Contrast-Enhanced Microtomographic Characterisation of Vessels in Native Bone and Engineered Vascularised Grafts Using Ink-Gelatin Perfusion and Phosphotungstic Acid

OBJECTIVES: Bone ischemia and necrosis are challenging to treat, requiring investigation of native and engineered bone revascularisation processes through advanced imaging techniques. This study demonstrates an experimental two-step method for precise bone and vessel analysis in native bones or vascularised bone grafts using X-ray microtomography (μCT), without interfering with further histological processing. METHODS: Distally ligated epigastric arteries or veins of 6 nude rats were inserted in central channels of porous hydroxyapatite cylinders and these pedicled grafts were implanted subcutaneously. One week later, the rats were perfused with ink-gelatin and euthanised and the femurs, tibias, and grafts were explanted. Samples were scanned using μCT, decalcified, incubated with phosphotungstic acid (PTA) for contrast enhancement, rescanned, and processed histologically. RESULTS: Contrast-enhanced μCT displayed the course and branching of native bone vessels. Histologically, both central (−17%) and epiphyseal vessels (−58%) appeared smaller than in μCT scans. Hydroxyapatite cylinders were thoroughly vascularised but did not display bone formation. Grafts with a central artery had more (+58%) and smaller (−52%) vessel branches compared to grafts with a vein. CONCLUSIONS: We present a relatively inexpensive and easy-to-perform two-step method to analyse bone and vessels by μCT, suitable to assess a variety of bone-regenerative strategies.