Effect of Anisotropic Structural Depth on Orientation and Differentiation Behavior of Skeletal Muscle Cells

[Image: see text] Extensive research has been conducted to examine how substrate topological factors are involved in modulating the cell behavior. Among numerous topological factors, the vital influence of the touchable depth of substrates on cell behaviors has already been extensively characterized, but the response of cells to the topological structure at untouchable depth is still elusive. Herein, the influences of substrate depth on myoblast behaviors are systematically investigated using substrates with depths ranging from touchable depth (microgrooved) to untouchable depth (microbridges). The results show that an increase in microgroove depth is accompanied by an inhibited cell spreading, an enhanced elongation, and a more obvious orientation along microgrooves. Interestingly, myoblasts located on microbridges show a more pronounced elongation with increasing culture time but a position-dependent orientation. Myoblasts on the center and parallel boundary of microbridges orient along the bridges, while myoblasts on the vertical boundary align perpendicular to the microbridges. Moreover, the differentiation results of the myoblasts indicate that the differentiated myotubes can maintain this position-dependent orientation. The simulation of the stress field in cell monolayers suggests that the position-dependent orientation is caused by the comprehensive response of myoblasts to the substrate discontinuity and substrate depth. These findings provide valuable insights into the mechanism of cell depth sensing and could inform the design of tissue engineering scaffolds for skeletal muscle and biohybrid actuation.