Unraveling the Mechanism behind Prophylactic Radiotherapy to Prevent Traumatic Heterotopic Ossification

Abstract

Purpose/Objective(s)

Heterotopic ossification (HO) is the formation of extra-skeletal bone in abnormal areas including muscle and soft tissue. Once HO forms, surgical excision is the only option which unfortunately frequently leads to recurrence. Radiation has been one of the most effective prophylactic treatments for HO, however, the mechanism remains unknown despite multiple randomized controlled trials demonstrating radiation efficacy. The purpose of this study is to recapitulate the use of radiation in preventing heterotopic ossification in an animal model to thereby mechanistically investigate radiation-induced changes in gene and protein expression changes at the single cell level.

Materials/Methods

We established a traumatic HO burn/tenotomy mouse model demonstrating decreased HO formation with radiation therapy. Single-cell RNA sequencing (scRNA-seq) of the Achilles tenotomy injury site at 7 days post-injury with and without radiation treatment was performed as an unbiased approach to determine radiation effects on gene expression at the single cell level. Further scRNA-seq analyses revealed distinct cell type clustering as well as differentially expressed genes and enriched pathways. Immunofluorescent histology of the injury site at 7 days post-injury was performed to confirm protein expression changes and scRNA-seq analyses.

Results

There was 7 Gy in one fraction delivered 72 hours perioperatively to the injury site decreased HO formation by approximately 50% compared to control group in a burn/tenotomy traumatic HO mouse model (P < 0.05, n = 10/group). Tendon-associated HO superior to the radiation field demonstrated no difference in HO volume between control and radiated groups (P = ns, n = 10/group). ScRNA-seq identified 10 distinct cell clusters in both control and radiated groups. Further analyses revealed decreased major transcription factors for osteogenic (Runx2) and chondrogenic (Sox9) gene expression in irradiated HO progenitor cells. Immunofluorescence of the injured hindlimb also reveal decreased RUNX2 and SOX9 signaling with radiation treatment (P < 0.05, n = 9/group). Our scRNA-seq analyses also demonstrated downregulated Alk4 and BMP HO signaling pathways with radiation treatment, leading to decreased HO formation.

Conclusion

Our study is the first to explore the mechanism of radiotherapy prophylaxis in the prevention of traumatic HO. Our findings reveal that radiation reduces aberrant osteochondral differentiation of HO progenitor cells, thereby decreasing overall HO and improving joint function. Future studies will further elucidate the key pathways and optimize the timing and dosage of radiation prophylaxis to mitigate HO.