Ultrasound-Activated Piezoelectric Scaffolds Target Dual Ca2+/NF-κB Signaling Pathways to Orchestrate Immunomodulation and Osteogenesis for Accelerated Bone Regeneration

Critical-sized bone defects fail to heal due to dysregulated inflammation and impaired osteogenesis. To overcome the limitations of static biomaterials, we engineered 3D-printed polycaprolactone scaffolds incorporating polydopamine coated BaTiO3/β-TCP nanoparticles (10 %PBT) for dynamic ultrasound (US) activation. Integrated in vitro/in vivo analyses revealed that US-activated piezoelectricity triggered two synergistic pathways: (1) Voltage gated calcium channel (VGCC) dependent Ca2+ influx specifically in osteoblasts (inhibited by ω-Hexatoxin-HV1A), activating Ca2+/NFAT signaling and direct mineralization; and (2) Suppression of NF-κB p65 phosphorylation and nuclear translocation in macrophages, driving anti-inflammatory M2 polarization. Crucially, M2 macrophages secreted pro-regenerative factors (BMP-2, VEGF), enhancing osteoblast differentiation and angiogenesis via paracrine signaling. In rat critical defects, 10 %PBT + US achieved 3 fold higher bone volume (BV/TV), mature collagen, increased CD31+ vessels, and elevated Runx2/BMP-2 expression. This work unveils a paradigm of targeted electro immunoengineering: wireless US dynamically orchestrates VGCC mediated Ca2+ osteoinduction and NF-κB inhibited M2 polarization with BMP-2/VEGF secretion, providing potent, growth factor free spatiotemporal control of Ca2+ and inflammatory signaling for precision bone regeneration.