Abstract
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.