Abstract
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.
Highlights
Bone formation and resorption depend on vessels to supply osteoblast and osteoclast progenitors, oxygen, and nutrients and guide bone formation by direct interaction with mature osteoblasts and osteoclasts [1]
Low blood supply or disturbed interaction between vessels and bone-forming cells leads to pathological conditions such as avascular bone necrosis (AVN) or fracture nonunion, in which bone does not heal after a fracture
In this study we demonstrated the use of ink-gelatin and phosphotungstic acid (PTA) as contrast enhancement for μCT analysis of bone vasculature
Summary
Bone formation and resorption depend on vessels to supply osteoblast and osteoclast progenitors, oxygen, and nutrients and guide bone formation by direct interaction with mature osteoblasts and osteoclasts [1]. Low blood supply or disturbed interaction between vessels and bone-forming cells leads to pathological conditions such as avascular bone necrosis (AVN) or fracture nonunion, in which bone does not heal after a fracture. In these cases, the required bone reconstruction is based on the use of autologous bone grafts, which generate additional morbidity and are limited by low availability [2, 3]. Analysis of spatial relationships between vessels and bone trabeculae, and calculation of blood flow are nontrivial Intravital observations such as two-photon microscopy are limited in depth and resolution and need a very thin bone cortex and sedation of animals [7].
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