Mechanosignaling and 3D morphological adaptation of MSCs in response to hydrogel rigidity underpin angiogenic and immunomodulatory efficacy for ischemic injury regeneration.

Activating angiogenic and immunomodulatory potential of stem cells through optimized cultivation strategies presents significant opportunities for cell-based tissue therapeutics. Among others, hydrogels with tunable chemo-mechanical properties offer optimal 3D environments for stem cell functions. Here, we report rigidity sensing and mechanoresponses of mesenchymal stem cells (MSC) in 3D hydrogels drive therapeutic effects in ischemic injury. We introduce a silk-collagen (SC) binary-protein system, engineered for high viscoelasticity and cell adhesion, to facilitate mechanosensing through integrins and the actin cytoskeleton. Notably, MSC mechanoresponses, such as actomyosin contractility and cell spreading in SC hydrogels, closely correlate with their pro-angiogenic and anti-inflammatory capacity. We identified key mechanotransduction pathways, including Rho/Rho-associated protein kinase and focal adhesion kinase (FAK)/proto-oncogene tyrosine-protein kinase Src (Src) signaling, as critical regulators of these therapeutic functions. Pharmacological intervention revealed FAK-Src signaling is essential for cytoskeletal remodeling and angiogenesis while simultaneously mediating anti-inflammatory effects. These findings underscore the interplay between cell mechanophenotype, morphology, and function, providing a strategy to optimize hydrogel-based MSC therapies. In a mouse model of ischemic hindlimb injury, mechano-primed MSCs delivered via SC hydrogels significantly improved blood reperfusion, cell survival, and anti-inflammatory responses, ultimately preventing limb loss. This study highlights the importance of controlling hydrogel mechanics and cellular mechanophenotype to enhance stem cell functions for regenerative therapies.