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Avascular necrosis of bone (AVN) leads to sclerosis and collapse of bone and joints. We have previously shown that axially vascularized osteogenic constructs, engineered by combining human stromal vascular fraction (SVF) cells and a ceramic scaffold, can revitalize necrotic bone of clinically relevant size in a rat model of AVN. For a clinical translation, the fetal bovine serum (FBS) used to generate such grafts should be substituted by a nonxenogeneic culture supplement. Human thrombin‐activated platelet‐rich plasma (tPRP) was evaluated in this context. SVF cells were cultured inside porous hydroxyapatite scaffolds with a perfusion‐based bioreactor system for 5 days. The culture medium was supplemented with either 10% FBS or 10% tPRP. The resulting constructs were inserted into devitalized bovine bone cylinders to mimic the treatment of a necrotic bone. A ligated vascular bundle was inserted into the constructs upon subcutaneous implantation in the groin of nude rats. After 1 and 8 weeks, constructs were harvested, and vascularization, host cell recruitment, and bone formation were analyzed. After 1 week in vivo, constructs were densely vascularized, with no difference between tPRP‐ and FBS‐based ones. After 8 weeks, bone formation and vascularization was found in both tPRP‐ and FBS‐precultured constructs. However, the amount of bone and the vessel density were respectively 2.2‐ and 1.8‐fold higher in the tPRP group. Interestingly, the density of M2, proregenerative macrophages was also significantly higher (6.9‐fold) following graft preparation with tPRP than with FBS. Our findings indicate that tPRP is a suitable substitute for FBS to generate vascularized, osteogenic grafts from SVF cells and could thus be implemented in protocols for clinical translation of this strategy towards the treatment of bone loss and AVN.

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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.