Composite hydrogel-microsphere delivery system promotes early nerve-mediated bone regeneration and late-stage mechanotransduction-driven bone remodeling via sequential release of NGF and Yoda1

Neuromodulatory signals play a critical role in initiating early vascularized bone regeneration following bone injury. Despite advancements in bone tissue engineering centered on mesenchymal stem cell regulation, the pivotal contributions of early innervation and late mechanical transduction in bone regeneration and remodeling are frequently overlooked. Nerve growth factor (NGF) facilitates neuronal axon regeneration in the initial stage of bone injury, while Yoda1, acting as a chemical agonist, triggers Piezo1-mediated mechanical transduction signals crucial for the mid-to-late stages of bone remodeling. This study developed a composite sequential delivery system utilizing GelMA hydrogel and PLA microspheres to enable the rapid release of NGF and delayed release of Yoda1, mimicking and expediting the natural bone repair process. The system was found to stimulate the migration and maturation of RSC96 and induce neuronal-like differentiation of PC-12, subsequently enhancing osteogenesis and angiogenesis within a neuromodulatory microenvironment. Notably, early neurovascularization and collagen fiber deposition were observed in a subcutaneous implantation model. Further investigations in a femur defect model confirmed that the rapid release of NGF initiates early neuro-vascular-osteogenic coupling, while sustained Yoda1 release in the mid-to-late phases activates and maintains bone regeneration and remodeling effects. In summary, this study underscores the critical roles of early innervation and late-stage mechanical transduction in bone regeneration, offering an innovative and precise therapeutic approach for bone defects.