Rotating strain field: Development of a novel mechanical stimulation device and orthogonal alignment of MC3T3-E1 cells in response to anisotropic stimulation.

Cyclic deformation of cell culture substrates induces various cellular responses, including alignment and cytoskeletal remodeling. However, most studies have focused on deformation in a fixed direction, which does not adequately replicate the complexity of in vivo mechanical environments. In this study, we propose a rotating strain field (RSF), in which the direction of extension continuously changes over time. We developed a device and culture chamber capable of applying this stimulus to cells and evaluated its performance. Deformation analysis showed that the RSF generated on the chamber bottom was anisotropic: strain along the diagonal directions was approximately twice that along the vertical and horizontal directions, owing to lateral deformation of the chamber walls. Mouse osteoblast-like MC3T3-E1 cells were exposed to this anisotropic RSF to assess morphological responses. Within 3 h, more than 70% of cells exhibited orthogonal alignment along the vertical and horizontal axes. Most cells reoriented from their initial angles toward the nearer of the vertical or horizontal axes, a behavior that may contribute to the rapid alignment. Fluorescent staining of actin filaments revealed that a small subpopulation of large cells, accounting for approximately 2% of the total cell population, showed orthogonal alignment of actin filaments within individual cells. These responses were not observed under uniaxial strain, indicating that anisotropic RSF elicits distinct cellular behaviors. Overall, our findings demonstrate that RSF profoundly influences cell orientation, providing new insights into mechanobiology, and suggest its potential as a novel strategy for controlling cell alignment in tissue engineering.