Soluble Sema4D From γδ T Cells Exerts Osteoblast Inhibition via Plexin-B/mTOR Signalling Contributing to Pathogenesis of Bisphosphonate-Related Osteonecrosis of the Jaws

Abstract

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a severe complication in patients undergoing long-term bisphosphonate therapy, while our knowledge on the pathogenesis of BRONJ is far from sufficient. Gamma delta (γδ) T cells predominantly distribute in mucosal tissues and play an important role in both immune modulation and bone metabolism; however, the mechanism of γδ T cells in the pathogenesis of BRONJ has not been elucidated. Here, we induced BRONJ-like lesions in wild-type (WT) and T-cell receptor delta-deficient (TCRδ^−/−) mice via intraperitoneal zoledronate injection. Our findings revealed that γδ T cells infiltrating BRONJ lesions suppressed osteoblast differentiation, whereas γδ T cell depletion in TCRδ^−/− mice restored osteogenic function and significantly reduced BRONJ lesion incidence. Mechanistically, we identified matrix metalloproteinase 3 (MMP3) secreted by activated γδ T cells as a critical enzyme cleaving membrane-bound Sema4D (mSema4D) into soluble Sema4D (sSema4D). This cleavage product bound to Plexin-B1/2 receptors on osteoblasts, activating the mTOR signalling pathway to inhibit osteogenic differentiation (ALP/Runx2 downregulation). To promote the repair of BRONJ lesions, we engineered a dual-functional composite hydrogel (Gel-BG@ab) combining PLGA-PEG-PLGA with mesoporous bioactive glass (BG) and anti-Sema4D antibodies. This composite hydrogel achieved sustained antibody release, effectively neutralising sSema4D, restoring osteoblast activity and reducing the formation of BRONJ-like lesions in vivo. This study provides evidence of MMP3-Sema4D-Plexin-B1/2/mTOR crosstalk in BRONJ and introduces a targeted biomaterial strategy to disrupt pathogenic feedback loops. The Gel-BG@ab is the integration of immunomodulation and regenerative medicine, providing both theoretical and technical insights for the immune-material combination therapy of BRONJ.

Keywords

BRONJ / gel-BG@ab / MMP3 / Sema4D / γδ T cells

References

[1] G. Cai, D. Aitken, L. L. Laslett, et al., “Effect of Intravenous Zoledronic Acid on Tibiofemoral Cartilage Volume Among Patients With Knee Osteoarthritis With Bone Marrow Lesions: A Randomized Clinical Trial,” Journal of the American Medical Association 323, no. 15 (2020): 1456–1466.

[2] S. Dhesy-Thind, G. G. Fletcher, P. S. Blanchette, et al., “Use of Adjuvant Bisphosphonates and Other Bone-Modifying Agents in Breast Cancer: A Cancer Care Ontario and American Society of Clinical Oncology Clinical Practice Guideline,” Journal of Clinical Oncology 35, no. 18 (2017): 2062–2081.

[3] M. Schiodt, S. Vadhan-Raj, M. S. Chambers, et al., “A Multicenter Case Registry Study on Medication-Related Osteonecrosis of the Jaw in Patients With Advanced Cancer,” Supportive Care in Cancer: Official Journal of the Multinational Association of Supportive Care in Cancer 26, no. 6 (2018): 1905–1915.

[4] J. Everts-Graber, D. Lehmann, J. P. Burkard, et al., “Risk of Osteonecrosis of the Jaw Under Denosumab Compared to Bisphosphonates in Patients With Osteoporosis,” Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research 37, no. 2 (2022): 340–348.

[5] G. Yang, R. Williams, L. Wang, et al., “Medication-Related Osteonecrosis of the Jaw in Cancer Patients: Result From the OneFlorida Clinical Research Consortium,” Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research 37, no. 12 (2022): 2466–2471.

[6] J. H. Park, S. H. Kong, J. Lee, et al., “Time Since Last Intravenous Bisphosphonate and Risk of Osteonecrosis of the Jaw in Osteoporotic Patients,” Nature Communications 16, no. 1 (2025): 4367.

[7] A. Spanou, K. Nelson, M. A. Ermer, D. Steybe, P. Poxleitner, and P. J. Voss, “Primary Wound Closure and Perioperative Antibiotic Therapy for Prevention of Bisphosphonate-Related Osteonecrosis of the Jaw After Tooth Extraction,” Quintessence International 51, no. 3 (2020): 220–228.

[8] H. G. Jeong, J. J. Hwang, J. H. Lee, Y. H. Kim, J. Y. Na, and S. S. Han, “Risk Factors of Osteonecrosis of the Jaw After Tooth Extraction in Osteoporotic Patients on Oral Bisphosphonates,” Imaging Science in Dentistry 47, no. 1 (2017): 45–50.

[9] S. L. Ruggiero, T. B. Dodson, J. Fantasia, et al., “American Association of Oral and Maxillofacial Surgeons Position Paper on Medication-Related Osteonecrosis of the Jaw—2014 Update,” Journal of Oral and Maxillofacial Surgery: Official Journal of the American Association of Oral and Maxillofacial Surgeons 72, no. 10 (2014): 1938–1956.

[10] N. Yarom, C. L. Shapiro, D. E. Peterson, et al., “Medication-Related Osteonecrosis of the Jaw: MASCC/ISOO/ASCO Clinical Practice Guideline,” Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology 37, no. 25 (2019): 2270–2290.

[11] Q. Zhang, I. Atsuta, S. Liu, et al., “IL-17-Mediated M1/M2 Macrophage Alteration Contributes to Pathogenesis of Bisphosphonate-Related Osteonecrosis of the Jaws,” Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 19, no. 12 (2013): 3176–3188.

[12] H. Liu, S. H. Wang, S. C. Chen, C. Y. Chen, J. L. Lo, and T. M. Lin, “Immune Modulation of CD4(+)CD25(+) Regulatory T Cells by Zoledronic Acid,” BMC Immunology 17, no. 1 (2016): 45.

[13] R. Elsayed, Z. Kurago, C. W. Cutler, et al., “Role of Dendritic Cell-Mediated Immune Response in Oral Homeostasis: A New Mechanism of Osteonecrosis of the Jaw,” FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology 34, no. 2 (2020): 2595–2608.

[14] A. H. Hovav, A. Wilharm, O. Barel, and I. Prinz, “Development and Function of γδT Cells in the Oral Mucosa,” Journal of Dental Research 99, no. 5 (2020): 498–505.

[15] M. A. Fischer, N. B. Golovchenko, and K. L. Edelblum, “γδ T Cell Migration: Separating Trafficking From Surveillance Behaviors at Barrier Surfaces,” Immunological Reviews 298, no. 1 (2020): 165–180.

[16] M. M. Nielsen, D. A. Witherden, and W. L. Havran, “γδ T Cells in Homeostasis and Host Defence of Epithelial Barrier Tissues,” Nature Reviews Immunology 17, no. 12 (2017): 733–745.

[17] K. Okamoto and H. Takayanagi, “Effect of T Cells on Bone,” Bone 168 (2023): 116675.

[18] O. Barel, Y. Aizenbud, Y. Tabib, et al., “γδ T Cells Differentially Regulate Bone Loss in Periodontitis Models,” Journal of Dental Research 101, no. 4 (2022): 428–436.

[19] D. X. Beringer, T. Straetemans, S. Minguet, et al., “Disrupting the Balance Between Activating and Inhibitory Receptors of γδT Cells for Effective Cancer Immunotherapy,” Nature Reviews Cancer 25 (2025): 590–612.

[20] X. Qu, Z. Wang, T. Zhou, and L. Shan, “Determination of the Molecular Mechanism by Which Macrophages and γδ-T Cells Contribute to ZOL-Induced ONJ,” Aging 12, no. 20 (2020): 20743–20752.

[21] S. Park, K. Kanayama, K. Kaur, et al., “Osteonecrosis of the Jaw Developed in Mice: Disease Variants Regulated by γδ T Cells in Oral Mucosal Barrier Immunity,” Journal of Biological Chemistry 290, no. 28 (2015): 17349–17366.

[22] A. Movila, H. Mawardi, K. Nishimura, et al., “Possible Pathogenic Engagement of Soluble Semaphorin 4D Produced by γδT Cells in Medication-Related Osteonecrosis of the Jaw (MRONJ),” Biochemical and Biophysical Research Communications 480, no. 1 (2016): 42–47.

[23] L. Ou, H. Wang, Q. Liu, et al., “Dichotomous and Stable Gamma Delta T-Cell Number and Function in Healthy Individuals,” Journal for Immunotherapy of Cancer 9, no. 5 (2021): e002274.

[24] L. Ou, H. Wang, H. Huang, et al., “Preclinical Platforms to Study Therapeutic Efficacy of Human γδ T Cells,” Clinical and Translational Medicine 12, no. 6 (2022): e814.

[25] H. Wang, H. Chen, S. Liu, et al., “Costimulation of γδTCR and TLR7/8 Promotes Vδ2 T-Cell Antitumor Activity by Modulating mTOR Pathway and APC Function,” Journal for Immunotherapy of Cancer 9, no. 12 (2021): e003339.

[26] K. H. G. Mills, “IL-17 and IL-17-Producing Cells in Protection Versus Pathology,” Nature Reviews Immunology 23, no. 1 (2023): 38–54.

[27] D. Xu, A. P. Robinson, T. Ishii, et al., “Peripherally Derived T Regulatory and γδ T Cells Have Opposing Roles in the Pathogenesis of Intractable Pediatric Epilepsy,” Journal of Experimental Medicine 215, no. 4 (2018): 1169–1186.

[28] R. Yan, R. Jiang, L. Hu, Y. Deng, J. Wen, and X. Jiang, “Establishment and Assessment of Rodent Models of Medication-Related Osteonecrosis of the Jaw (MRONJ),” International Journal of Oral Science 14, no. 1 (2022): 41.

[29] D. W. Williams, C. Lee, T. Kim, et al., “Impaired Bone Resorption and Woven Bone Formation Are Associated With Development of Osteonecrosis of the Jaw-Like Lesions by Bisphosphonate and Anti-Receptor Activator of NF-κB Ligand Antibody in Mice,” American Journal of Pathology 184, no. 11 (2014): 3084–3093.

[30] Y. Bi, Y. Gao, D. Ehirchiou, et al., “Bisphosphonates Cause Osteonecrosis of the Jaw-Like Disease in Mice,” American Journal of Pathology 177, no. 1 (2010): 280–290.

[31] F. Tang, J. Li, W. Xie, et al., “Bioactive Glass Promotes the Barrier Functional Behaviors of Keratinocytes and Improves the Re-Epithelialization in Wound Healing in Diabetic Rats,” Bioactive Materials 6, no. 10 (2021): 3496–3506.

[32] L. Zheng, C. Li, X. Huang, et al., “Thermosensitive Hydrogels for Sustained-Release of Sorafenib and Selenium Nanoparticles for Localized Synergistic Chemoradiotherapy,” Biomaterials 216 (2019): 119220.

[33] S. Itohara, P. Mombaerts, J. Lafaille, et al., “T Cell Receptor Delta Gene Mutant Mice: Independent Generation of Alpha Beta T Cells and Programmed Rearrangements of Gamma Delta TCR Genes,” Cell 72, no. 3 (1993): 337–348.

[34] R. P. Hunter and R. Isaza, “Concepts and Issues With Interspecies Scaling in Zoological Pharmacology,” Journal of Zoo and Wildlife Medicine: Official Publication of the American Association of Zoo Veterinarians 39, no. 4 (2008): 517–526.

[35] V. Rollason, A. Laverrière, L. C. MacDonald, T. Walsh, M. R. Tramèr, and N. B. Vogt-Ferrier, “Interventions for Treating Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ),” Cochrane Database of Systematic Reviews 2, no. 2 (2016): Cd008455.

[36] H. Okawa, T. Kondo, A. Hokugo, et al., “Mechanism of Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ) Revealed by Targeted Removal of Legacy Bisphosphonate From Jawbone Using Competing Inert Hydroxymethylene Diphosphonate,” eLife 11 (2022): 11.

[37] D. Zhao, D. Xiao, M. Liu, et al., “Tetrahedral Framework Nucleic Acid Carrying Angiogenic Peptide Prevents Bisphosphonate-Related Osteonecrosis of the Jaw by Promoting Angiogenesis,” International Journal of Oral Science 14, no. 1 (2022): 23.

[38] M. L. Bouxsein, S. K. Boyd, B. A. Christiansen, R. E. Guldberg, K. J. Jepsen, and R. Müller, “Guidelines for Assessment of Bone Microstructure in Rodents Using Micro-Computed Tomography,” Journal of Bone and Mineral Research: the Official Journal of the American Society for Bone and Mineral Research 25, no. 7 (2010): 1468–1486.

[39] S. Krishnan, I. E. Prise, K. Wemyss, et al., “Amphiregulin-Producing γδ T Cells Are Vital for Safeguarding Oral Barrier Immune Homeostasis,” Proceedings of the National Academy of Sciences of The United States of America 115, no. 42 (2018): 10738–10743.

[40] A. Wilharm, Y. Tabib, M. Nassar, et al., “Mutual Interplay Between IL-17-Producing γδT Cells and Microbiota Orchestrates Oral Mucosal Homeostasis,” Proceedings of the National Academy of Sciences of The United States of America 116, no. 7 (2019): 2652–2661.

[41] Y. Duan, G. Li, M. Xu, et al., “CFTR Is a Negative Regulator of γδ T Cell IFN-γ Production and Antitumor Immunity,” Cellular & Molecular Immunology 18, no. 8 (2021): 1934–1944.

[42] X. Xia, G. Cao, G. Sun, et al., “GLS1-Mediated Glutaminolysis Unbridled by MALT1 Protease Promotes Psoriasis Pathogenesis,” Journal of Clinical Investigation 130, no. 10 (2020): 5180–5196.

[43] L. Zhao, Z. Zhang, H. Su, et al., “Molecular Docking-QSAR-Kronecker-Regularized Least Squares-Based Multiple Machine Learning for Assessment and Prediction of PFAS-Protein Binding Interactions,” Journal of Hazardous Materials 492 (2025): 138069.

[44] P. Lesclous, S. Abi Najm, J. P. Carrel, et al., “Bisphosphonate-Associated Osteonecrosis of the Jaw: A Key Role of Inflammation?,” Bone 45, no. 5 (2009): 843–852.

[45] C. Tan, N. N. Lu, C. K. Wang, et al., “Endothelium-Derived Semaphorin 3G Regulates Hippocampal Synaptic Structure and Plasticity via Neuropilin-2/PlexinA4,” Neuron 101, no. 5 (2019): 920–37.e13.

[46] Y. Ke, E. Dang, S. Shen, et al., “Semaphorin4D Drives CD8(+) T-Cell Lesional Trafficking in Oral Lichen Planus via CXCL9/CXCL10 Upregulations in Oral Keratinocytes,” Journal of Investigative Dermatology 137, no. 11 (2017): 2396–2406.

[47] M. Bonneville, “Semaphorins: New Cues for Skin Healing by γδ T Cells,” Immunity 37, no. 2 (2012): 194–196.

[48] A. Kumanogoh and H. Kikutani, “Immunological Functions of the Neuropilins and Plexins as Receptors for Semaphorins,” Nature Reviews Immunology 13, no. 11 (2013): 802–814.

[49] L. Chen, S. Chen, Y. Rong, et al., “Identification and Evaluation of Antioxidant Peptides From Highland Barley Distiller's Grains Protein Hydrolysate Assisted by Molecular Docking,” Food Chemistry 434 (2024): 137441.

[50] P. Mou, Z. Zeng, Q. Li, et al., “Identification of a Calmodulin-Binding Domain in Sema4D That Regulates Its Exodomain Shedding in Platelets,” Blood 121, no. 20 (2013): 4221–4230.

[51] A. Das, A. Barai, M. Monteiro, S. Kumar, and S. Sen, “Nuclear Softening Is Essential for Protease-Independent Migration,” Matrix Biology: Journal of the International Society for Matrix Biology 82 (2019): 4–19.

[52] J. Liu, G. Zhu, N. Jia, et al., “CD9 Regulates Keratinocyte Migration by Negatively Modulating the Sheddase Activity of ADAM17,” International Journal of Biological Sciences 15, no. 2 (2019): 493–506.

[53] J. Reißing, P. Lutz, M. Frissen, et al., “Immunomodulatory Receptor VSIG4 Is Released During Spontaneous Bacterial Peritonitis and Predicts Short-Term Mortality,” JHEP Reports: Innovation in Hepatology 4, no. 1 (2022): 100391.

[54] M. Chen, Y. Tan, J. Hu, et al., “Injectable Immunotherapeutic Thermogel for Enhanced Immunotherapy Post Tumor Radiofrequency Ablation,” Small (Weinheim an Der Bergstrasse, Germany) 17, no. 52 (2021): e2104773.

[55] W. Li, J. Qiu, X. L. Li, et al., “BBB Pathophysiology-Independent Delivery of siRNA in Traumatic Brain Injury,” Science Advances 7, no. 1 (2021): eabd6889.

[56] D. Hadaya, A. Soundia, I. Gkouveris, et al., “Antiresorptive-Type and Discontinuation-Timing Affect ONJ Burden,” Journal of Dental Research 100, no. 7 (2021): 746–753.

[57] Y. Chen, Y. Guo, J. Li, et al., “Endoplasmic Reticulum Stress Remodels Alveolar Bone Formation After Tooth Extraction,” Journal of Cellular and Molecular Medicine 24, no. 21 (2020): 12411–12420.

[58] I. Roato, R. Mauceri, V. Notaro, T. Genova, V. Fusco, and F. Mussano, “Immune Dysfunction in Medication-Related Osteonecrosis of the Jaw,” International Journal of Molecular Sciences 24, no. 9 (2023): 7948.

[59] P. A. Romagnoli, B. S. Sheridan, Q. M. Pham, L. Lefrançois, and K. M. Khanna, “IL-17A-Producing Resident Memory γδ T Cells Orchestrate the Innate Immune Response to Secondary Oral Listeria monocytogenes Infection,” Proceedings of the National Academy of Sciences of The United States of America 113, no. 30 (2016): 8502–8507.

[60] A. H. Verma, J. P. Richardson, C. Zhou, et al., “Oral Epithelial Cells Orchestrate Innate Type 17 Responses to Candida albicans Through the Virulence Factor Candidalysin,” Science Immunology 2, no. 17 (2017): eaam8834.

[61] D. A. Witherden, M. Watanabe, O. Garijo, et al., “The CD100 Receptor Interacts With Its Plexin B2 Ligand to Regulate Epidermal γδ T Cell Function,” Immunity 37, no. 2 (2012): 314–325.

[62] T. Negishi-Koga, M. Shinohara, N. Komatsu, et al., “Suppression of Bone Formation by Osteoclastic Expression of Semaphorin 4D,” Nature Medicine 17, no. 11 (2011): 1473–1480.

[63] B. Huang, Y. Wang, W. Wang, et al., “mTORC1 Prevents Preosteoblast Differentiation Through the Notch Signaling Pathway,” PLoS Genetics 11, no. 8 (2015): e1005426.

[64] J. H. Wu, Y. N. Li, A. Q. Chen, et al., “Inhibition of Sema4D/PlexinB1 Signaling Alleviates Vascular Dysfunction in Diabetic Retinopathy,” EMBO Molecular Medicine 12, no. 2 (2020): e10154.

[65] S. Yang, L. Wang, W. Pan, et al., “MMP2/MMP9-Mediated CD100 Shedding Is Crucial for Inducing Intrahepatic Anti-HBV CD8 T Cell Responses and HBV Clearance,” Journal of Hepatology 71, no. 4 (2019): 685–698.

RIGHTS & PERMISSIONS

2025 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.