Mesenchymal stromal cells-loaded 3D radially aligned composite scaffold with potentiated paracrine signaling for sequential bone regeneration.

Critical-sized bone defects remain a major clinical challenge because they lack the intrinsic capacity to heal and cannot orchestrate the sequential processes of inflammation, angiogenesis, and osteogenesis required for regeneration. Mesenchymal stromal cells (MSCs) offer potent paracrine signaling, yet therapeutic efficacy is constrained by poor survival in the early inflammatory milieu and the intrinsic plasticity of MSCs, which leads to attenuation of paracrine activity as inflammation resolves, limiting sustained support across all regenerative phases. Here, we present a 3D radially aligned nanofiber platform comprising hydroxyapatite (HAp)-incorporated fibers with a GelMA hydrogel encapsulating bone marrow-derived MSCs (BMSCs). The radial architecture promotes early centripetal cell infiltration, GelMA preserves encapsulated BMSCs viability and residency to extend paracrine signaling, and HAp provides durable osteoconductive cues while amplifying encapsulated BMSC paracrine output, thereby expediting the immune-angiogenic-osteogenic transition. In vitro metabolomic and transcriptomic analyses revealed that upregulation of glycerophospholipid metabolism supports early BMSC proliferation, while activation of PI3K/Akt signaling drives osteogenic commitment. In rat subcutaneous implant and critical-sized cranial defect model, the composite scaffold attenuated early inflammation and markedly enhanced bone regeneration. Overall, this paracrine-enhancing platform integrates radial topography, sustained paracrine amplification, and persistent osteoconductive support to achieve temporally coordinated regulation of cranial bone repair with high translational potential.