Static mechanical stretch induces collective alignment of C2C12 myoblasts

Cell alignment is a fundamental process in tissue morphogenesis. While density-dependent collective cell alignment has been widely observed, its underlying mechanisms remain poorly understood. Here, using C2C12 myoblasts, we demonstrate that static uniaxial mechanical stretch induces collective cell alignment in a density-dependent manner: densely populated cultures align robustly, whereas sparse populations do not. We reveal a biphasic alignment process, comprising an initial passive phase and a subsequent active phase. The passive phase, driven by substrate deformation, transiently biases cell orientation along the stretch axis regardless of density. In the active phase, initial alignment progressively dissipates in low-density cultures, but is sustained and reinforced in high-density cultures. Supported by coarse-grained agent-based simulations, we propose that self-generated cellular forces facilitate cell reorientation, enabling cells to explore orientational states, whereas cell-cell interactions provide a thermodynamic bias that stabilizes the locally aligned state. In dense cultures, strong intercellular interactions promote this stabilization, enabling persistent alignment. In contrast, sparse cultures lack sufficient cell-cell interaction, leading to alignment dissipation. Within this C2C12 system, our findings highlight the cooperative roles of cellular forces and intercellular interactions in orchestrating multicellular ordering, offering new insights into mechanobiology of tissue morphogenesis. STATEMENT OF SIGNIFICANCE: Cell alignment is essential to build functional tissues, yet how multicellular groups achieve and maintain this order remains unclear. We show that a brief uniaxial stretch can "write" long-lived orientation into myoblast sheets through two phases: a passive phase where cells deform with the substrate, and an active, density-dependent phase in which cell-cell adhesion stabilizes order against stochastic forces. By separating passive deformation from adhesion-mediated reinforcement and ruling out substrate artifacts, we provide a thermodynamic perspective on how active cellular forces and adhesion cooperate to pattern living tissues. This work links biomechanics, material science, and thermodynamics, and offers a scalable design rule to program persistent anisotropy without patterned scaffolds or continuous loading, advancing strategies for tissue engineering applications.