3D-Printing-Assisted, Microfabricated Devices Reveal Hierarchical and Temporal Mechanosensing in High-Density Fibroblast Culture.

Understanding how cells integrate mechanical forces across multiple directions, length scales, and time scales remains a fundamental challenge in mechanobiology. This is particularly important in the context of wound healing, where the timing and duration of the fibroblast-to-myofibroblast transition can determine the healing outcomes. Here, we discovered that fibroblasts in tissue equivalents respond to directional anisotropy in stress through a hierarchical temporal cascade, with individual cell elongation (24 h) preceding collective alignment (48 h), which then drives α-smooth muscle actin expression and myofibroblast transition (96 h). To enable this discovery, we developed a modified hydrogel-assisted stereolithographic elastomer (HASTE) prototyping platform to incorporate a detergent that improves the wettability of template agar hydrogels by poly(dimethylsiloxane) elastomer. This allowed rapid prototyping of intricate three-dimensional (3D) micropost arrays with microscale precision. Using these with engineered microtissues with isotropic (8-post) versus anisotropic (4-post) boundary conditions, we found that cells sense and respond to stress directionality before bulk tissue reorganization occurs. Computational modeling predicted steady-state activation patterns based on initial stress anisotropy rather than magnitude, and our experiments reveal that reaching this state requires sequential mechanosensitive processes operating across distinct time scales. This temporal hierarchy persists even when extensive cell-cell contacts might be expected to mask matrix-mediated mechanical signals. Our findings demonstrate that fibroblast mechanosensing involves mechanical memory encoded through progressive cell and tissue reorganization. Results provide insight into how nanoscale mechanosensing scales up to direct tissue-level organization, with implications for understanding wound healing, fibrosis, and engineering functional tissue replacements.