Inducing Neurite Outgrowth by Mechanical Cell Stretch

Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen- coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation

Inducing Neurite Outgrowth by Mechanical Cell Stretch

Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen- coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation

Focal adhesion kinase regulation in stem cell alignment and spreading on nanofibers

While electrospun nanofibers have demonstrated the potential for novel tissue engineering scaffolds, very little is known about the molecular mechanism of how cells sense and adapt to nanofibers. Here, we revealed the role of focal adhesion kinase (FAK), one of the key molecular sensors in the focal adhesion complex, in regulating mesenchymal stem cell (MSC) shaping on nanofibers. We produced uniaxially aligned and randomly distributed nanofibers from poly(L-lactic acid) to have the same diameters (about 130 nm) and evaluated MSC behavior on these nanofibers comparing with that on flat PLLA control. C3H10T1/2 murine MSCs exhibited upregulations in FAK expression and phosphorylation (pY397) on nanofibrous cultures as assessed by immunoblotting, and this trend was even greater on aligned nanofibers. MSCs showed significantly elongated and well-spread morphologies on aligned and random nanofibers, respectively. In the presence of FAK silencing via small hairpin RNA (shRNA), cell elongation length in the aligned nanofiber direction (cell major axis length) was significantly decreased, while cells still showed preferred orientation along the aligned nanofibers. On random nanofibers, MSCs with FAK-shRNA showed impaired cell spreading resulting in smaller cell area and higher circularity. Our study provides new data on how MSCs shape their morphologies on aligned and random nanofibrous cultures potentially via FAK-mediated mechanism

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., ªflowtaxisº, 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

Osteoblast recruitment in the flow direction and motility under shear are increased with ROCK interference.

<p>(a) Osteoblasts with ROCK-shRNA (ROCK-sh) under FF25 showed significantly greater number of cells migrating with the flow direction. (b) Even after the initial period, the speeds of ROCK-silenced cells were greater for both static and sheared conditions (e.g., 60 min). (c) The displacement of ROCK-shRNA FF25 group assessed after 120 min was significantly greater compared with other conditions. (d) The ROCK-shRNA static group was less confined in migration path. (e) The ROCK-shRNA cells paused significantly less during the migration. (f) The RMS displacement shows that the collective migration of the ROCK-shRNA FF25 group was continued throughout the measurement time resulting in greater RMS displacement at 120 min. The dashed vertical line marks 30 min after the flow onset. *: comparison with vector control static. ‡: comparison with vector control FF25. +: comparison with ROCK-shRNA static. Single, double, and triple symbols represent p < 0.05, 0.01, and 0.001, respectively.</p

The displacement length and arrest coefficient of osteoblast migration show fluid shear sensitivity but only for a short period after the flow onset.

<p>Data were presented for the short-term (from 0 to 30 min) and long-term (the entire tracking from 0 to 120 min) durations. (a) The short-term displacement increased with shear. FF25 migrated significantly further than the static control. These differences were not observed in the long-term data. (b) There was no significant difference in the confinement ratio (directness of the migration path) with respect to shear stress. The ratio generally decreased for all test conditions as time increased, indicating reduced path efficiency with time. (c) Flow groups had significantly smaller arrest coefficients (less time paused) compared with the static control in the short-term data, which was not observed in the long-term result. *, **, and ***: p < 0.05, 0.01, and 0.001 compared with static control; ##: p < 0.01 with FF2; ψ: p < 0.05 with FF15.</p

Motility coefficient obtained as a slope of the RMS displacement vs. t^1/2 plot.

<p>Motility coefficient obtained as a slope of the RMS displacement vs. t^1/2 plot.</p