Mechanical Behavior and Indentation-Induced Injury of Soft Microtissues with Different Densification Levels

Tissue densification is a fundamental biological process involved in development, regeneration, and disease, significantly influencing tissue mechanics and cellular mechanical microenvironments. However, the effects of tissue densification on mechanical properties, the roles of key cytoskeletal components, and their responses to external mechanical stress remain poorly understood. In this study, fibroblast-collagen microtissues were cultured on polydimethylsiloxane (PDMS) microstring scaffolds with different mechanical constraints to generate low- and high-densification microtissues. High-precision indentation force sensor analysis demonstrated that increased densification enhanced microtissue stiffness, accelerated stress relaxation, and elevated energy dissipation. Pharmacological disruption of cytoskeletons revealed the critical role of F-actin in regulating stiffness and viscoelastic resistance in a densification-dependent manner: in high-densification microtissues, F-actin had a greater impact on stiffness and dissipated energy, but a reduced effect on stress relaxation. Furthermore, under 50% strain indentation, high-densification microtissues exhibited irreversible deformation and increased cellular injury. Injured regions showed reduced stiffness, shorter stress relaxation time constants, and higher energy dissipation, indicating structural and cellular damage. These results enhance our understanding of tissue mechanobiology by elucidating the interplay between tissue densification, cytoskeletal mechanics, and injury responses, providing valuable insights for optimizing tissue mechanical microenvironments and improving the mechanical compatibility of biomaterials.