Icariin-loaded GelMa hydrogel encapsulated potassium sodium niobate biomimetic piezoelectric scaffold regulates macrophage polarization to accelerate bone defect repair

Large bone defects present significant clinical challenges due to limitations of current treatments including immune rejection, infection, and poor osteoinductive capacity. This study developed a novel biomimetic piezoelectric scaffold system combining 3D-printed potassium sodium niobate/nano-hydroxyapatite/polylactic acid (KNN/nHA/PLA) scaffolds with icariin-loaded gelatin methacryloyl hydrogel (ICA@G/NHP) to synergistically promote bone regeneration and immunomodulation. The composite scaffolds demonstrated excellent mechanical properties and stable piezoelectric output under ultrasonic activation. In vitro studies revealed that ultrasonic stimulation was essential for activating piezoelectric effects, significantly enhancing osteogenic differentiation of bone marrow mesenchymal stem cells through upregulation of ALP, RUNX2, and COL1 expression. The incorporated icariin effectively promoted endothelial cell migration and induced M2 macrophage polarization via C-type lectin receptor signaling pathway, creating a pro-regenerative immune microenvironment. In vivo validation using rat cranial and rabbit femoral condyle defect models demonstrated superior bone regeneration with enhanced bone mineral density, bone volume fraction, and mature trabecular architecture compared to controls. Immunofluorescence analysis confirmed sustained M2 macrophage dominance and suppressed inflammatory responses. RNA sequencing identified PI3K/Akt as the central mechanotransduction pathway mediating scaffold effects. This integrated platform addresses dual challenges of osteogenesis and immune regulation, offering a promising therapeutic strategy for critical-sized bone defect repair through ultrasound-activated piezoelectric stimulation combined with targeted immunomodulation. • Novel biomimetic scaffold design: 3D-printed KNN/nHA/PLA scaffold integrated with icariin-loaded gelatin hydrogel combines piezoelectric stimulation with controlled drug release for enhanced bone regeneration. • Dual-functional bone regeneration mechanism: ICA@G/NHP scaffold simultaneously promotes osteogenic differentiation of BMSCs and regulates macrophage polarization toward anti-inflammatory M2 phenotype, creating a synergistic osteoimmune microenvironment. • Piezoelectric-enhanced cellular responses: Ultrasound-activated piezoelectric properties significantly amplify osteogenic marker expression (2.4-2.9fold) and mineralization without compromising biocompatibility or inducing cytotoxicity. • Controlled drug delivery optimization: Scaffold architecture enables sustained icariin release (94.6% over 28 days) compared to rapid burst release (98.2% in standalone hydrogel), ensuring prolonged therapeutic effects throughout bone healing phases. • Comprehensive in vivo efficacy: ICA@G/NHP treatment achieved significant bone defect repair with enhanced M2 macrophage infiltration, reduced apoptosis, and improved bone regeneration confirmed by micro-CT and histological analyses at 12 weeks post-surgery.