Tissue engineering of a vascularized bone graft of critical size with an osteogenic and angiogenic factor-based in vivo bioreactor.

Engineering a large vascularized bone graft is a much greater challenge than engineering small bone tissues. Although this is essentially feasible through an osteogenic factor-based in vivo bioreactor technique, the ossification needs improving. This study was aimed to investigate the possibility and efficacy of ectopic cultivation of sizeable bone grafts with large angiogenic and osteogenic factor-loaded natural bovine bone mineral (NBBM) scaffolds. For this purpose, six groups of sizeable composite scaffolds were constructed, consisting of a titanium mesh cage of NBBM or a mixture of NBBM/autogenous bone particles (AB), which were preloaded with 660 μg recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or 4 μg recombinant human vascular endothelial growth factor165 (rhVEGF165). The scaffolds were implanted in bilateral latissimus dorsi muscles in eight pigs to construct in vivo bioreactors. Sequential fluorescence labeling was then applied to trace bone formation at the early stage. The implants were retrieved 12 weeks later. The undecalcified sections were observed in turn under the fluorescence microscope and light microscope to investigate early stage osteogenesis and histology. Moreover, new bone density (BD) was measured with histomorphometry. Compared with rhBMP-7-delivered NBBM scaffolds, rhVEGF165/rhBMP-7-delivered NBBM scaffolds were with more intense intra-scaffold osteogenesis at the early stage and the ultimate sizeable bone grafts of microstructurally more lamellae and trabeculae, and quantitatively higher BD (31.93% vs. 22.37%, p<0.01). This study demonstrated that as for the endocultivation of a large bone graft with bioactive factor-based in vivo bioreactor technique, dual delivery of rhVEGF165/rhBMP-7 has synergic effects on improving early stage bone formation and subsequently bone quality and quantity of the bone grafts.