Uniaxial Stretch Research Articles

AFM and Fluorescence Microscopy of Single Cells with Simultaneous Mechanical Stimulation via Electrically Stretchable Substrates.

We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.

Effect of Uniaxial Stretching on Molecular Orientation, Crystallinity and Oxygen Permeability of Ethylene Vinyl Alcohol

Abstract This work studies the effects of uniaxial stretching on molecular orientation, crystallinity, crystallography and oxygen permeability of high barrier multilayer films based on EVOH barrier layer. Film samples were prepared using two different blow-up ratios (BUR) of 1 and 3. Uniaxial stretching was applied at 100°C using a tensile machine equipped with an environmental chamber. Fourier transform infrared (FTIR) spectroscopy was used to examine the molecular orientation in the EVOH layer before and after stretching and confirmed a considerable increase in the molecular orientation in the EVOH layer. Dynamic scanning calorimetry (DSC) and wide-angle X-ray diffraction (XRD) results point to the perfection of EVOH crystal structure by applying uniaxial stretching at high temperatures. Oxygen permeability results showed a considerable 25% decrease after stretching of the sample with lower BUR while a significant 80% reduction in oxygen permeability was observed for the sample prepared at higher BUR. The mechanism involved in increasing oxygen permeability due to the uniaxial stretching at high temperature is discussed in detail.

Uniaxial Cyclic Stretching Promotes Chromatin Accessibility of Gene Loci Associated With Mesenchymal Stem Cells Morphogenesis and Osteogenesis.

It has been previously demonstrated that uniaxial cyclic stretching (UCS) induces differentiation of mesenchymal stem cells (MSCs) into osteoblasts in vitro. It is also known that interactions between cells and external forces occur at various aspects including cell–matrix, cytoskeleton, nucleus membrane, and chromatin. However, changes in chromatin landscape during this process are still not clear. The present study was aimed to determine changes of chromatin accessibility under cyclic stretch. The influence of cyclic stretching on the morphology, proliferation, and differentiation of hMSCs was characterized. Changes of open chromatin sites were determined by assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Our results showed that UCS induced cell reorientation and actin stress fibers realignment, and in turn caused nuclear reorientation and deformation. Compared with unstrained group, the expression of osteogenic and chondrogenic marker genes were the highest in group of 1 Hz + 8% strain; this condition also led to lower cell proliferation rate. Furthermore, there were 2022 gene loci with upregulated chromatin accessibility in 1 Hz + 8% groups based on the analysis of chromatin accessibility. These genes are associated with regulation of cell morphogenesis, cell–substrate adhesion, and ossification. Signaling pathways involved in osteogenic differentiation were found in up-regulated GO biological processes. These findings demonstrated that UCS increased the openness of gene loci associated with regulation of cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, the findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation, and deformation. Our study may provide reference for directed differentiation of stem cells induced by mechanical microenvironments.

The Effect of Mechanical Stretch on Myotube Growth Suppression by Tumor‐derived Factors

The mechanisms of cancer-induced muscle wasting are widely investigated with in vivo preclinical models and in vitro experiments examining effects of tumor-derived factors. Myogenic differentiation is associated with rapid increases in myotube size and contractile properties, which tumor-derived factors can suppress. Specifically, colon-26 (C26) conditioned media induces catabolic signaling in cultured myotubes. While mechanical stimuli can induce anabolic signaling and myotube growth, the effect of mechanical stretch on myotubes treated with C26 conditioned media (CM) is not known. Therefore, we examined if uniaxial mechanical stretch could reverse myotube growth suppression caused by C26 conditioned media. Cachectic C26 or non-cachectic EL4 lymphoma CM was collected after 48h (~90% confluence). C2C12 myoblasts were seeded on type I collagen-coated polystyrene or Flexcell uniaxial stretch plates, proliferated, then placed in differentiation media for 5-7 days. On day 5 of differentiation, myotubes were treated with 50% control, C26, or EL4 CM for 48h. An additional experiment stretched myotubes 5% for the last 24h of the C26 incubation, and myotubes were collected immediately post stretch. Myotube diameter quantification used digital images. For all comparisons, at least three replicates from 2 independent experiments were used. Myotubes increased diameter (28%, p<0.0001) and myosin heavy chain-fast (MHC-F) protein expression (IOD/ug protein; 127%, p<0.0001) between days 5 and 7 of differentiation. Myotube growth was associated with increased AktT308 phosphorylation and suppressed E3 ligase expression. C26, but not EL4 suppressed myotube diameter (C26: -18%, p<0.0001; EL4: 1%, p=0.708) and MHC-F protein expression (C26: -62%, p<0.0001; EL4: 0%, p=0.999). C26 suppressed AktS473, ERK1/2, and 4E-BP1phosphorylation and increased MuRF1 and Atrogin1 expression. The C26 decrease in myotube diameter was not rescued by 24h of stretch (C26 control: -18%, p<0.0001; C26 stretch: -12%, p=0.0005). Stretch prevented C26 suppression of MHC-F protein expression (C26 control: -18%, p=0.001; C26 stretch: 11%, p=0.389). In summary, MHC-F protein concentration was rescued by mechanical stretch in C26 treated myotubes independent of myotube size. Additional research is warranted to determine how mechanical stretch prevents myosin's catabolic regulation in myotubes by tumor-derived factors.

Quantifying Lymphatic Endothelial Cell Morphological Changes in Response to Fluid Shear Stress, Cyclic Strain, or Combined Stress and Strain In Vitro

The lymphatic system performs vital transport of fluid, lipids, immune cells, and macromolecules. Lymphatic endothelial cells (LECs), which are highly mechanosensitive, are involved in modulating lymphatic contraction, permeability, etc. LECs are sensitive to the fluid shear stress (FSS) caused by lymph flow and the cyclic strain caused by wall contraction and relaxation. The effects of these mechanical forces on LECs are less understood compared to blood vascular endothelial cells, (BVEC), yet the relative changes in these forces on LECs are considerably greater in situ. To the best of our knowledge, no previous in vitro studies have applied simultaneous FSS and cyclic stretching to LECs. To address this information gap, we tested the hypothesis that both FSS and cyclic stretching will alter LEC structure and function. Toward this goal, we developed a novel bioreactor to apply simultaneous, independently-controlled FSS and cyclic uniaxial stretching to LECs on silicone membranes, and a second bioreactor for applying only uniaxial cyclic stretch. After subjecting LECs to FSS alone, stretch alone, or combined FSS and stretch in these systems, we imaged the cells using conventional or confocal fluorescence techniques and measured alterations in orientation and actin fiber alignment of the cultured LECs. Our results show statistically significant changes in LEC orientation compared to controls, aligning in the direction of low steady FSS (0.2 dynes/cm^2) after 12-24 hours. LECs subjected to the low steady flow combined with a 7.5% cyclic stretch, exhibited a more dramatic alignment that was parallel to flow and perpendicular to stretch after only 6 hours. These data indicate that, like BVEC, LEC morphological alignment is sensitive to both FSS and stretching, with a synergistic response when both are applied simultaneously. Similar levels of alignment were seen in LECs subjected to only cyclic stretch after 6 or 12 hours, but rather than aligning perpendicular to stretch, the distribution of cell angles was bimodal, with most cells aligning about +/- 15 degrees from perpendicular. This slightly off-axis alignment could be a function of applying stretch without fluid flow, or may be a response to the higher magnitude of stretch in those studies (40% compared to 7.5%). There was some visual indication of alignment of actin fibers in LEC subjected to combined FSS and stretching, but it was more difficult to determine clear trends due to spatial variation when imaging different regions of the membrane, and further study is therefore required. Taken together, our results show that application of FSS and strain differentially alter LEC alignment and morphology and provides an important platform to further investigate the mechanisms underlying the LEC response to complex physiologically relevant mechanical environments.

Time-Dependent Elastic Tensor of Cellulose Nanocrystal Probed by Hydrostatic Pressure and Uniaxial Stretching.

The elastic properties of crystals are fundamental for structural material. However, in the absence of macroscopic single crystals, the experimental determination of the elastic tensor is challenging because the measurement depends on the transmission of stress inside the material. To avoid arbitrary hypotheses about stress transfer, we combine hydrostatic pressure and uniaxial-stretching experiments to investigate the elastic properties of cellulose Iβ. Three orthogonal compressibilities are 50.0, 6.6, and 1.71 TPa-1. Combining Poisson's ratios from a uniaxial stretching experiment directly gives the Young's modulus along the chain direction (E33). However, Poisson's ratio depends on the deformation rate leading to apparent modulus E33 = 113 GPa using a slow cycle (hours) and 161 GPa using a fast cycle (minutes). The lattice deformation along the chain is not time-dependent, so the off-diagonal elements are time-dependent on the scale of minutes to hours.

Experimental Study on Axial Tensile Stress-Strain Relationship of High Crack Resistance Concrete

For demand of high crack resistance of concrete, the calcium-magnesium expansion agents and basalt fiber are chosen. The study of the full stress-strain curve of concrete under uniaxial tensile can comprehensively reflect its mechanical properties. The uniaxial stretch tests are used to study the concrete axial tensile constitutive model, and then the corresponding curve and formula are obtained in this paper. The influence of different dosages and water-binder ratio on the characteristic points of the curve is studied, and a suitable mathematical expression for the axial tensile stress-strain curve is proposed. Studies have shown that by adopting the method of dual admixture of expansive agent and basalt fiber, the toughness, tensile strength, ductility, and even other properties of concrete can be significantly improved. This shows that the dual admixture of the two materials has a strong toughening effect on concrete. With the amount of fiber content, the amount of expansion agent and the water-binder ratio is respectively 1%, 7% and 0.42, the performance of concrete shows the best state. The proposed calculation formula is in good agreement with the experimental results, which can provide a theoretical basis for the design and nonlinear analysis of concrete structures.

In Vitro Models of Traumatic Brain Injury: A Systematic Review.

Traumatic brain injury (TBI) is a major public health challenge that is also the third leading cause of death worldwide. It is also the leading cause of long-term disability in children and young adults worldwide. Despite a large body of research using predominantly in vivo and in vitro rodent models of brain injury, there is no medication that can reduce brain damage or promote brain repair mainly due to our lack of understanding in the mechanisms and pathophysiology of the TBI. The aim of this review is to examine in vitro TBI studies conducted from 2008-2018 to better understand the TBI in vitro model available in the literature. Specifically, our focus was to perform a detailed analysis of the in vitro experimental protocols used and their subsequent biological findings. Our review showed that the uniaxial stretch is the most frequently used way of load application, accounting for more than two-thirds of the studies reviewed. The rate and magnitude of the loading were varied significantly from study to study but can generally be categorized into mild, moderate, and severe injuries. The in vitro studies reviewed here examined key processes in TBI pathophysiology such as membrane disruptions leading to ionic dysregulation, inflammation, and the subsequent damages to the microtubules and axons, as well as cell death. Overall, the studies examined in this review contributed to the betterment of our understanding of TBI as a disease process. Yet, our review also revealed the areas where more work needs to be done such as: 1) diversification of load application methods that will include complex loading that mimics in vivo head impacts; 2) more widespread use of human brain cells, especially patient-matched human cells in the experimental set-up; and 3) need for building a more high-throughput system to be able to discover effective therapeutic targets for TBI.

3D Printed Metamaterial Capacitive Sensing Array for Universal Jamming Gripper and Human Joint Wearables

Existing stretchable capacitive sensor arrays face challenges in decoupling normal forces and stretch stimuli which restrict its applications into soft robotics. Herein, a metamaterial capacitive sensor array with 6 × 6 sensors is developed for the detection of normal forces on curved deforming surfaces common to both the soft universal jamming gripper and human elbow. The fabrication process involves the 3D printing of carbon black thermoplastic polyurethane (PI–ETPU) electrodes and thermoplastic polyurethane (TPU) insulation based on commercially available multimaterial fused deposition modeling (FDM). This metamaterial capacitive sensor array is unaffected by a uniaxial stretch of up to 21.6% and remains capable of detecting normal forces. The sensor array structure possesses a negative Poisson's ratio which is desirable for improving compliance and conformability to adhered expanding surfaces. Consequently, normal force‐distribution and proximity detection capabilities on curved deforming surfaces are demonstrated on a soft universal jamming gripper for measuring gripping forces and identifying object shapes. Conformal force‐sensing joint wearable on a human elbow with an individualized Poisson's ratio is explored as another application. The possibility of programming Poisson's ratio of a metamaterial capacitive sensing array opens new avenues for potential sensing applications into soft robotics and personalized wearables.

Cooling rate-dependent microstructure and mechanical properties of 2D surface polycrystals in liquid AuSi thin films

Molecular dynamics simulations are performed to investigate the influence of cooling rate on the microstructures and mechanical properties of 2D surface crystals of liquid AuSi thin films. It is found that the increasing cooling rate significantly reduces the grain size of polycrystallization, and its formation mechanism is explored by tracing the evolution of nanoclusters. We develop a bulk-surface framework to extract the Young’s modulus and Poisson’s ratio of single crystal surface from uniaxial and biaxial stretch deformation. Our simulations results real that polycrystalline surface shows a large drop of stiffness and ultimate strength due to the existence of grain boundaries.

Development of Inorganic Particle-Filled Polypropylene/High Density Polyethylene Membranes via Multilayer Co-Extrusion and Stretching.

This work is made to ascertain the effects of mineral fillers, namely calcium carbonate and talc, on the morphology and properties of multilayer polypropylene (PP)/high-density polyethylene (HDPE) porous membranes. Multilayer membranes were prepared using the three-stage Melt-Extrusion, Annealing and Uniaxial Stretching (MEAUS) process. The orientation of PP’s crystalline phase was affected by both the flow-induced crystallization and the heterogeneous nucleation promoted by the fillers. A synergistic effect was observed in the filled samples due to the generation of pores after the stretching-induced lamellae separation and the debonding of mineral fillers from the polymeric matrix. The fillers increased the porous surface, leading to an increase of permeance to air, being this effect more marked at higher filler contents. Talc showed a higher efficiency to create porous surfaces when compared to calcium carbonate. The thermal stability of the membranes increased with filler addition, as well as their stiffness and strength.

Relationship Between Formation of Kink Structure and Necking of a Specimen Comprising Hard and Soft Lamellar Microdomains Under Uniaxial Stretching

Relationship Between Formation of Kink Structure and Necking of a Specimen Comprising Hard and Soft Lamellar Microdomains Under Uniaxial Stretching

Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli.

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-β3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-β3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-β3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration.

Mechano-chemo signaling interactions modulate matrix production by cardiac fibroblasts

Extracellular matrix remodeling after myocardial infarction occurs in a dynamic environment in which local mechanical stresses and biochemical signaling species stimulate the accumulation of collagen-rich scar tissue. It is well-known that cardiac fibroblasts regulate post-infarction matrix turnover by secreting matrix proteins, proteases, and protease inhibitors in response to both biochemical stimuli and mechanical stretch, but how these stimuli act together to dictate cellular responses is still unclear. We developed a screen of cardiac fibroblast-secreted proteins in response to combinations of biochemical agonists and cyclic uniaxial stretch in order to elucidate the relationships between stretch, biochemical signaling, and cardiac matrix turnover. We found that stretch significantly synergized with biochemical agonists to inhibit the secretion of matrix metalloproteinases, with stretch either amplifying protease suppression by individual agonists or antagonizing agonist-driven upregulation of protease expression. Stretch also modulated fibroblast sensitivity towards biochemical agonists by either sensitizing cells towards agonists that suppress protease secretion or de-sensitizing cells towards agonists that upregulate protease secretion. These findings suggest that the mechanical environment can significantly alter fibrosis-related signaling in cardiac fibroblasts, suggesting caution when extrapolating in vitro data to predict effects of fibrosis-related cytokines in situations like myocardial infarction where mechanical stretch occurs.

Mechanoadaptive organization of stress fiber subtypes in epithelial cells under cyclic stretches and stretch release

Cyclic stretch applied to cells induces the reorganization of stress fibers. However, the correlation between the reorganization of stress fiber subtypes and strain-dependent responses of the cytoplasm and nucleus has remained unclear. Here, we investigated the dynamic involvement of stress fiber subtypes in the orientation and elongation of cyclically stretched epithelial cells. We applied uniaxial cyclic stretches at 5%, 10%, and 15% strains to cells followed by the release of the mechanical stretch. Dorsal, transverse arcs, and peripheral stress fibers were mainly involved in the cytoplasm responses whereas perinuclear cap fibers were associated with the reorientation and elongation of the nucleus. Dorsal stress fibers and transverse arcs rapidly responded within 15 min regardless of the strain magnitude to facilitate the subsequent changes in the orientation and elongation of the cytoplasm. The cyclic stretches induced the additional formation of perinuclear cap fibers and their increased number was almost maintained with a slight decline after 2-h-long stretch release. The slow formation and high stability of perinuclear cap fibers were linked to the slow reorientation kinetics and partial morphology recovery of nucleus in the presence or absence of cyclic stretches. The reorganization of stress fiber subtypes occurred in accordance with the reversible distribution of myosin II. These findings allowed us to propose a model for stretch-induced responses of the cytoplasm and nucleus in epithelial cells based on different mechanoadaptive properties of stress fiber subtypes.

Inhomogeneous deformation in hyperspace

Geometrical approach to continuum mechanics allowing an atomistic treatment is developed. It suggests a body as a surface immersed into ambient space. Four kinds of surfaces inherent to basic deformations determine classical dynamics of the atoms alongside their spatial coordinates. For uniaxial stretch, this surface is isomorphic to the cylinder. For a simple shear, it is the twisted cylinder, for a bending — a cone, and for a torsion — a helicoid. Scalar parameters of the metric of these surfaces that are stretch ratios, shear, cone, and torsion angles used instead of the strain tensor constitute extra coordinates of the particles in hypersurface. Correspondingly, vector-valued tensions are used instead of stress. In the case of pure deformation, the normal and extra coordinates of atoms obey the classical equation of motion admitting periodic boundary conditions. It is shown how the employing of artificial orders of freedom leads to a minimum of elastic energy. In boundary problems, the system of governing equations unambiguously reproduce the stress–strain–displacement relations with the help of using the tension boundary theorem instead of the Cauchy traction principle. The derived direct method of elastostatic makes the system of governing equations closed and the compatibility condition unnecessary. Commonest examples of bending problem normally following from the Airy stress function illustrate the new concept.

Understanding the Morphological and Structural Evolution of α- and γ-Poly(vinylidene fluoride) During High Temperature Uniaxial Stretching by In Situ Synchrotron X-ray Scattering

Poly(vinylidene fluoride) (PVDF) is rich in different polymorphs. Among them, β-phase PVDF is of technical importance because of its strong spontaneous polarization and desired piezoelectric proper...

Correlative Study on Plastic Response and Formability of Ti-6Al-4V Sheets under Hot Forming Conditions

Abstract In this study, the formability behavior of Ti-6Al-4V alloy sheets under uniaxial and multi-axial loading conditions at 923 K (650 °C), which has received less attention till now, has been extensively investigated by experiments and numerical modeling. Uniaxial tension tests at various strain rates and limit dome height (LDH) tests at the elevated temperature, covering the uniaxial stretch, plane strain, and biaxial stretch deformation modes, were conducted, and the corresponding constitutive models were constructed. Based on the experimentally characterized mechanical properties and associated constitutive laws, finite element simulations of the LDH tests were conducted, and the formability of the investigated material was analyzed using the Marciniak-Kuczyinski theory by employing the von-Mises isotropic yield function and Voce hardening law, including strain-rate sensitivity. At the elevated temperature, a strong positive rate sensitivity effect was observed. Finally, the predicted load-displacement responses of the LDH tests were in good agreement with the measured data. The corresponding formability analysis can contribute to the design of optimal hot forming processes for Ti-6Al-4V sheets.

Investigating Passive Muscle Mechanics With Biaxial Stretch.

IntroductionThe passive stiffness of skeletal muscle can drastically affect muscle function in vivo, such as the case for fibrotic tissue or patients with cerebral palsy. The two constituents of skeletal muscle that dominate passive stiffness are the intracellular protein titin and the collagenous extracellular matrix (ECM). However, efforts to correlate stiffness and measurements of specific muscle constituents have been mixed, and thus the complete mechanisms for changes to muscle stiffness remain unknown. We hypothesize that biaxial stretch can provide an improved approach to evaluating passive muscle stiffness.MethodsWe performed planar biaxial materials testing of passively stretched skeletal muscle and identified three previously published datasets of uniaxial materials testing. We developed and employed a constitutive model of passive skeletal muscle that includes aligned muscle fibers and dispersed ECM collagen fibers with a bimodal von Mises distribution. Parametric modeling studies and fits to experimental data (both biaxial and previously published) were completed.ResultsBiaxial data exhibited differences in time dependent behavior based on orientation (p < 0.0001), suggesting different mechanisms supporting load in the direction of muscle fibers (longitudinal) and in the perpendicular (transverse) directions. Model parametric studies and fits to experimental data exhibited the robustness of the model (<20% error) and how differences in tissue stiffness may not be observed in uniaxial longitudinal stretch, but are apparent in biaxial stretch.ConclusionThis work presents novel materials testing data of passively stretched skeletal muscle and use of constitutive modeling and finite element analysis to explore the interaction between stiffness, constituent variability, and applied deformation in passive skeletal muscle. The results highlight the importance of biaxial stretch in evaluating muscle stiffness and in further considering the role of ECM collagen in modulating passive muscle stiffness.

3D cellular alignment and biomimetic mechanical stimulation enhance human adipose-derived stem cell myogenesis

Determination of a stem cell source with sufficient myogenic differentiation capacity that can be easily obtained in large quantities is of great importance in skeletal muscle regeneration therapies. Adipose-derived stem cells (ASCs) are readily available, can be isolated from fat tissue with high yield and possess myogenic differentiation capacity. Consequently, ASCs have high applicability in muscle regenerative therapies. However, a key challenge is their low differentiation efficiency. In this study, we have explored the potential of mimicking the natural microenvironment of the skeletal muscle tissue to enhance ASC myogenesis by inducing 3D cellular alignment and using dynamic biomimetic culture. ASCs were entrapped and 3D aligned in parallel within fibrin-based microfibers and subjected to uniaxial cyclic stretch. 3D cell alignment was shown to be necessary for achieving and maintaining the stiffness of the construct mimicking the natural tissue (12 ± 1 kPa), where acellular aligned fibers and cell-laden random fibers had stiffness values of 4 ± 1 and 5 ± 2 kPa, respectively, at the end of 21 d. The synergistic effect of 3D cell alignment and biomimetic dynamic culture was evaluated on cell proliferation, viability and the expression of muscle-specific markers (immunofluorescent staining for MyoD1, myogenin, desmin and myosin heavy chain). It was shown that the myogenic markers were only expressed on the aligned-dynamic culture samples on day 21 of dynamic culture. These results demonstrate that 3D skeletal muscle grafts can be developed using ASCs by mimicking the structural and physiological muscle microenvironment.