Biphasic Mechanical Loading Disrupts Cytoskeletal Symmetry in 3D Architected Scaffolds

Cells in load-bearing tissues experience both solid deformation and interstitial fluid flow during physiological loading, but the mechanisms by which they integrate these biphasic mechanical signals remain poorly understood. Here, we develop a porous, nanoarchitected 3D scaffold that allows simultaneous delivery and control of matrix strain and fluid shear stress. We validated the platform through fatigue loading experiments and simulations of fluid-structure interactions. In static culture, osteoblast-like cells adopted shapes, cytoskeletal architectures, and focal adhesion patterns templated by scaffold geometry. Under cyclic compression, the combined influence of matrix deformation and induced fluid flow disrupted this alignment, producing disordered actin structures and reduced focal adhesion eccentricity. These changes emerged even under low-frequency loading, within the drained poroelastic regime, indicating a high sensitivity of cytoskeletal organization to fluid-solid coupling. Our findings establish a tractable and tunable platform to investigate how cells sense and respond to dynamic biphasic mechanical environments in 3D. Significance StatementCells in tissues such as bone experience mechanical inputs from both matrix deformation and interstitial fluid flow. However, existing in vitro systems often isolate one type of input or lack the ability to control both independently. We engineered a nanoarchitected 3D scaffold that delivers tunable biphasic mechanical inputs by combining structural compression and fluid flow. Without external loads, cells align their cytoskeleton and focal adhesions to the scaffold geometry. When subjected to dynamic loading, they transition to disordered morphologies and less mature focal adhesions, suggesting a transition to migratory states. These results highlight the sensitivity of cells to even subtle biphasic cues and provide a new platform to study how cells integrate multiple mechanical signals in 3D environments.