Active and passive crowding pathways drive topological and mechanical divergence in myoblasts

Cell crowding is closely associated with many physiological processes, yet our understanding of crowded tissue organization has been developed largely in polygonal epithelia, leaving intrinsically anisotropic cell collectives comparatively poorly understood. Using C2C12 myoblasts, we investigate this problem by decoupling the final crowded state from the pathway taken to reach it, comparing active crowding, arising from cell proliferation during incubation, with passive crowding, imposed directly through high-density seeding. Unlike polygonal epithelia, crowded myoblast monolayers do not undergo epithelial-like kinetic arrest: cell motion is strongly suppressed, yet local rearrangements persist and packing remains structurally irregular. However, the two crowding pathways generate distinct collective states, differing in nematic order and junctional coupling. By contrasting these states, we reveal a close interplay among cell geometry, intercellular coupling, tissue-scale nematic order, and force transmission. This mechanical coordination ultimately shapes physiological function, preserving sensitivity to topographical cues and promoting efficient, spatially organized myogenic fusion. These findings broaden the physical framework of collective organization in active matter and establish key mechanical principles for in vitro tissue engineering.