Uniaxial Cyclic Stretch Research Articles

Bioreactor development for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain: proof of concept

Skeletal muscles overcome terrestrial, gravitational loading by producing tensile forces that produce movement through joint rotation. Conversely, the microgravity of spaceflight reduces tensile loads in working skeletal muscles, causing an adaptive muscle atrophy. Unfortunately, the design of stable, physiological bioreactors to model skeletal muscle tensile loading during spaceflight experiments remains challenging. Here, we tested a bioreactor that uses initiation and cessation of cyclic, tensile strain to induce hypertrophy and atrophy, respectively, in murine lineage (C2C12) skeletal muscle myotubes. Uniaxial cyclic stretch of myotubes was conducted using a StrexCell® (STB-1400) stepper motor system (0.75 Hz, 12% strain, 60 min day^-1). Myotube groups were assigned as follows: (a) quiescent over 2- or (b) 5-day (no stretch), (c) experienced 2-days (2dHY) or (d) 5-days (5dHY) of cyclic stretch, or (e) 2-days of cyclic stretch followed by a 3-day cessation of stretch (3dAT). Using ß-sarcoglycan as a sarcolemmal marker, mean myotube diameter increased significantly following 2dAT (51%) and 5dAT (94%) vs. matched controls. The hypertrophic, anabolic markers talin and Akt phosphorylation (Thr308) were elevated with 2dHY but not in 3dAT myotubes. Inflammatory, catabolic markers IL-1ß, IL6, and NF-kappaB p65 subunit were significantly higher in the 3dAT group vs. all other groups. The ratio of phosphorylated FoxO3a/total FoxO3a was significantly lower in 3dAT than in the 2dHY group, consistent with elevated catabolic signaling during unloading. In summary, we demonstrated proof-of-concept for a spaceflight research bioreactor, using uniaxial cyclic stretch to produce myotube hypertrophy with increased tensile loading, and myotube atrophy with subsequent cessation of stretch.

Optimized Biomanufacturing for Treatment of Volumetric Muscle Loss Enables Physiomimetic Recovery.

Volumetric muscle loss (VML) injuries are defined by loss of sufficient skeletal muscle to produce persistent deficits in muscle form and function, with devastating lifelong consequences to both soldiers and civilians. There are currently no satisfactory treatments for VML injuries. The work described herein details the implementation of a fully enclosed bioreactor environment (FEBE) system that efficiently interfaces with our existing automated bioprinting and advanced biomanufacturing methods for cell deposition on sheet-based scaffolds for our previously described tissue-engineered muscle repair (TEMR) technology platform. Briefly, the TEMR technology consists of a porcine bladder acellular matrix seeded with skeletal muscle progenitor cells and preconditioned via 10% uniaxial cyclic stretch in a bioreactor. Overall, TEMR implantation in an established rat tibialis anterior (TA) VML injury model can result in 60 to ∼90% functional recovery. However, our original study documented >50% failure rate. That is, more than half of the implanted TEMR constructs produced no functional improvement beyond no treatment/repair. The high failure rate was attributed to the untoward mechanical disruption of TEMR during surgical implantation. In a follow-up study, adjustments were made to the geometry of both the VML injury and the TEMR construct, and the "nonresponder" group was reduced from over half the TEMR-treated animals to just 33%. Nonetheless, additional improvement is needed for clinical applicability. The main objectives of the current study were twofold: (1) explore the use of advanced biomanufacturing methods (i.e., FEBE bioreactor) to further improve TEMR reliability (i.e., increase functional response rate), (2) determine if previously established bioprinting methods, when coupled to the customized FEBE system would further improve the rate, magnitude or amplitude of functional outcomes following TEMR implantation in the same rat TA VML injury model. The current study demonstrates the unequivocal benefits of a customized bioreactor system that reduces manipulation of TEMR during cell seeding and maturation via bioprinting while simultaneously maximizing TEMR stability throughout the biofabrication process. This new biomanufacturing strategy not only accelerated the rate of functional recovery, but also eliminated all TEMR failures. In addition, implementation of bioprinting resulted in more physiomimetic skeletal muscle characteristics of repaired muscle tissue.

Vascular Smooth Muscle Cell Mechanical Stretch Modulates Tissue Transglutaminase Activity and Cytoskeletal Dynamics

Vascular smooth muscle cells (VSMCs) are exposed to a wide range of mechanical stimuli in both physiological and pathological conditions. These mechanical stimuli are converted into biochemical signals that regulate VSMC contractility and cytoskeletal structures in a process known as mechanotransduction. Thus, the ability of VSMCs to sense and respond to mechanical signals is essential for proper regulation of their structural characteristics and the modulation of vascular tone. We had previously shown that increased tissue transglutaminase 2 (TG2) activity modulates actin dynamics via alterations of the phosphorylation state of cofilin leading to VSMC stiffening and remodeling of the vascular wall. However, whether mechanical forces act through TG2 to modulate VSMC stiffness remains unknown. Herein, we tested the hypothesis that mechanical stretch increases VSMC stiffness by increasing TG2 activity, reducing cofilin activity, and increasing actin cytoskeletal stress fiber formation. To this end, immortalized human aortic vascular smooth muscle cells (HAoSMCs) were cultured and seeded on Uniflex culture plates. HAoSMCs were left unstretched or exposed to cyclic uniaxial stretch using a Flexcell tension system with 11% elongation at 1 Hz for 6 hrs. HAoSMCs were treated without or with the nitric oxide (NO) donor s-nitrosoglutathione (400μmol/L) or cystamine (200μmol/L) under stretched and unstretched conditions, as NO and cystamine are known inhibitors of TG2. All differences reported herein are significant at p<0.05. Our results show that cyclic uniaxial stretch for 6 hrs increases VSMC TG2 content and activity, actin stress fibers (polymerization), and phospho-cofilin/cofilin ratios (cofilin inactivation), relative to the unstretched condition. Notably, the increase in TG2 activity and F-actin content occurring in response to stretch was inhibited by the NO donor or cystamine. As it has been previously shown that cellular stiffness is associated with stress fiber formation, this work suggests that stretch-induced cellular stiffening is potentially mediated by TG2 activity in VSMC. National Institutes of Health grant: R01HL153264 to LM-L and JP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Uniaxial cyclic stretch enhances osteogenic differentiation of OPLL-derived primary cells via YAP-Wnt/β-catenin axis.

The pathogenesis of posterior longitudinal ligament ossification (OPLL) remains inadequately understood. Mechanical stimulation is one of the important pathogenic factors in OPLL. As one of the mechanical stimulation transduction signals, the yes-associated protein (YAP) interacts with the Wnt/β-catenin signalling pathway, which plays an important role in osteogenic differentiation. This study aimed to demonstrate the role of YAP-Wnt/β-catenin axis in cell differentiation induced by mechanical stress. Primary cells extracted from posterior longitudinal ligament tissues from OPLL or non-OPLL patients were subjected to sinusoidal uniaxial cyclic stretch (5 %, 0.5 Hz, 3 d). The expression of runt-related transcription factor 2, collagen I, osterix, osteocalcin and alkaline phosphatase were compared between the static and the experimental groups. In addition, the cytoskeleton was detected using phalloidin staining while YAP phosphorylation states and nuclear location were identified using immunofluorescence. The results showed that mechanical stretching loading increased the expression of osteogenic genes and proteins in the OPLL group, while it had no significant effect on the control group. When OPLL cells were stretched, YAP exhibited an obvious nuclear translocation and the Wnt/β-catenin pathway was activated. Knocking down YAP or β-catenin could weaken the impact upon osteogenic differentiation induced by mechanical stimulation. YAP-mediated mechanical stimulation promoted osteogenic differentiation of OPLL cells through Wnt/β-catenin pathway and this progress was independent of the Hippo pathway.

Nuclear pore complexes concentrate on Actin/LINC/Lamin nuclear lines in response to mechanical stress in a SUN1 dependent manner

Formation of robust actomyosin stress fibers (SF) in response to cell stretch plays a key role in the transfer of information from the cytoplasm into the nucleus. Actin/LINC/Lamin (ALL) nuclear lines provide mechanical linkage between the actin cytoskeleton and the lamin nucleoskeleton across the nuclear envelope. To understand the establishment of ALL lines, we used live cell imaging of cells exposed to cyclic stretch. We discovered that nuclear pore complexes (NPCs) concentrate along ALL lines that are generated in response to uniaxial cyclic stretch. The ALL-associated NPCs display increased fluorescence intensity of nucleoporins Pom121, TPR and Nup153 relative to nucleoporins that are distal to the ALL lines. Here we test the hypothesis that a LINC complex component of ALL lines, SUN1 is involved in the integration of NPCs with ALL lines. We generated CRISPR SUN1 knockdown and knockout cell lines and show that SUN1 is essential for normal integration of NPCs to ALL lines. Loss or elimination of SUN1 significantly diminishes NPC/ALL line integration, demonstrating a key role for SUN1 in the recruitment or stabilization of NPCs to a discrete subdomain of the nuclear envelope at ALL lines. This work provides new insight into the mechanism by which cells respond to mechanical force through nuclear envelope remodeling.

MFN2 Prevents Neointimal Hyperplasia in Vein Grafts via Destabilizing PFK1.

Mechanical forces play crucial roles in neointimal hyperplasia after vein grafting; yet, our understanding of their influences on vascular smooth muscle cell (VSMC) activation remains rudimentary. A cuff mouse model was used to study vein graft hyperplasia. Fifteen percent to 1 Hz uniaxial cyclic stretch (arterial strain), 5% to 1 Hz uniaxial cyclic stretch or a static condition (venous strain) were applied to the cultured VSMCs. Metabolomics analysis, cell proliferation and migration assays, immunoblotting, co-immunoprecipitation, mutagenesis, pull-down and surface plasmon resonance assays were employed to elucidate the potential molecular mechanisms. RNA-sequencing in vein grafts and the controls identified changes in metabolic pathways and downregulation of mitochondrial protein MFN2 (mitofusin 2) in the vein grafts. Exposure of VSMCs to 15% stretch resulted in MFN2 downregulation, mitochondrial fragmentation, metabolic shift from mitochondrial oxidative phosphorylation to glycolysis, and cell proliferation and migration, as compared with that to a static condition or 5% stretch. Metabolomics analysis indicated an increased generation of fructose 1,6-bisphosphate, an intermediate in the glycolytic pathway converted by PFK1 (phosphofructokinase 1) from fructose-6-phosphate, in cells exposed to 15% stretch. Mechanistic study revealed that MFN2 physically interacts through its C-terminus with PFK1. MFN2 knockdown or exposure of cells to 15% stretch promoted stabilization of PFK1, likely through interfering the association between PFK1 and the E3 ubiquitin ligase TRIM21 (E3 ubiquitin ligase tripartite motif [TRIM]-containing protein 21), thus, decreasing the ubiquitin-protease-dependent PFK1 degradation. In addition, study of mechanotransduction utilizing pharmaceutical inhibition indicated that the MFN2 downregulation by 15% stretch was dependent on inactivation of the SP1 (specificity protein 1) and activation of the JNK (c-Jun N-terminal kinase) and ROCK (Rho-associated protein kinase). Adenovirus-mediated MFN2 overexpression or pharmaceutical inhibition of PFK1 suppressed the 15% stretch-induced VSMC proliferation and migration and alleviated neointimal hyperplasia in vein grafts. MFN2 is a mechanoresponsive protein that interacts with PFK1 to mediate PFK1 degradation and therefore suppresses glycolysis in VSMCs.

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 inNIH 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.

Controlled Growth Factor Delivery and Cyclic Stretch Induces a Smooth Muscle Cell-like Phenotype in Adipose-Derived Stem Cells

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-β1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology.

Combination of parathyroid hormone pretreatment and mechanical stretch promotes osteogenesis of periodontal ligament fibroblasts

Parathyroid hormone (PTH) potentiates the mechanical loading induced bone formation in fracture healing and orthodontics. This study aimed to gain insight into the underlying mechanisms in periodontal ligament fibroblasts (PDLFs). Human PDLFs were cultured and subjected to uniaxial cyclic stretch at 0.5Hz and 2000μ for 0, 6, 12, and 24hours, respectively. 10nM PTH was preadministered for 30minutes before loading. The expression of PTH1R and osteogenic biomarkers Runx2, osteopontin, collagen type 1, alkaline phosphatase was assessed via immunofluorescence staining, quantitative polymerase chain reaction, or Western blot. Transfection of siPTH1R was applied, and alterations of osteogenic biomarkers were examined by Western blot. The expression of essential Wnt signal components Wnt3a, β-catenin, low-density lipoprotein receptor-related protein 5, Wnt5a, receptor tyrosine kinase-like orphan receptor 2 were examined, and the influence of dickkopf-related protein 1 on osteogenic biomarkers was evaluated. The expression of PTH1R was instantaneously upregulated with PTH pretreatment and maintained a gradual increase until 24hours. PTH synergistically enhanced the increase of Runx2, osteopontin, collagen type 1, and alkaline phosphatase under cyclic stretch, which was substantially attenuated by siPTH1R transfection. As for Wnt signal components, synergistic upregulation was detected on Wnt3a, β-catenin, and low-density lipoprotein receptor-related protein 5, whereas Wnt5a and receptor tyrosine kinase-like orphan receptor 2 increased relatively mildly. Blockage of the canonical Wnt/β-catenin pathway by dickkopf-related protein 1 impaired the boost of osteogenic biomarkers under the combined action of PTH and cyclic stretch. The combination of PTH pretreatment and cyclic stretch promotes osteogenesis of PDLFs synergistically, and the canonical Wnt/β-catenin pathway is crucially involved in the underlying mechanism.

Crosstalk Between CD11b and Piezo1 Mediates Macrophage Responses to Mechanical Cues.

Macrophages are versatile cells of the innate immune system that perform diverse functions by responding to dynamic changes in their microenvironment. While the effects of soluble cues, including cytokines and chemokines, have been widely studied, the effects of physical cues, including mechanical stimuli, in regulating macrophage form and function are less well understood. In this study, we examined the effects of static and cyclic uniaxial stretch on macrophage inflammatory and healing activation. We found that cyclic stretch altered macrophage morphology and responses to IFNγ/LPS and IL4/IL13. Interestingly, we found that both static and cyclic stretch suppressed IFNγ/LPS induced inflammation. In contrast, IL4/IL13 mediated healing responses were suppressed with cyclic but enhanced with static stretch conditions. Mechanistically, both static and cyclic stretch increased expression of the integrin CD11b (αM integrin), decreased expression of the mechanosensitive ion channel Piezo1, and knock down of either CD11b or Piezo1 through siRNA abrogated stretch-mediated changes in inflammatory responses. Moreover, we found that knock down of CD11b enhanced the expression of Piezo1, and conversely knock down of Piezo1 enhanced CD11b expression, suggesting the potential for crosstalk between integrins and ion channels. Finally, stretch-mediated differences in macrophage activation were also dependent on actin, since pharmacological inhibition of actin polymerization abrogated the changes in activation with stretch. Together, this study demonstrates that the physical environment synergizes with biochemical cues to regulate macrophage morphology and function, and suggests a role for CD11b and Piezo1 crosstalk in mechanotransduction in macrophages.

Electrical performance evolution and fatigue mechanisms of silver-filled polymer ink under uniaxial cyclic stretch

Flexible hybrid electronics rely upon compliant interconnects in order to maintain performance integrity in cases that require repeated elongation, including repeated stretching. A class of such conductive interconnects are composites of polymer with conductive particles that can be stretched at high strains without circuit failure. However, their fatigue response has so far remained largely unexplored and is essential prior to using in health monitoring applications. In this research, a stretchable silver-filled conductor is evaluated under high-strain cycling. In-situ techniques, including four-point resistance measurement and laser profilometry, are used to correlate changes in electrical performance to the fatigue response. Surface crack formation is extensive upon stretching during the first loading cycle, forming a heavily interconnected crack network at higher strains that does not immediately result in open circuit failure. Resistance increase with cycling is attributed to a gradual deepening of these cracks until their depths approach the film thickness, eventually leading to electrical failure. Fatigue life, the number of cycles required to reach a predetermined electrical performance limit, is shown to be most influenced by the applied strain amplitude. Using a normalized resistance increase limit of R/R 0 = 500, it is found that 500 μm wide conductive lines endure 23 cycles at 35% strain amplitude, but this becomes over 500 cycles when the amplitude is dropped to 5%. Sensitivity to mean strain, ϵ m, is relevant to strain amplitudes below 15%. In this manner, a composite conductor was shown to exhibit crack evolution behavior distinctly different from homogeneous metallic films.

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.

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.

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.

Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue.

In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs.

As the endothelial cell reorients, its tensile forces stabilize

When adherent cells are subjected to uniaxial sinusoidal stretch at frequencies close to physiological, their body and their contractile stress fibers realign nearly perpendicularly to the stretch axis. A common explanation for this phenomenon is that stress fibers reorient along the direction where they are unaffected by the applied cyclic stretch and thus can maintain optimal (homeostatic) tensile force. The ability of cells to achieve tensional homeostasis in response to external disturbances is important for normal physiological functions of cells and tissues and it provides protection against diseases, including cancer and atherosclerosis. However, quantitative experimental data that support the idea that stretch-induced reorientation is associated with tensional homeostasis are lacking. We observed previously that in response to uniaxial cyclic stretch of 10% strain amplitudes, traction forces of single endothelial cells reorient in the direction perpendicular to the stretch axis. Here we carried out a secondary analysis of those data to investigate whether this reorientation of traction forces is associated with tensional homeostasis. Our analysis showed that stretch-induced reorientation of traction forces was accompanied by attenuation of temporal variability of the traction field to the level that was observed in the absence of stretch. These findings represent a quantitative experimental evidence that stretch-induced reorientation of cellular traction forces is associated with the cell’s tendency to achieve tensional homeostasis.

Cyclic uniaxial mechanical stretching of cells using a LEGO® parts-based mechanical stretcher system.

Mechanical cues are essential for the regulation of cell and tissue physiology. Hence, it has become an utmost necessity for cell biologists to account for those mechanical parameters when investigating biological processes and they need devices to manipulate cells accordingly. Here, we report a simple mechanical cell-stretching system that can generate uniaxial cyclic mechanical stretch on cells in tissue culture. This system is based upon a low-cost battery-powered uniaxial cyclic mechanical stretcher exclusively built out of LEGO® parts combined with a stretchable poly(dimethylsiloxane) tissue culture plate in order to grow and stretch cells. We characterize the system and show that it can be used in a wide variety of downstream applications, including immunofluorescence, western blotting and biochemical assays. We also illustrate how this system can be useful in a study as we investigated the behavior of integrin adhesion complexes upon cell stretching. We therefore present a cost-effective, multipurpose cell-stretching system that should help to increase understanding of mechanical signaling.This article has an associated First Person interview with the first author of the paper.

Combination of Mechanical and Chemical Methods Improves Gene Delivery in Cell-based HIV Vaccines.

Novel vaccination approaches are required to control human immunodeficiency virus (HIV) infections. The membrane proximal external region (MPER) of Env gp41 subunit and the V3/glycans of Env gp120 subunit were known as potential antigenic targets for anti-HIV-1 vaccines. In this study, we prepared the modified dendritic cells (DCs) and mesenchymal stem cells (MSCs) with HIV-1 MPER-V3 gene using mechanical and chemical approaches. At first, MPER-V3 fusion DNA delivery was optimized in dendritic cells (DCs) and mesenchymal stem cells (MSCs) using three mechanical (i.e., uniaxial cyclic stretch, equiaxial cyclic stretch and shear stress bioreactors), and two chemical (i.e., TurboFect or Lipofectamine) methods. Next, the modified DCs and MSCs with MPER-V3 antigen were compared to induce immune responses in vivo. Our data showed that the combination of equiaxial cyclic stretch loading and lipofectamine twice with 48 h intervals increased the efficiency of transfection about 60.21 ± 1.05 % and 65.06 ± 0.09 % for MSCs and DCs, respectively. Moreover, DCs and MSCs transfected with MPER-V3 DNA in heterologous DC or MSC prime/ peptide boost immunizations induced high levels of IgG2a, IgG2b, IFN-γ and IL-10 directed toward Th1 responses as well as an increased level of Granzyme B. Indeed, the modified MSCs and DCs with MPER-V3 DNA could significantly enhance the MPER/V3-specific T-cell responses compared to MPER/V3 peptide immunization. These findings showed that the modified MSC-based immunization could elicit effective immune responses against HIV antigen similar to the modified DC-based immunization.

Cyclic stretch-induced mechanical stress to the cell nucleus inhibits ultraviolet radiation-induced DNA damage.

Ultraviolet (UV) radiation exerts adverse effects on genome stability, alters the normal state of life, and causes several diseases by inducing DNA damage. Although mechanical stimulation such as stretching has significant effects on the prevention and treatment of diseases, its influence on nuclear morphology and/or intranuclear functions involving resistance to DNA damage remains unknown. Here, we investigated the effects of mechanical stimulation by cyclic stretching on nuclear morphology and resistance of DNA to UV damage in NIH3T3 fibroblasts. Adherent cells on silicone elastic membranes were subjected to ~ 10% cyclic uniaxial stretch at a frequency of 0.5Hz for 12h. As a result, the intracellular actin cytoskeleton and nucleus were found to be elongated and aligned in the direction of zero normal strain (~ 62° with respect to the stretch direction) in an actomyosin tension-dependent manner. The nuclei of the stretched cells were dramatically compressed by the reorganized actin stress fibers located on their apical and both sides, and a significant increase in the intranuclear DNA density was observed. Intercellular tension, as assessed with live cell atomic force microscopy imaging, also increased following exposure to cyclic stretch. The UV radiation-induced DNA damage, estimated from the fluorescence intensity of the phospho-histone γ-H2AX, significantly decreased in these stretched cells. These results indicate that the cyclic stretch-induced morphological changes in the nucleus may improve the UV radiation resistance of cells, probably owing to the intracellular force-induced condensation of chromatin. To our knowledge, this is the first study to demonstrate the inhibition of the UV radiation-induced DNA damage by mechanical stimulation.