ID3-directed sonogenetics to enhance osteogenesis and bone defect healing.

Sonogenetics, a noninvasive approach combining ultrasound physics with genetic engineering, has shown clinical potential in promoting bone repair. However, its therapeutic efficacy remains suboptimal due to the scarcity of bone-specific ultrasound-responsive targets and insufficient modulation of osteogenic signaling pathways. Herein, we investigated the role of the transcriptional repressor inhibitor of DNA binding/differentiation 3 (Id3) in bridging sonogenetics and bone regeneration. RNA sequencing and PCR array analyses revealed significant downregulation of Id3 during low-intensity pulsed ultrasound (LIPUS)-induced bone formation, suggesting Id3 acts as a "brake" on ultrasound-mediated osteogenesis. As a member of the ID protein family, Id3 binds to basic helix-loop-helix (bHLH) transcription factors (e.g., E-proteins) to suppress their DNA-binding capacity and osteogenic gene transcription-particularly in bone marrow-derived mesenchymal stem cells (BMSCs), a key cell type for bone repair. Functional experiments demonstrated that Id3 knockdown enhanced LIPUS-induced osteogenic differentiation of BMSCs in vitro (upregulating RUNX2, OCN) and promoted in vivo bone formation in rodent bone defect models, while Id3 overexpression reversed these effects. Chromatin immunoprecipitation (ChIP) assays confirmed that Id3 directly targets bHLH proteins to inhibit the Bmp/Smad pathway, a core cascade for osteogenesis. Based on these findings, we propose an ID3-based sonogenetic strategy: we engineered an Id3-inhibitory peptide (rID3) targeting the Id3/E2A interface, which would synergize with LIPUS to enhance defect healing of cranial bone. Our work delineates a novel CaN/NFATC3/Id3/E2A/Bmp4 pathway for ultrasound mechanobiology and provides a promising combinatorial therapeutic strategy for precision sonogenetics bone regeneration.