differentiation but is also crucial for understanding brain pathology. In this study, we constructed an in vitro system to assess neuronal responses to cyclic strain as a mechanical signal. The selected strain amplitudes mimicked physiological as well as pathological conditions. By subjecting embryonic neuronal cells to cyclic uniaxial strain we could steer the direction of neuronal outgrowth perpendicular to strain direction for all applied amplitudes. A long-term analysis proved maintained growth direction. Moreover, stretched neurons showed an enhanced length, growth, and formation of nascent side branches with most elevated growth rates subsequent to physiological straining. Application of cyclic strain to already formed neurites identified retraction bulbs with destabilized microtubule structures as spontaneous responses. Importantly, neurons were able to adapt to the mechanical signals without induction of cell death and showed a triggered growth behavior when compared to unstretched neurons. The data suggest that cyclic strain plays a critical role in neuronal development.

Cyclic Uniaxial Tensile Strain Enhances the Mechanical Properties of Engineered, Scaffold-Free Tendon Fibers.

The treatment of injured tendon is an ever-increasing clinical and financial burden, for which tissue-engineered replacements have shown great promise. Recently, there has been growing interest in a more regenerative approach to tissue engineering, in which the cells' abilities to self-assemble and create matrix are harnessed to create tissue constructs without the use of a scaffold. Herein, utilizing our scaffold-free technique to engineer tendon at the single fiber level, we study how applied mechanical loading, namely cyclic uniaxial strain, influences the mechanical properties and nuclear alignment of developing tendon fiber constructs. Engineered fibers were subjected to 1, 3, and 7 days of intermittent uniaxial loading (0.0-0.7% sinusoidal strain), and then characterized mechanically by constant-rate elongation to failure to obtain tensile properties and histologically to examine cytoskeletal arrangement and nuclear shape, and characterized using real-time polymerase chain reaction to measure the expression of tendon-specific makers, scleraxis and tenomodulin. Fiber peak stress, elastic modulus, toughness, and nuclear aspect ratio increased with the presence and duration of loading, while failure strain, toe-in strain, and nuclear area were unchanged. These biomechanical results suggest that cyclic strain promotes matrix deposition in a manner that increases the fiber resistance to stretch, but preserves fiber extensibility over the 7-day loading period. Over 7 days of loading, the scleraxis and tenomodulin expression increased drastically. Histologically, while there was no immediate difference in nuclear area with the addition of loading, nuclear aspect ratio significantly increased with loading duration, such that nuclei became progressively more elongated to the long axis of the fiber. Together with our biomechanical findings, such nuclear deformation suggests that cyclic strain elicits a mechanotransductive response, particularly one that modulates gene expression to promote matrix deposition during fiber development.

Morphology and contractile gene expression of adipose-derived mesenchymal stem cells in response to short-term cyclic uniaxial strain and TGF-β1.

Previous studies have shown smooth muscle induction in adipose-derived mesenchymal stem cells (ASCs) caused by long-term cyclic stretch. Here we examined the capability of the short-term straining with time steps of 4, 8, 16 and 24 h alone or combined with TGF-β1 on smooth muscle induction of rabbit ASCs. Alterations in cell morphology were quantified through the cell shape index and orientation angle, and expression levels of α-SMA, SM22-α, h-caldesmon and calponin3 markers were examined using the real-time polymerase chain reaction (PCR) method. Moreover, F-actin cytoskeleton organization was observed by fluorescence staining. Mechanical strain either alone or combined with growth factor treatment caused significant up-regulation of both early and intermediate smooth muscle cells (SMCs) specific markers during the initial hours of stimulation peaking in 8 to 16 h. Furthermore, gradual alignment of cells perpendicular to the strain direction during loading time, and cell elongation resembling contractile SMC phenotype, together with alignment and reorganization of F-actin fibers were observed. Considering previously reported protein up-regulation in following days of straining, the effects of short-term cyclic stretch on smooth muscle induction of ASCs were revealed which can be helpful in achieving functional contractile SMCs through synergistic mechano-chemical regulation of ASCs as an appealing cell source for vascular tissue engineering.

Modelling The Combined Effects Of Collagen and Cyclic Strain On Cellular Orientation In Collagenous Tissues

Adherent cells are generally able to reorient in response to cyclic strain. In three-dimensional tissues, however, extracellular collagen can affect this cellular response. In this study, a computational model able to predict the combined effects of mechanical stimuli and collagen on cellular (re)orientation was developed. In particular, a recently proposed computational model (which only accounts for mechanical stimuli) was extended by considering two hypotheses on how collagen influences cellular (re)orientation: collagen contributes to cell alignment by providing topographical cues (contact guidance); or collagen causes a spatial obstruction for cellular reorientation (steric hindrance). In addition, we developed an evolution law to predict cell-induced collagen realignment. The hypotheses were tested by simulating bi- or uniaxially constrained cell-populated collagen gels with different collagen densities, subjected to immediate or delayed uniaxial cyclic strain with varying strain amplitudes. The simulation outcomes are in agreement with previous experimental reports. Taken together, our computational approach is a promising tool to understand and predict the remodeling of collagenous tissues, such as native or tissue-engineered arteries and heart valves.

The Regulation of Differentiation of Mesenchymal Stem-cells into Skeletal Muscle: A Look at Signalling Molecules Involved in Myogenesis.

Mesenchymal Stem Cells (MSCs) are an attractive option for the development of treatment for musculoskeletal pathologies due to their wide availability, clinical safety and multiple techniques available. Understanding the control of MSC differentiation into skeletal muscle is vital for developing protocols and therapeutic applications that are safe and effective. This paper therefore aims to review the current understanding of factors that regulate the differentiation of MSCs into skeletal muscle. Medline, Embase, Pubmed and Web of Science were searched for December 2015 using the terms 'differentia*, skeletal*, skeleton*, myocyt*, myogen* and mesenchym* stem-cell*. This returned a total of 1215 results. 48 papers were included in the review. Forty-eight studies were reviewed. Eight related to external signalling molecules, sixteen related to local environmental factors and twenty-four related to intracellular signalling pathways. Uniaxial strain, medium stiffness of the extracellular matrix and submicron grooved topography were identified as promoting myogenesis. TGF-β was identified as a main inhibitor of myogenesis. Smad and Pax signalling were identified as important intracellular pathways and the relationship of menin, Setdb1, GEFT, PAX3-FOXO1, IGF-II, TAZ and PRDM2 with MyoD and MyoG was explored. Further research into the effect of the inflammatory response on skeletal muscle differentiation is suggested. Clarification of the mechanism of action of TGF-β, the role of submicron grooves and cyclic uniaxial strain, and two important factors in the development of tissue scaffolds.

Myogenic Differentiation of ASCs Using Biochemical and Biophysical Induction.

Adipose-derived stem/stromal cells (ASCs) constitute a very promising source for cell therapy and tissue engineering approaches as they can be easily obtained in large quantities with comparatively minimal patient discomfort. Moreover, ASCs have multilineage differentiation capacity. Among these, differentiation capacity along the myogenic lineage is of particular interest since myogenic precursors are scarce and obtaining a large number of cells from skeletal muscle biopsies is difficult. Here, we describe a method to effectively induce ASC myogenesis through the combination of biochemical (cocktail including 5-azacytidine and horse serum) and biophysical (dynamic culture via uniaxial cyclic strain) stimulation. This method results in multinucleated cells that are positive in myogenic markers including Pax 3/7, desmin, myoD, and myosin heavy chain.

Focal Adhesion Kinase Activation Is Necessary for Stretch-Induced Alignment and Enhanced Differentiation of Myogenic Precursor Cells.

Myogenic precursors sense and dynamically respond to mechanical stimulation through complex integrin-mediated mechanotransduction, in which focal adhesion kinase (FAK) is a fundamental intracellular signaling mediator. When skeletal myoblasts are exposed to uniaxial cyclic tensile strain (UCTS), they display uniform alignment and an enhanced rate of differentiation. In this work, we explored the role of FAK activation by using C2C12 myoblasts that were grown on flexible culture plates and exposed to UCTS during the early differentiation phase. After 24 h, the cells oriented perpendicularly to the direction of strain and exhibited an enhanced differentiation profile. Next, the cells were exposed to a strain field that was either kept in the same direction or rotated 90°, in the presence or not of an FAK phosphorylation inhibitor. On reorientation of the strain field by 90°, the cells reassembled their focal adhesions and actin cytoskeleton to regain the perpendicular position with respect to the engaging stress. After blocking the FAK, however, the cells failed to respond to the reoriented strain field and their differentiation was abrogated. Interestingly, when the strain field remained in the same direction, the FAK inhibitor compromised the differentiation, even though there was no evident change in cell orientation. Our data indicate that during exposure to UCTS, the activation of FAK is necessary for the myoblasts to undergo alignment and enhanced differentiation.

Inhibitory effect of JAK inhibitor on mechanical stress-induced protease expression by human articular chondrocytes.

To investigate whether janus kinase (JAK) inhibitor exhibits a chondro-protective effect against mechanical stress-induced expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinase (MMPs) in human chondrocytes. Normal human articular chondrocytes were seeded onto stretch chambers and incubated with or without tofacitinib (1000nM) for 12h before mechanical stimulation or cytokine stimulation. Uni-axial cyclic tensile strain (CTS) (0.5Hz, 10% elongation, 30min) was applied and the gene expression levels of type II collagen α1 chain (COL2A1), aggrecan (ACAN), ADAMTS4, ADAMTS5, MMP13, and runt-related transcription factor 2 (RUNX-2) were examined by real-time polymerase chain reaction. Nuclear translocation of RUNX-2 and nuclear factor-κB (NF-κB) was examined by immunocytochemistry, and phosphorylation of mitogen-activated protein kinase (MAPK) and signaling transducer and activator of transcription (STAT) 3 was examined by western blotting. The concentration of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in the supernatant was examined by enzyme-linked immunosorbent assay. COL2A1 and ACAN gene expression levels were decreased by CTS, but these catabolic effects were canceled by tofacitinib. Tofacitinib significantly down-regulated CTS-induced expression of ADAMTS4, ADAMTS5, MMP13, and RUNX2, and the release of IL-6 in supernatant by chondrocytes. Tofacitinib also reduced CTS-induced nuclear translocation of RUNX-2 and NF-κB, and phosphorylation of MAPK and STAT3. Tofacitinib suppressed mechanical stress-induced expression of ADAMTS4, ADAMTS5, and MMP13 by human chondrocytes through inhibition of the JAK/STAT and MAPK cascades.

Abstract 373: Effects of Coronary Wall Mechanics on Smooth Muscle Cell Phenotypic Switch and CD44 + Mesenchymal Cell Repopulation in Saphenous Vein Grafts

Background: Despite the preferred application of arterial conduits, the greater saphenous vein (SV) remains indispensable for bypass grafting, especially in multi-vessel coronary artery disease. Early remodeling induced by altered wall mechanics has been recognized to play a key role in SV graft disease. The mechanism remains, however, unknown. Aim: To investigate mechanical factors involved in early graft remodeling, we characterized SV-derived smooth muscle cells (SMCs) after both ex vivo coronary-like mechanical stimulation of SV segments and in vitro unidirectional strain. Methods: SV segments from patients receiving coronary artery bypass grafts were stimulated in a custom-made coronary pulse-duplicator bioreactor. After 7 (n=6) or 14 (n=5) days, stretched and control SVs were fixed and stained for immunofluorescence. Additionally, SMCs isolated from SVs of 7 patients undergoing saphenectomy were subjected to uniaxial cyclic strain (10% elongation, 1 Hz) for 24 or 72 hours using a Flexcell platform. SMCs analysis was performed by western blotting and mass spectrometry-based secretome analysis. Results: Coronary stimulation elevated apoptosis of SV medial cells after 7 days, and consistently reduced the percentage of cells positive for contractile markers α-SMA and calponin. Conversely, synthetic phenotype marker tropomyosin-4 (TM4) and early contractile marker SM22α were elevated at T14. Mesenchymal marker CD44 was markedly upregulated in cells populating the media after 14 days of stimulation. In accordance, strained SMCs displayed decreased α-SMA and SM22α, and increased TM4 protein expression after 72h. Analysis of the supernatant showed a significant increase of plasminogen activator inhibitor-1 and thrombospondin 1. Conclusions: Mechanical stimulation of SVs leads to apoptosis of medial cells and a decrease of contractile SMC markers, followed by repopulation with cells expressing the mesenchymal marker CD44. In addition, unidirectional strain induces a switch of SMC phenotype and secretion of proteins related to vascular remodeling. We are currently investigating whether CD44 + cells derive from SMCs undergoing phenotypic switch, or from progenitor cells localized in the adventitia.

Abstract 475: Human Saphenous Vein Progenitor Cells Are Susceptible to Mechanical Stimulation. Novel Insights in Pathologic Programming of Saphenous Vein Bypass Graft Disease

Background: Implantation of saphenous vein (SV) grafts into coronary position determines structural vessel wall remodeling and intimal hyperplasia. The role of the altered wall mechanics and cell-based mechanosensing has been recently implicated in the priming of this pathologic process. We investigated the effects of cyclic uniaxial strain on human saphenous vein progenitor cells (SVPs), a cell type endowed with pericyte stem cell characteristics resident in the adventitia. Methods: CD34 + CD31 - SVPs were isolated with immuno-magnetic sorting (MACS) from SVs of patients (age 58±12.6, Mean±SD) undergoing saphenectomy. Cells were subject to uniaxial strain (10% elongation; 1Hz) for 24 and 72 hrs using the FlexCell system. Cell orientation, immunofluorescence and western analyses were performed to assess the effects of strain on cell orientation/shape, cell cycle and activity of the YAP/Hippo-dependent mechanotransduction machinery. RNA-sequencing from control vs. strained SVPs was performed at both time points using RNA from cells of 5 independent donors. Results: Results indicated an increase in the expression of the cell cycle-associated markers Ki67 and pHH3 in mechanically stimulated vs. control SVPs at 24 hrs, followed by a significant reduction at 72 hrs of stimulation. Variations in cell shape was observed as verified by a significant change in the nuclei orientation in the strain field as well as in the cell shape index/spread areas at both time points. Immunofluorescence revealed a significant increase in cells showing a nuclear localization of the YAP transcription factor at 72hrs. In line with these findings Western analyses indicated a significant decrease in the ratio between phosphorylated/total YAP and its upstream kinase LATS in mechanically stimulated vs. control SVPs, suggesting an inhibition of the HIPPO kinase pathway by mechanosensing. RNASeq gene expression analyses showed a coherent modification of gene expression pathways and the upregulation of a specific HIPPO/YAP/TEAD gene expression signature in SVPs mechanically stimulated for 72hrs. Conclusions: These findings demonstrate the direct susceptibility of human SVPs to pathologic strain and identifies this cell population as mechano-perceptors in the vein wall.

The effect of a cyclic uniaxial strain on urinary bladder cells

PurposePre-conditioning of a cell seeded construct may improve the functional outcome of a tissue engineered construct for augmentation cystoplasty. The precise effects of mechanical stimulation on urinary bladder cells in vitro are not clear. In this study we investigate the effect of a cyclic uniaxial strain culture on urinary bladder cells which were seeded on a type I collagen scaffold.MethodsIsolated porcine smooth muscle cells or urothelial cells were seeded on a type I collagen scaffolds and cultured under static and dynamic conditions. A uniform cyclic uniaxial strain was applied to the seeded scaffold using a Bose Electroforce Bio-Dynamic bioreactor. Cell proliferation rate and phenotype were investigated, including SEM analysis, RT-PCR and immunohistochemistry for α-Smooth muscle actin, calponin-1, desmin and RCK103 expression to determine the effects of mechanical stimulation on both cell types.ResultsDynamic stimulation of smooth muscle cell seeded constructs resulted in cell alignment and enhanced proliferation rate. Additionally, expression of α-Smooth muscle actin and calponin-1 was increased suggesting differentiation of smooth muscle cells to a more mature phenotype.ConclusionsMechanical stimuli did not enhance the proliferation and differentiation of urothelial cells. Mechanical stimulation, i.e., preconditioning may improve the functional in vivo outcome of smooth muscle cell seeded constructs for flexible organs such as the bladder.

Modeling Stem Cell Myogenic Differentiation

The process of stem cell myogenesis (transformation into skeletal muscle cells) includes several stages characterized by the expression of certain combinations of myogenic factors. The first part of this process is accompanied by cell division, while the second part is mainly associated with direct differentiation. The mechanical cues are known to enhance stem cell myogenesis, and the paper focuses on the stem cell differentiation under the condition of externally applied strain. The process of stem cell myogenic differentiation is interpreted as the interplay among transcription factors, targeted proteins and strain-generated signaling molecule, and it is described by a kinetic multi-stage model. The model parameters are optimally adjusted by using the available data from the experiment with adipose-derived stem cells subjected to the application of cyclic uniaxial strains of the magnitude of 10%. The modeling results predict the kinetics of the process of myogenic differentiation, including the number of cells in each stage of differentiation and the rates of differentiation from one stage to another for different strains from 4% to 16%. The developed model can help better understand the process of myogenic differentiation and the effects of mechanical cues on stem cell use in muscle therapies.

Cellular strain avoidance is mediated by a functional actin cap - observations in an Lmna-deficient cell model.

In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.

The driving force governing room temperature grain coarsening in thin gold films

Strong room-temperature grain coarsening in gold films on polyimide induced by cyclic uniaxial mechanical strain is demonstrated. Detailed electron backscatter diffraction analysis revealed that, in contrast to the predictions of shear-coupled grain boundary migration model, the grain coarsening is isotropic and coarsened grains do not exhibit any specific crystallographic orientations or misorientations to the neighboring grains. It is shown that a thermodynamic model where the driving force appears due to the difference in yield stresses between the grains with different sizes provides an adequate explanation of the experimental data.

Heading in the Right Direction: Understanding Cellular Orientation Responses to Complex Biophysical Environments.

The aim of cardiovascular regeneration is to mimic the biological and mechanical functioning of tissues. For this it is crucial to recapitulate the in vivo cellular organization, which is the result of controlled cellular orientation. Cellular orientation response stems from the interaction between the cell and its complex biophysical environment. Environmental biophysical cues are continuously detected and transduced to the nucleus through entwined mechanotransduction pathways. Next to the biochemical cascades invoked by the mechanical stimuli, the structural mechanotransduction pathway made of focal adhesions and the actin cytoskeleton can quickly transduce the biophysical signals directly to the nucleus. Observations linking cellular orientation response to biophysical cues have pointed out that the anisotropy and cyclic straining of the substrate influence cellular orientation. Yet, little is known about the mechanisms governing cellular orientation responses in case of cues applied separately and in combination. This review provides the state-of-the-art knowledge on the structural mechanotransduction pathway of adhesive cells, followed by an overview of the current understanding of cellular orientation responses to substrate anisotropy and uniaxial cyclic strain. Finally, we argue that comprehensive understanding of cellular orientation in complex biophysical environments requires systematic approaches based on the dissection of (sub)cellular responses to the individual cues composing the biophysical niche.

On-chip assessment of human primary cardiac fibroblasts proliferative responses to uniaxial cyclic mechanical strain.

Cardiac cell function is substantially influenced by the nature and intensity of the mechanical loads the cells experience. Cardiac fibroblasts (CFs) are primarily involved in myocardial tissue remodeling: at the onset of specific pathological conditions, CFs activate, proliferate, differentiate, and critically alter the amount of myocardial extra-cellular matrix with important consequences for myocardial functioning. While cyclic mechanical strain has been shown to increase matrix synthesis of CFs in vitro, the role of mechanical cues in CFs proliferation is unclear. We here developed a multi-chamber cell straining microdevice for cell cultures under uniform, uniaxial cyclic strain. After careful characterization of the strain field, we extracted human heart-derived CFs and performed cyclic strain experiments. We subjected cells to 2% or 8% cyclic strain for 24 h or 72 h, using immunofluorescence to investigate markers of cell morphology, cell proliferation (Ki67, EdU, phospho-Histone-H3) and subcellular localization of the mechanotransduction-associated transcription factor YAP. Cell morphology was affected by cyclic strain in terms of cell area, cell and nuclear shape and cellular alignment. We additionally observed a strain intensity-dependent control of cell growth: a significant proliferation increase occurred at 2% cyclic strain, while time-dependent effects took place upon 8% cyclic strain. The YAP-dependent mechano-transduction pathway was similarly activated in both strain conditions. These results demonstrate a differential effect of cyclic strain intensity on human CFs proliferation control and provide insights into the YAP-dependent mechano-sensing machinery of human CFs.