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Angiogenesis is essential for skeletal development, bone healing, and regeneration. Improved non-destructive, three-dimensional (3D) imaging of the vasculature within bone tissue would benefit many research areas, especially implantology and tissue engineering.

X-ray microtomography (microCT) is a well-suited non-destructive 3D imaging technique for bone morphology. For microCT-based detection of vessels, it is paramount to use contrast enhancement. Limited differences in radiopacity between perfusion agents and mineralized bone make their distinct segmentation problematic and have been a major drawback of this approach. A decalcification step resolves this issue but inhibits the simultaneous assessment of bone microstructure and vascular morphology. The problem of contrasting becomes further compounded in samples with metal implants.

This study describes μAngiofil-enhanced microCT-based visualization of vasculature within bone tissue in small and large animal models, with and without decalcification. We present simultaneous microvascular and bone imaging in murine tibia, a murine bone metastatic model, the pulp chamber, gingiva, and periodontal ligaments. In a large animal model (minipig), we perform visualization and segmentation of different tissue types and vessels in the hemimandible containing metal implants. Moreover, we show the potential of the dual-energy approach in facilitating the distinction between bone tissue and the applied contrast agent.

Our manuscript introduces the first non-destructive approach for 3D imaging of the vasculature within soft and hard tissues in the vicinity of metal implants in a large animal model.