YAP mechanotransduction under cyclic mechanical stretch loading for mesenchymal stem cell osteogenesis is regulated by ROCK.

While yes-associated protein (YAP) is now recognized as a potent mechanosensitive transcriptional regulator to affect cell growth and differentiation including the osteogenic transcription of mesenchymal stem cells (MSCs), most studies have reported the YAP mechanosensing of static mechanophysical cues such as substrate stiffness. We tested MSC response to dynamic loading, i.e., cyclic mechanical stretching, and assessed YAP mechanosensing and resultant MSC osteogenesis. We showed that cyclic stretching at 10% strain and 1 Hz frequency triggered YAP nuclear import in MSCs. YAP phosphorylation at S127 and S397, which is required for YAP cytoplasmic retention, was suppressed by cyclic stretch. We also observed that anti-YAP-regulatory Hippo pathway, LATS phosphorylation, was significantly decreased by stretch. We confirmed the stretch induction of MSC osteogenic transcription and differentiation, and this was impaired under YAP siRNA suggesting a key role of YAP dynamic mechanosensing in MSC osteogenesis. As an underlying mechanism, we showed that the YAP nuclear transport by cyclic stretch was abrogated by ROCK inhibitor, Y27632. ROCK inhibitor also impaired the stretch induction of F-actin formation and MSC osteogenesis, thus implicating the role of the ROCK-F-actin cascade in stretch-YAP dynamic mechanosensing-MSC osteogenesis. Our results provide insight into bone tissue engineering and skeletal regenerative capacity of MSCs especially as regards the role of dynamic mechanical loading control of YAP-mediated MSC osteogenic transcription.

The Role of Fluid Shear and Metastatic Potential in Breast Cancer Cell Migration.

During the migration of cancer cells for metastasis, cancer cells can be exposed to fluid shear conditions. We examined two breast cancer cell lines, MDA-MB-468 (less metastatic) and MDA-MB-231 (more metastatic), and a benign MCF-10A epithelial cell line for their responsiveness in migration to fluid shear. We tested fluid shear at 15 dyne/cm2 that can be encountered during breast cancer cells traveling through blood vessels or metastasizing to mechanically active tissues such as bone. MCF-10A exhibited the least migration with a trend of migrating in the flow direction. Intriguingly, fluid shear played a potent role as a trigger for MDA-MB-231 cell migration, inducing directional migration along the flow with significantly increased displacement length and migration speed and decreased arrest coefficient relative to unflowed MDA-MB-231. In contrast, MDA-MB-468 cells were markedly less migratory than MDA-MB-231 cells, and responded very poorly to fluid shear. As a result, MDA-MB-468 cells did not exhibit noticeable difference in migration between static and flow conditions, as was distinct in root-mean-square (RMS) displacement-an ensemble average of all participating cells. These may suggest that the difference between more metastatic MDA-MB-231 and less metastatic MDA-MB-468 breast cancer cells could be at least partly involved with their differential responsiveness to fluid shear stimulatory cues. Our study provides new data in regard to potential crosstalk between fluid shear and metastatic potential in mediating breast cancer cell migration.

The Role of Microenvironmental Cues and Mechanical Loading Milieus in Breast Cancer Cell Progression and Metastasis.

Cancer can disrupt the microenvironments and mechanical homeostatic actions in multiple scales from large tissue modification to altered cellular signaling pathway in mechanotransduction. In this review, we highlight recent progresses in breast cancer cell mechanobiology focusing on cell-microenvironment interaction and mechanical loading regulation of cells. First, the effects of microenvironmental cues on breast cancer cell progression and metastasis will be reviewed with respect to substrate stiffness, chemical/topographic substrate patterning, and 2D vs. 3D cultures. Then, the role of mechanical loading situations such as tensile stretch, compression, and flow-induced shear will be discussed in relation to breast cancer cell mechanobiology and metastasis prevention. Ultimately, the substrate microenvironment and mechanical signal will work together to control cancer cell progression and metastasis. The discussions on breast cancer cell responsiveness to mechanical signals, from static substrate and dynamic loading, and the mechanotransduction pathways involved will facilitate interdisciplinary knowledge transfer, enabling further insights into prognostic markers, mechanically mediated metastasis pathways for therapeutic targets, and model systems required to advance cancer mechanobiology.

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation.

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

The LINC complex, mechanotransduction, and mesenchymal stem cell function and fate.

Mesenchymal stem cells (MSCs) show tremendous promise as a cell source for tissue engineering and regenerative medicine, and are understood to be mechanosensitive to external mechanical environments. In recent years, increasing evidence points to nuclear envelope proteins as a key player in sensing and relaying mechanical signals in MSCs to modulate cellular form, function, and differentiation. Of particular interest is the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that includes nesprin and SUN. In this review, the way in which cells can sense external mechanical environments through an intact nuclear envelope and LINC complex proteins will be briefly described. Then, we will highlight the current body of literature on the role of the LINC complex in regulating MSC function and fate decision, without and with external mechanical loading conditions. Our review and suggested future perspective may provide a new insight into the understanding of MSC mechanobiology and related functional tissue engineering applications.

Enhanced cardiomyogenic induction of mouse pluripotent cells by cyclic mechanical stretch.

The cardiac milieu is mechanically active with spontaneous contraction beginning from early development and persistent through maturation and homeostasis, suggesting that mechanical loading may provide a biomimetic myocardial developmental signal. In this study, we tested the role of cyclic mechanical stretch loading in the cardiomyogenesis of pluripotent murine embryonic (P19) stem cells. A Flexcell tension system was utilized to apply equiaxial stretch (12% strain, 1.25 Hz frequency) to P19 cell-derived embryoid bodies (EBs). Interestingly, while control EBs without any further stimulation did not exhibit cardiomyogenesis, stretch stimulation alone could induce P19-derived EBs to become spontaneously beating cardiomyocytes (CMs). The beating colony number, average contracting area, and beating rate, as quantified by video capturing and framed image analysis, were even increased for stretch alone case relative to those from known biochemical induction with 5-Azacytidine (5-Aza). Key CM differentiation markers, GATA4 and Troponin T, could also be detected for the stretch alone sample at comparable levels as with 5-Aza treatment. Stretch and 5-Aza co-stimulation produced in general synergistic effects in CM developments. Combined data suggest that stretch loading may serve as a potent trigger to induce functional CM development in both beating dynamics and genomic development, which is still a challenge for myocardial regenerative medicine.

Flowtaxis of osteoblast migration under fluid shear and the effect of RhoA kinase silencing.

Despite the important role of mechanical signals in bone remodeling, relatively little is known about how fluid shear affects osteoblastic cell migration behavior. Here we demonstrated that MC3T3-E1 osteoblast migration could be activated by physiologically-relevant levels of fluid shear in a shear stress-dependent manner. Interestingly, shear-sensitive osteoblast migration behavior was prominent only during the initial period after the onset of the steady flow (for about 30 min), exhibiting shear stress-dependent migration speed, displacement, arrest coefficient, and motility coefficient. For example, cell speed at 1 min was 0.28, 0.47, 0.51, and 0.84 µm min-1 for static, 2, 15, and 25 dyne cm-2 shear stress, respectively. Arrest coefficient (measuring how often cells are paused during migration) assessed for the first 30 min was 0.40, 0.26, 0.24, and 0.12 respectively for static, 2, 15, and 25 dyne cm-2. After this initial period, osteoblasts under steady flow showed decreased migration capacity and diminished shear stress dependency. Molecular interference of RhoA kinase (ROCK), a regulator of cytoskeletal tension signaling, was found to increase the shear-sensitive window beyond the initial period. Cells with ROCK-shRNA had increased migration in the flow direction and continued shear sensitivity, resulting in greater root mean square displacement at the end of 120 min of measurement. It is notable that the transient osteoblast migration behavior was in sharp contrast to mesenchymal stem cells that exhibited sustained shear sensitivity (as we recently reported, J. R. Soc. Interface. 2015; 12:20141351). The study of fluid shear as a driving force for cell migration, i.e., "flowtaxis", and investigation of molecular mechanosensors governing such behavior (e.g., ROCK as tested in this study) may provide new and improved insights into the fundamental understanding of cell migration-based homeostasis.

Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

Mechanical stretching for tissue engineering: two-dimensional and three-dimensional constructs.

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols.

Macro and microfluidic flows for skeletal regenerative medicine.

Fluid flow has a great potential as a cell stimulatory tool for skeletal regenerative medicine, because fluid flow-induced bone cell mechanotransduction in vivo plays a critical role in maintaining healthy bone homeostasis. Applications of fluid flow for skeletal regenerative medicine are reviewed at macro and microscale. Macroflow in two dimensions (2D), in which flow velocity varies along the normal direction to the flow, has explored molecular mechanisms of bone forming cell mechanotransduction responsible for flow-regulated differentiation, mineralized matrix deposition, and stem cell osteogenesis. Though 2D flow set-ups are useful for mechanistic studies due to easiness in in situ and post-flow assays, engineering skeletal tissue constructs should involve three dimensional (3D) flows, e.g., flow through porous scaffolds. Skeletal tissue engineering using 3D flows has produced promising outcomes, but 3D flow conditions (e.g., shear stress vs. chemotransport) and scaffold characteristics should further be tailored. Ideally, data gained from 2D flows may be utilized to engineer improved 3D bone tissue constructs. Recent microfluidics approaches suggest a strong potential to mimic in vivo microscale interstitial flows in bone. Though there have been few microfluidics studies on bone cells, it was demonstrated that microfluidic platform can be used to conduct high throughput screening of bone cell mechanotransduction behavior under biomimicking flow conditions.