Orientation of Smooth Muscle-Derived A10 Cells in Culture by Cyclic Stretching: Relationship between Stress Fiber Rearrangement and Cell Reorientation

Abstract

Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1-3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretchinduced cell reorientation was blocked by 1 µM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.

Mechanical Stretching for Tissue Engineering: Two-Dimensional and Three-Dimensional Constructs

“…[38][39][40][41] Cells receive varying degrees of compressive strain in a direction perpendicular to the stretch axis dependent on the Poisson ratio of the membrane or scaffold. 42,43 Work in 3D FE has shown that the strain that cells actually receive can be much larger than that applied to the matrix due to variations in matrix microarchitecture. 44 On the other hand, cell strain may be less than the given substrate elongation since cells are randomly distributed at arbitrary orientations with respect to the stretch direction.…”

“…These provided valuable insight into frequency-dependent orientation responses observed experimentally. 42,[62][63][64] Although strain homeostasis varies with cell type and is also influenced by cellular adaptive response to substrate stiffness, the saturation in frequency response seemed to occur around 1 Hz for various cells. 62,65 Stretch frequency has also been shown to affect other cell behaviors than orientation, such as proliferation, apoptosis, and membrane permeability.…”

A novel platform for in situ investigation of cells and tissues under mechanical strain

“…However they do not allow live imaging with high numerical aperture optics because of the incompatibility with the short working distance (100–200 µm) objectives. This is typically due to either limitations of the stretching device itself [31, 32, 33, 34, 35] or the thickness of the stretchable substrate [36, 37, 38, 39]. Stretching systems, which are compatible with high resolution optics, tend to be complicated and require integration with a specific microscopy system [40, 41].…”

Effect of Focal Adhesion Proteins on Endothelial Cell Adhesion, Motility and Orientation Response to Cyclic Strain

“…7,20,25 It is also wellestablished that these responses are highly dependent on actin assembly and disassembly since drugs that inhibit stress fiber polymerization also inhibit the morphologic and orientation changes. 12,14,28,31 Despite the key role of actin stress fibers, the specific contributions of the many actin binding and focal adhesion proteins that serve to anchor and reinforce the ventral actin filaments are not well understood. For example, focal adhesion proteins such as FAK and paxillin may play a pivotal role in the cell orientation changes to cyclic strain since they are known to affect cell migration, spreading and adhesion.…”