Researchers from Capital Medical University, Beijing, China, have identified a CaN/NFATC3/Id3/E2A/Bmp4 signaling pathway that helps explain how low-intensity pulsed ultrasound (LIPUS) promotes bone regeneration. Id3 was identified as a molecular “brake” that limits LIPUS-induced osteogenesis and was significantly downregulated following ultrasound stimulation. The researchers developed an Id3 inhibitory peptide, rID3, which, when combined with LIPUS, enhanced osteogenesis and bone-defect healing in mice, suggesting a potential strategy for improving ultrasound-based bone regeneration .
Sonogenetics, an emerging non-invasive technology that combines ultrasound with genetic engineering, has shown promise for promoting bone repair. Low-intensity pulsed ultrasound (LIPUS) provides a non-invasive mechanical stimulus that can activate cellular signaling pathways involved in bone formation. However, its therapeutic efficacy in bone defect repair remains suboptimal, highlighting the need to identify molecular targets that could improve its regenerative effects.
A team of researchers from the Beijing Laboratory of Oral Health and Beijing Stomatological Hospital, Capital Medical University, Beijing, China, including corresponding authors Professor Songlin Wang and Associate Professor Lei Hu, has uncovered a key molecular pathway underlying LIPUS-induced bone regeneration. Their work was published in the International Journal of Oral Science on 14 September, 2026. “ If materialized, this could be a convenient non-invasive strategy for bone healing. Moreover, it wouldn’t have the systemic effects of drugs either ”, says Prof. Wang.
To identify molecular regulators that might limit LIPUS-induced bone formation, the team first used RNA sequencing to examine gene-expression changes in LIPUS-treated mouse tibiae. They then screened 44 transcriptional repressors using a customized PCR array and identified Id3 as a key candidate based on its strong downregulation and association with bone-related processes. They found that Id3 expression decreased during LIPUS-induced bone formation in both mouse bone tissue and human and mouse bone marrow-derived mesenchymal stem cells (BMSCs). When Id3 was overexpressed, LIPUS-induced osteogenic differentiation was suppressed. Id3 (inhibitor of DNA binding/differentiation 3) is a transcriptional repressor that binds to proteins involved in gene regulation, including the bHLH protein E2A, thereby limiting the expression of certain osteogenic genes.
To further investigate the role of Id3, the researchers studied Id3 knockout (KO) mice and BMSCs isolated from these animals. Id3-deficient BMSCs showed increased proliferation, osteogenic differentiation, and mineralization, while Id3-deficient mice showed greater bone mass. Conversely, when Id3 was overexpressed in bone tissue using a bone-targeting recombinant adeno-associated virus (rAAV9.DSS-Nter-Id3), bone formation was reduced. These findings supported Id3 as a negative regulator of bone formation.
RNA-seq pathway-enrichment analysis was then used to investigate how Id3 regulates LIPUS-induced bone formation. The researchers found that LIPUS increased Bmp4 signaling. Further experiments showed that LIPUS increased Bmp4 and activated downstream Smad1/5 phosphorylation and the osteogenic transcription factors Runx2 and Osx. In contrast, Id3 overexpression blocked activation of the Bmp4-related pathway. When the researchers blocked BMP signaling using DMH-1, LIPUS-induced Bmp4–Smad1/5 signaling was blocked and Runx2 and Osx returned to baseline levels, supporting the importance of this pathway in LIPUS-induced osteogenic differentiation.
Further experiments showed that Id3 normally restrains E2A, a transcription factor that can activate the Bmp4 gene. When LIPUS reduces Id3 levels, E2A can bind the Bmp4 promoter more effectively, increasing Bmp4 signaling and activating Smad1/5, Runx2, and Osx to promote osteogenesis.
“ We now knew how Id3 acted as a molecular brake on LIPUS induced bone formation. But how LIPUS suppressed Id3 was to be known for a comprehensive understanding and to target Id3 for any future therapeutic interventions ”, says Assoc.Prof. Hu.
RNA-seq analysis revealed that LIPUS activated calcium-related signalling. Further experiments showed that LIPUS increased intracellular Ca²⁺, calcineurin activity, and NFATC3 nuclear translocation. NFATC3 directly suppressed Id3 expression, establishing the Ca²⁺/CaN/NFATC3 pathway as an upstream regulator of Id3. “ Previous literature confirmed that NFATC3 inhibited Id3, connecting the dots ,” explains Dr. Liu.
Finally, the researchers developed rID3, an Id3 inhibitory peptide that disrupts the Id3–E2A interaction. Combined with LIPUS, rID3 enhanced osteogenesis and improved bone-defect healing in mice.
Overall, the study identifies Id3 as a molecular brake on LIPUS-induced bone formation and highlights rID3 as a potential strategy for enhancing ultrasound-based bone regeneration. Further studies are needed to establish its safety and clinical applicability.
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Reference
DOI: 10.1038/s41368-026-00456-1
About Capital Medical University
Founded in 1960, Capital Medical University (CMU) is a key medical higher-education institution in Beijing, China. The university offers education and research programs across medicine and related health sciences and is affiliated with a network of clinical medical schools and hospitals. CMU supports multidisciplinary research spanning basic medical sciences, clinical medicine, public health, and biomedical research. Beijing Stomatological Hospital is among the university’s affiliated clinical medical institutions.
Website: https://www.ccmu.edu.cn/index.htm
About Professor Songlin Wang from Capital Medical University, China
Professor Songlin Wang is a Chair Professor and Chief Professor and Chairman of the Beijing Laboratory of Oral Health at Capital Medical University, Beijing, China. He is also Dean of the School of Medicine at Southern University of Science and Technology and a member of the Chinese Academy of Sciences. His research focuses on tooth development and regeneration, dental stem cells, homeostatic medicine, and oral-systemic health.
About Associate Professor Lei Hu from Capital Medical University, China
Associate Professor Lei Hu is a clinician-scientist at Beijing Stomatological Hospital, Capital Medical University. His research focuses on tooth development and regeneration, dental stem cells, tooth-root regeneration, and oral-systemic health, with research spanning dental pulp stem-cell regeneration, bio-root regeneration, cementum regeneration, and the effects of salivary nitrate on bone health.
Funding information
This work was supported by the Beijing Municipal Government grant (Beijing Laboratory of Oral Health, PXM2021-014226-000041), the National Natural Science Foundation of China (82350003, 82030031, 81991504, 92149301, 82001067 and L2224038), Beijing Municipal Natural Science Foundation (7232071), Beijing Nova Program (2024009), the Beijing Municipal Government (Beijing Scholar Program, PXM2021_014226_000020), the Beijing Municipal Education Commission (119207020201), the Innovation Research Team Project of Beijing Stomatological Hospital, Capital Medical University (CXTD202201), the Chinese Research Unit of Tooth Development and Regeneration, Academy of Medical Sciences (2019-12M-5-031), the Beijing Municipal Colleges and Universities High Level Talents Introduction and Cultivate Project-Beijing Great Wall Scholar Program (CIT&TCD 20180332), Scientific Research Common Program of Beijing Municipal Commission of Education (KM202110025009), the excellent youth talent program of Capital Medical University (KCB2311), and the Young Scientist Program of Beiing Stomatological Hospital, Capital Medical University (NO.YSP202408).
International Journal of Oral Science
Experimental study
Animals
ID3-directed sonogenetics to enhance osteogenesis and bone defect healing
14-Sep-2026
The authors declare no competing interests.