Radiation Modulates Macrophage Polarization and Osteoclast Differentiation as a Potential Mechanism for Preventing Heterotopic Ossification

Summary: Heterotopic ossification (HO) is the pathological formation of bone in extraskeletal tissues, including muscles, nerves, and soft tissues. It commonly arises after trauma or invasive orthopedic procedures and is strongly associated with inflammation. Radiation therapy (RT) has been proven effective in preventing HO following orthopedic surgeries, as demonstrated by multiple randomized controlled trials. However, its application has been limited, primarily due to uncertainties regarding its mechanisms, efficacy, and potential toxicities, such as impaired bone health and delayed wound healing. In a trauma- and burn-relevant mouse model, we previously demonstrated that postoperative administration of 7 Gy RT at the injury site reduced HO formation by approximately 50%. The present study investigates the cellular and molecular mechanisms underlying this effect, focusing on immune cell populations due to their central roles in promoting HO inflammation and radiation-associated toxicities. Male C57BL/6J mice underwent a 30% TBSA dorsal burn combined with an Achilles tendon transection of the left hindlimb. On POD3, mice were randomized to receive either no RT (control) or 7 Gy RT at the injury site. To examine RT-induced changes in the trauma microenvironment, we performed single-cell RNA sequencing of injury/HO sites at 7 days post-injury. Cell-cell interaction networks were analyzed using CellChat. Immunohistochemistry was employed to validate macrophage population shifts, phenotypic changes, and osteoclast numbers, while multiplex ELISA was used to assess chemokine and cytokine profiles in irradiated and non-irradiated hindlimbs. ScRNA-seq identified 10 distinct cell clusters in both control and irradiated groups. Analysis of the immune microenvironment revealed significant changes in macrophage populations and function following RT, including reduced circulating macrophages (F4/80), increased numbers of mature tissue-resident macrophages (CD169), and enhanced polarization of monocyte-derived macrophages toward M1-like phenotypes. Immunofluorescence staining confirmed these changes (p < 0.05, n = 9/group). CellChat analysis revealed enhanced intercellular communication among macrophages and between macrophages and other cell types in the irradiated microenvironment. These immune changes were associated with increased osteoclast differentiation. Specifically, injury sites with RT exhibited higher osteoclast numbers one-week post-injury, accompanied by upregulation of key osteoclast differentiation transcription factors, including Tnf, Tnfrs11a, Fos, Jun, and Nfkb1, as identified by scRNA-seq. Despite these alterations, multiplex cytokine and chemokine analysis showed no significant differences with and without RT at the same time point. Our findings suggest that RT modulates the immune microenvironment by altering macrophage polarization and promoting osteoclast differentiation, potentially contributing to reduced HO formation. Future studies aim to validate these mechanisms and leverage these insights to optimize RT protocols and to develop safer, more effective HO prevention strategies. Meng-Lun Hsieh (she/her/hers), meng-lun.hsieh@utsouthwestern.edu (Presenting Author) - UTSW; Sneha Korlakunta, BS (Co-Author) - University of Texas Southwestern Medical Center; Ji Hae Choi, BS (Co-Author) - University of Texas Southwestern Medical Center; Stefanie Moye, BS (Co-Author) - University of Texas Southwestern Medical Center; Ciara Newman, BS (Co-Author) - University of Texas Southwestern Medical Center; Benjamin Levi, MD (Co-Author) - University of Texas Southwestern Medical Center; Yuanyuan Zhang, MD (Co-Author) - University of Texas Southwestern Medical Center