Uniaxial Stretch Research Articles

Viscoelastic Properties of Vascular Endothelial Cells Exposed to Uniaxial Stretch

Vascular endothelial cells (VECs) that line the interior of blood vessels are continually stretched as the vessel walls expand and contract. It is widely accepted that VECs will remodel themselves in response to this mechanical stimuli, leading to changes in cell function. Few studies, however, have analyzed the mechanical properties of these cells under stretch. We hypothesize that uniaxial stretch will cause an anisotropic realignment of actin filaments, and a change in the viscoelastic properties of the cell. To test this hypothesis, VECs were grown on a thin, transparent membrane mounted on a microscope. The membrane was stretched, consequently stretching the cells. Time-lapse sequences of the cells were taken every hour with a time resolution of 10 Hz. The random trajectories of intracellular endogenous particles were tracked using in-house algorithms. Using a novel particle tracking microrheology formulation, that takes into account the anisotropic nature of the cytoplasm, these trajectories were analyzed and the mechanical properties of VECs subjected to various stretching conditions were calculated and compared.

8D36 間葉系幹細胞に対する伸縮刺激が細胞外マトリクスの発現量と構造に及ぼす影響(OS07 軟組織およびその構成要素のバイオメカニクス1)

8D36 間葉系幹細胞に対する伸縮刺激が細胞外マトリクスの発現量と構造に及ぼす影響(OS07 軟組織およびその構成要素のバイオメカニクス1)

Time-Resolved Synchrotron X-ray Scattering Study on Propylene–1-Butylene Random Copolymer Subjected to Uniaxial Stretching at High Temperatures

Synchrotron wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) were used to characterize the structure evolution of propylene–1-butylene (P–B) random copolymer subjected to uniaxial tensile deformation at 100 °C. Polymorphism and preferred orientation of the crystal phases were examined quantitatively by 2D WAXD. The results indicated that three ensembles of crystalline modifications with distinctive orientation modes coexisted during stretching. The orthorhombic γ-form adopted a tilted cross-β configuration, in which the c-axis had a tilt angle with respect to the fiber axis. The monoclinic α-form in the mother lamellae had a c-axis orientation with polymer chains parallel to the fiber axis. In the α-phase daughter lamellae, the unit cell assumed an a-axis orientation, where the c-axis had an 80° angle with respect to the fiber axis. Stretching transformed the γ-phase into the energetically more stable α-phase. In the late stage, the system was dominated by the α-phase with parallel chain packing. Complemented by qualitative SAXS analysis, simultaneous inter- and intralamellar chain slips were observed during the early stage of stretching. After yielding, a fibrillation process followed. The formation of fibril bundles together with a cross-linked network after yielding might account for the stress-hardening behavior in the late stage of stretching.

Optimizing an Intermittent Stretch Paradigm Using ERK1/2 Phosphorylation Results in Increased Collagen Synthesis in Engineered Ligaments

Dynamic mechanical input is believed to play a critical role in the development of functional musculoskeletal tissues. To study this phenomenon, cyclic uniaxial mechanical stretch was applied to engineered ligaments using a custom-built bioreactor and the effects of different stretch frequency, amplitude, and duration were determined. Stretch acutely increased the phosphorylation of p38 (3.5±0.74-fold), S6K1 (3.9±0.19-fold), and ERK1/2 (2.45±0.32-fold). The phosphorylation of ERK1/2 was dependent on time, rather than on frequency or amplitude, within these constructs. ERK1/2 phosphorylation was similar following stretch at frequencies from 0.1 to 1 Hz and amplitudes from 2.5% to 15%, whereas phosphorylation reached maximal levels at 10 min of stretch and returned toward basal within 60 min of stretch. Following a single 10-min bout of cyclic stretch, the cells remained refractory to a second stretch for up to 6 h. Using the phosphorylation of ERK1/2 as a guide, the optimum stretch paradigm was hypothesized to be 10 min of stretch at 2.5% of resting length repeated every 6 h. Consistent with this hypothesis, 7 days of stretch using this optimized intermittent stretch program increased the collagen content of the grafts more than a continuous stretch program (CTL=3.1%±0.44%; CONT=4.8%±0.30%; and INT=5.9%±0.56%). These results suggest that short infrequent bouts of loading are optimal for improving engineered tendon and ligament physiology.

The Effect of Dynamical Strain on the Maturation of Collagen-Based Cell-Containing Scaffolds for Vascular Tissue Engineering

Diseases occurring to blood vessel are preferentially solved by replacing the vessel by an autologous graft. When it is not available, a synthetic graft is used which has low patency rates for small diameter (<6 mm) vessels. Tissue engineering of blood vessel aims to improve the performance of vascular substitutes. Bioreactors are used in vascular tissue engineering to mimic the mechanical and biochemical environment of blood vessel. A 2D bioreactor was custom made in order to impose a dynamical strain to silicone membrane receiving the collagen cell-based construct. Collagen gels with vascular smooth muscle cells cultured inside were subdued to maturation under dynamical uniaxial stretch regimes at 1Hz for 48 hours. The percentage of deformation encountered by the silicone membrane was measured by ImageJ. Collagen fibrils and porcine smooth muscle cells (PSMC) orientations were assessed by scanning electron microscopy (SEM). Results show that the study of mechanical conditioning on cell activity is an important issue for enhancing the alignment of collagen fibrils.

Microtubule Dynamics Regulate Cyclic Stretch-Induced Cell Alignment in Human Airway Smooth Muscle Cells

Microtubules are structural components of the cytoskeleton that determine cell shape, polarity, and motility in cooperation with the actin filaments. In order to determine the role of microtubules in cell alignment, human airway smooth muscle cells were exposed to cyclic uniaxial stretch. Human airway smooth muscle cells, cultured on type I collagen-coated elastic silicone membranes, were stretched uniaxially (20% in strain, 30 cycles/min) for 2 h. The population of airway smooth muscle cells which were originally oriented randomly aligned near perpendicular to the stretch axis in a time-dependent manner. However, when the cells treated with microtubule disruptors, nocodazole and colchicine, were subjected to the same cyclic uniaxial stretch, the cells failed to align. Lack of alignment was also observed for airway smooth muscle cells treated with a microtubule stabilizer, paclitaxel. To understand the intracellular mechanisms involved, we developed a computational model in which microtubule polymerization and attachment to focal adhesions were regulated by the preexisting tensile stress, pre-stress, on actin stress fibers. We demonstrate that microtubules play a central role in cell re-orientation when cells experience cyclic uniaxial stretching. Our findings further suggest that cell alignment and cytoskeletal reorganization in response to cyclic stretch results from the ability of the microtubule-stress fiber assembly to maintain a homeostatic strain on the stress fiber at focal adhesions. The mechanism of stretch-induced alignment we uncovered is likely involved in various airway functions as well as in the pathophysiology of airway remodeling in asthma.

Comparing mechano-transduction in fibroblasts deficient of focal adhesion proteins

Mechano-transduction was studied in wildtype and focal adhesion (FA) protein-deficient mouse embryonic fibroblasts (MEFs). Using a cell stretcher, we determined the effect of stretch on cell morphology, apoptosis, and phosphorylation of ERK1/2. After 20% cyclic, uniaxial stretch, FA-deficient MEFs showed morphological changes and levels of apoptosis of the order: focal adhesion kinase>p130Cas>vinculin compared to wildtype cells. ERK1/2 phosphorylation peaked in wildtype cells at around 10min, and in all FA-deficient cells at around 5min. The relative change in strain energy of FA-deficient cells compared to wildtype cells was of the order: vinculin>FAK>p130Cas. Taken together, FAK and p130Cas are more important in the stretch-mediated downstream signaling and cell survival pathway, while vinculin is more critical in maintaining cell contractility.

Combining Dynamic Stretch and Tunable Stiffness to Probe Cell Mechanobiology In Vitro

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.

A Transition Model for Finite Element Simulation of Kinematics of Central Nervous System White Matter

Mechanical damage to axons is a proximal cause of deficits following traumatic brain injury and spinal cord injury. Axons are injured predominantly by tensile strain, and identifying the strain experienced by axons is a critical step toward injury prevention. White matter demonstrates complex nonlinear mechanical behavior at the continuum level that evolves from even more complex, dynamic, and composite behavior between axons and the "glial matrix" at the microlevel. In situ, axons maintain an undulated state that depends on the location of the white matter and the stage of neurodevelopment. When exposed to tissue strain, axons do not demonstrate pure affine or non-affine behavior, but instead transition from non-affine-dominated kinematics at low stretch levels to affine kinematics at high stretch levels. This transitional and predominant kinematic behavior has been linked to the natural coupling of axons to each other via the glial matrix. In this paper, a transitional kinematic model is applied to a micromechanics finite element model to simulate the axonal behavior within a white matter tissue subjected to uniaxial tensile stretch. The effects of the transition parameters and the volume fraction of axons on axonal behavior is evaluated and compared to previous experimental data and numerical simulations.

Effects of stress fiber contractility on uniaxial stretch guiding mitosis orientation and stress fiber alignment

It has been documented that mitosis orientation (MO) is guided by stress fibers (SFs), which are perpendicular to exogenous cyclic uniaxial stretch. However, the effect of mechanical forces on MO and the mechanism of stretch-induced SFs reorientation are not well elucidated to date. In the present study, we used murine 3T3 fibroblasts as a model, to investigate the effects of uniaxial stretch on SFO and MO utilizing custom-made stretch device. We found that cyclic uniaxial stretch induced both SFs and mitosis directions orienting perpendicularly to the stretch direction. The F-actin and myosin II blockages, which resulted in disoriented SFs and mitosis directions under uniaxial stretch, suggested a high correlation between SFO and MO. Y27632 (10μM), ML7 (50μM, or 75μM), and blebbistatin (50μM, or 75μM) treatments resulted in SFO parallel to the principle stretch direction. Upon stimulating and inhibiting the phosphorylation of myosin light chain (p-MLC), we observed a monotonic proportion of SFO to the level of p-MLC. These results suggested that the level of cell contraction is crucial to the response of SFs, either perpendicular or parallel, to the external stretch. Showing the possible role of cell contractility in tuning SFO under external stretch, our experimental data are valuable to understand the predominant factor controlling SFO response to exogenous uniaxial stretch, and thus helpful for improving mechanical models.

Chronic oscillatory strain induces MLCK associated rapid recovery from acute stretch in airway smooth muscle cells

A deep inspiration (DI) temporarily relaxes agonist-constricted airways in normal subjects, but in asthma airways are refractory and may rapidly renarrow, possibly due to changes in the structure and function of airway smooth muscle (ASM). Chronic largely uniaxial cyclic strain of ASM cells in culture causes several structural and functional changes in ASM similar to that in asthma, including increases in contractility, MLCK content, shortening velocity, and shortening capacity. However, changes in recovery from acute stretch similar to a DI have not been measured. We have therefore measured the response and recovery to large stretches of cells modified by chronic stretching and investigated the role of MLCK. Chronic, 10% uniaxial cyclic stretch, with or without a strain gradient, was administered for up to 11 days to cultured cells grown on Silastic membranes. Single cells were then removed from the membrane and subjected to 1 Hz oscillatory stretches up to 10% of the in situ cell length. These oscillations reduced stiffness by 66% in all groups (P < 0.05). Chronically strained cells recovered stiffness three times more rapidly than unstrained cells, while the strain gradient had no effect. The stiffness recovery in unstrained cells was completely inhibited by the MLCK inhibitor ML-7, but recovery in strained cells exhibiting increased MLCK was slightly inhibited. These data suggest that chronic strain leads to enhanced recovery from acute stretch, which may be attributable to the strain-induced increases in MLCK. This may also explain in part the more rapid renarrowing of activated airways following DI in asthma.

Oriented Morphology and Anisotropic Transport in Uniaxially Stretched Perfluorosulfonate Ionomer Membranes

Relations between morphology and transport sensitively govern proton conductivity in perfluorsulfonate ionomers (PFSIs) and thus determine useful properties of these technologically important materials. In order to understand such relations, we have conducted a broad systematic study of H+-form PFSI membranes over a range of uniaxial extensions and water uptakes. On the basis of small-angle X-ray scattering (SAXS) and 2H NMR spectroscopy, uniaxial deformation induces a strong alignment of ionic domains along the stretching direction. We correlate ionic domain orientation to transport using pulsed-field-gradient 1H NMR measurements of water diffusion coefficients along the three orthogonal membrane directions. Intriguingly, we observe that uniaxial deformation enhances water transport in one direction (parallel-to-draw direction) while reducing it in the other two directions (two orthogonal directions relative to the stretching direction). We evaluate another important transport parameter, proton conductiv...

Angiotensin II induces microtubule reorganization mediated by a deacetylase SIRT2 in endothelial cells

Angiotensin II has been implicated in vascular remodeling. Microtubule composed of tubulins regulates cell shape, migration and survival. Tubulin acetylation has an important role in the control of microtubule structure and microtubule-based cellular functions. In this study, angiotensin II induced disassembly and deacetylation of α-tubulin, which were blocked by pretreatment with an angiotensin II type 1 receptor blocker losartan and a sirtuin class deacetylase inhibitor sirtinol, and by depletion of a deacetylase SIRT2 using RNA interference. We investigated the involvement of SIRT2 in angiotensin II-induced endothelial cell migration using the Boyden chamber method. Angiotensin II caused a significant increase in cell migration, which was blocked by pretreatment with sirtinol and SIRT2 depletion. It has been reported that angiotensin II is involved in cytoskeletal reorganization stimulated by mechanical stretch in endothelial cells. We also demonstrated that endothelial cells subjected to a 10% uniaxial stretch showed vertical alignment to the direction of tension and tubulin deacetylation in the peripheral side of cells, in comparison with control static cells. The mechanical stretch-induced changes of microtubules were blocked by pretreatment with sirtinol and SIRT2 depletion. Immunofluorescence microscopy showed that acetylated tubulin was decreased in platelet-endothelial cell adhesion molecule-1-positive cells in the intima of the aortic walls in mice loaded with angiotensin II, in comparison with mice loaded with control vehicle. These data show that angiotensin II and mechanical stretch stimulate microtubule redistribution and deacetylation via SIRT2 in endothelial cells, suggesting the emerging role of SIRT2 in hypertension-induced vascular remodeling.

Temperature Fall and Rise of Plastics During Uniaxial Stretching

Dumbbell specimens of common plastics—polyethylene, polypropylene, and polyamide—were uniaxially stretched and the surface temperature was measured by thermography. The surface temperature decreased at small strain below the yield point and then increased at larger strain. The endothermic deformation in the elastic regime was unexpected. It might be a characteristic of polymer material, which is possessed of free volume. The endotherm is interpreted by the volume increment with stretching as represented by Poisson's ratio. The temperature rise at larger strain is not surprising for plastic deformation over the yield point. The exotherm is interpreted in terms of the melting of crystallites and re-crystallization induced by large deformation.

The Effect of Uniaxial Stretch on the Tensile Properties and Collagen Content of Tissue-Engineered Ligaments

The Effect of Uniaxial Stretch on the Tensile Properties and Collagen Content of Tissue-Engineered Ligaments

Real-Time Structure Changes during Uniaxial Stretching of Poly(ω-pentadecalactone) by in Situ Synchrotron WAXD/SAXS Techniques

Poly(ω-pentadecalactone) (PPDL), a model polymer in the poly(ω-hydroxyl fatty acids) family, is a new biopolymer with monomer synthesized by yeast-catalyzed ω-hydroxylation of fatty acids. In this study, deformation-induced structural changes in two PPDL samples with different molecular weights were studied by in situ wide-angle X-ray diffraction (WAXD) and small-angle X-ray scattering (SAXS) techniques. The high molecular weight PPDL (PPDL-high) sample exhibited notable strain hardening, while the low molecular weight PPDL (PPDL-low) sample did not. The behavior can be explained by the entanglement density concept. The evolution of crystallinity (from WAXD) as a function of strain could be divided into four distinct regions, but their respective mechanisms differ slightly in each sample. During stretching, a mesomorphic phase formed in both samples, bridging between the amorphous and strain-induced crystal phases. The SAXS data verified the effect of molecular weight (or the entanglement density) on the deformation-induced structure of PPDL. The parameters of chain orientation factor (f) calculated from the orthorhombic crystal cell as well as the nonorthorhombic crystal cell proposed by Wilchinsky were used to follow the orientation process during stretching of PPDLs. It was found that the different molecular entanglement network (i.e., PPDL-low versus PPDL-high) led to different crystal orientation behavior, especially in the low strain range.

Finite amplitude, horizontal motion of a load symmetrically supported between isotropic hyperelastic springs

The undamped, finite amplitude horizontal motion of a load supported symmetrically between identical incompressible, isotropic hyperelastic springs, each subjected to an initial finite uniaxial static stretch, is formulated in general terms. The small amplitude motion of the load about the deformed static state is discussed; and the periodicity of the arbitrary finite amplitude motion is established for all such elastic materials for which certain conditions on the engineering stress and the strain energy function hold. The exact solution for the finite vibration of the load is then derived for the classical neo-Hookean model. The vibrational period is obtained in terms of the complete Heuman lambda-function whose properties are well-known. Dependence of the period and hence the frequency on the physical parameters of the system is investigated and the results are displayed graphically.

Biophysical Regulation of Histone Acetylation in Mesenchymal Stem Cells

Histone deacetylation and acetylation are catalyzed by histone deacetylase (HDAC) and histone acetyltransferase, respectively, which play important roles in the regulation of chromatin remodeling, gene expression, and cell functions. However, whether and how biophysical cues modulate HDAC activity and histone acetylation is not well understood. Here, we tested the hypothesis that microtopographic patterning and mechanical strain on the substrate regulate nuclear shape, HDAC activity, and histone acetylation. Bone marrow mesenchymal stem cells (MSCs) were cultured on elastic membranes patterned with parallel microgrooves 10 μm wide that kept MSCs aligned along the axis of the grooves. Compared with MSCs on an unpatterned substrate, MSCs on microgrooves had elongated nuclear shape, a decrease in HDAC activity, and an increase of histone acetylation. To investigate anisotropic mechanical sensing by MSCs, cells on the elastic micropatterned membranes were subjected to static uniaxial mechanical compression or stretch in the direction parallel or perpendicular to the microgrooves. Among the four types of loads, compression or stretch perpendicular to the microgrooves caused a decrease in HDAC activity, accompanied by the increase in histone acetylation and slight changes of nuclear shape. Knocking down nuclear matrix protein lamin A/C abolished mechanical strain-induced changes in HDAC activity. These results demonstrate that micropattern and mechanical strain on the substrate can modulate nuclear shape, HDAC activity, and histone acetylation in an anisotropic manner and that nuclear matrix mediates mechanotransduction. These findings reveal a new mechanism, to our knowledge, by which extracellular biophysical signals are translated into biochemical signaling events in the nucleus, and they will have significant impact in the area of mechanobiology and mechanotransduction.

Real-time PCR analysis of mechanical strain and BMPs in human periosteal cells: an in vitro model of entheseal stress

Background and objectivesEnthesitis with new cartilage and bone formation, which leads to joint ankylosis, is a hallmark of spondyloarthritis (SpA). Ankylosis is thought to occur initially in entheseal sites, however,...

Differentiation of Embryonic Stem Cells into Cardiomyocytes in a Compliant Microfluidic System

The differentiation process of murine embryonic stem cells into cardiomyocytes was investigated with a compliant microfluidic platform which allows for versatile cell seeding arrangements, optical observation access, long-term cell viability, and programmable uniaxial cyclic stretch. Specifically, two environmental cues were examined with this platform--culture dimensions and uniaxial cyclic stretch. First, the cardiomyogenic differentiation process, assessed by a GFP reporter driven by the α-MHC promoter, was enhanced in microfluidic devices (µFDs) compared with conventional well-plates. The addition of BMP-2 neutralizing antibody reduced the enhancement observed in the µFDs and the addition of exogenous BMP-2 augmented the cardiomyogenic differentiation in well plates. Second, 24 h of uniaxial cyclic stretch at 1 Hz and 10% strain on day 9 of differentiation was found to have a negative impact on cardiomyogenic differentiation. This microfluidic platform builds upon an existing design and extends its capability to test cellular responses to mechanical strain. It provides capabilities not found in other systems for studying differentiation, such as seeding embryoid bodies in 2D or 3D in combination with cyclic strain. This study demonstrates that the microfluidic system contributes to enhanced cardiomyogenic differentiation and may be a superior platform compared with conventional well plates. In addition to studying the effect of cyclic stretch on cardiomyogenic differentiation, this compliant platform can also be applied to investigate other biological mechanisms.