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Optimizing Quantification Of H-Vessel Proliferation In An Irradiated Mandibular Fracture Callus Following Deferoxamine Administration
Nathan Sheppard, B.Arch., Melissa Daniel, MD, MPH, Noah Nelson, MPH, Alex Donneys, MD, Steven Buchman, MD;
University of Michigan, Ann Arbor, MI, USA

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.

Statistical Analysis of H-vessel Area Fraction using Tukey Multiple Comparisons Test

	Fx, n=5	XFx, n=5	XFx-DFO, n=5
Mean H-vessel Area Fraction	.000325370	0.000093582	0.003785198
	Difference	95% CI	p-value
Fx vs. XFx	0.000232	-0.002447 to 0.002911	p=0.9711
XFx vs. XFx-DFO	0.003692	0.001012 to 0.006371	p=0.0082
Fx vs. XFx-DFO	0.003460	0.000781 to 0.006139	p=0.0125

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