Hierarchical micro-/nanostructured hydroxyapatite scaffolds promote osteoporotic bone regeneration via activation of hedgehog and HIF-1α signaling

Osteoporotic bone defects remain a major clinical challenge due to impaired osteogenesis, disrupted angiogenesis, and poor scaffold integration. To overcome these limitations, we developed hierarchical micro-/nanostructured hydroxyapatite (nwHA) scaffolds by integrating morphology-specific nanohydroxyapatite (nHA) onto whisker-reinforced hydroxyapatite (wHA) scaffolds. This modular strategy decouples mechanical strength from interfacial bioactivity, enabling programmable topographical control. Five distinct nHA morphologies were used to functionalize wHA scaffolds, which were systematically evaluated both in vitro and in osteoporotic rat models. Among them, nanofiber-coated scaffolds (nwHA1) significantly enhanced bone volume fraction, mineral apposition rate, mechanical strength, and neovascularization. Histological analysis identified three distinct ossification patterns-type I (wall-penetrating), type II (surface-appositional), and a hybrid endochondral-intramembranous mode-whose distribution varied with nHA morphology and the local microenvironment. Mechanistically, nwHA1 activated canonical Hedgehog signaling and upregulated HIF-1α in both MSCs and HUVECs, thereby promoting coordinated osteogenic and angiogenic responses. Pharmacological inhibition with cyclopamine, as well as siRNA-mediated knockdown of GLI1 or HIF-1α, significantly attenuated these pro-osteoangiogenic markers, confirming functional crosstalk between Hedgehog and hypoxia signaling pathways in response to scaffold-induced topographic cues. These findings establish nHA morphology as a critical topographical regulator of bone regeneration and provide a versatile platform for designing adaptive bioceramics tailored to osteoporotic bone repair.