Optimizing Quantification of H-Vessel Proliferation in an Irradiated Mandibular Fracture Callus Following Deferoxamine Administration

PURPOSE: A recent discovery identified H-vessels as an essential capillary subtype in the process of angiogenic-osteogenic coupling, making them an important biomarker of bone healing. H-vessels are critically deficient in the setting of radiation, which is associated with pathologic fractures and chronic non-union. We hypothesize that injected deferoxamine (DFO) will promote upregulation of type H vasculature within the callus of an irradiated mandible fracture and thereby enhance angiogenic-mediated bone healing. With this study, we demonstrate the use of an optimized FFPE immunofluorescent staining protocol to quantify the presence of H-vessels in a mandibular fracture callus during irradiated bone healing. METHODS: We examined FFPE specimens in a rat model of mandibular bone healing across a range of fracture conditions, including healthy bone (Fx), irradiated bone (XFx), and irradiated bone treated with DFO (XFx-DFO). H-vessel area fraction was determined by quantifying pixel-positive regions of double EMCN+/CD31+ signal. RESULTS: Quantitative analysis revealed an association between DFO administration during fracture repair and H-vessel abundance within the callus. No significant difference was found between the Fx and XFx groups, but the relative diminution of H-vessels in the radiated setting was a clear trend. CONCLUSION: Results of this study support our hypothesis that injected DFO promotes upregulation of type H vasculature within the fracture callus of irradiated bone, portending therapeutic benefit to patients with radiation-induced bone injury.