Elucidating the Mechanism of Radiation Therapy on Mesenchymal Cell Fate in Preventing Heterotopic Ossification

OBJECTIVE: To recapitulate the use of radiation in preventing heterotopic ossification (HO) in an animal model to thereby mechanistically investigate radiation-induced changes at the single-cell level. BACKGROUND: HO is the formation of extra-skeletal bone in abnormal areas including muscle and soft tissue. Radiation therapy is a clinically proven, localized preventive measure for HO. Despite its efficacy, there is a lack of standardization of radiation prescription; however, the mechanism of the impact of radiation on HO prevention remains unknown. METHODS: C57BL6J male mice underwent burn/tenotomy with and without perioperative radiation treatment. Single-cell RNA sequencing was performed to analyze downstream signaling after HO-forming injury. Immunofluorescence microscopy was used to visualize protein expression changes in HO progenitor cells. In vivo range of motion analyses, histological staining, and micro-computerized tomography were performed to investigate mature HO's effect on joint function and to characterize total HO structure and volume. RESULTS: In one fraction, 7 Gy delivered to the injury site within 72 hours postoperatively significantly decreases HO formation and improves hindlimb range of motion. In-depth single-cell transcriptomic analyses with immunofluorescent staining demonstrate decreased cellular numbers, as well as aberrant endochondral differentiation and downregulation of associated upstream BMP and ALK4 signaling pathways in irradiated mesenchymal progenitor cells. CONCLUSIONS: Our study is the first to explore the mechanism of radiotherapy prophylaxis in the prevention of traumatic HO. Not only does radiation decreases total HO progenitor cell numbers but also reduces aberrant osteochondral differentiation at the injury site, thereby decreasing overall HO and improving joint function.