Modulating iMSC Spheroid Function with Mechanically Tunable Hydrogels to Strengthen the Bone-Muscle Axis

Bone fractures often involve both bone and muscle damage, representing a critical need for regenerative strategies that can repair both tissues. We developed a gelatin-based hydrogel system with tunable stiffness to guide spheroids formed from induced pluripotent stem cell (iPSC)-derived mesenchymal stromal cells (iMSCs) for simultaneous muscle and bone repair. Hydrogels were formed using hydrogen peroxide with horseradish peroxidase as a catalyst and substrate stiffness ranged from 1.6 to 9.3 kPa. Hydrogels entrapping iMSC spheroids enabled localized release of the iMSC secretome that exhibited biological effects. iMSC spheroids in soft hydrogels enhanced myogenic differentiation of C2C12 myoblasts via paracrine signaling. Conversely, iMSC spheroids exposed to the myoblast secretome and cultured in stiffer hydrogels exhibited enhanced osteogenic differentiation. This study demonstrates the synergistic influence of stiffness and reciprocal secretome signaling on iMSC behavior, offering a clinically relevant "two-in-one" platform for musculoskeletal regeneration.