Fig. 4
Combined delivery of bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor A (VEGFA) induced ectopic bone formation in scaffold explants in a subcutaneous NOD/SCID mouse model. The ability of VEGFA alone or in combination with BMP2 to induce ectopic bone formation was investigated in NOD/SCID mice by subcutaneously implanting scaffolds seeded with 5 × 105 BMSC from different experimental groups. Scaffold explants at 8 weeks were examined by μCT to evaluate ectopic bone formation. Negligible amounts of radiopaque bone-like structures were detected in a untransduced, b ad-GFP, and c ad-VEGFA BMSC. In contrast, numerous bony-like radiopaque structures were found at the periphery as well as within the scaffolds of ad-BMP2 + VEGFA explants (d, cross sectional view in e). f Quantification of the radiopaque bone-like structures in the scaffold explants revealed significant amounts of bone formation in ad-BMP2 + VEGFA scaffolds. g–n Representative images of H&E stained FFPE sections of scaffold explants from different experimental groups at 2 and 8 weeks. g–l Formation of bony structures was not detected in untransduced, ad-GFP, or ad-VEGFA groups at either 2 or 8 weeks. m In the ad-BMP2 + VEGFA BMSC group, formation of bony structures (black arrows) could be seen as early as 2 weeks, mostly at the periphery of the scaffold explants. n Extensive bone formation (green arrows) with bony trabeculae extending inside the scaffolds was found at 8 weeks in ad-BMP2 + VEGFA BMSC. The structure consisted of numerous osteocyte-like cells at both 2 (black arrowheads, inset in m) and 8 weeks (green arrowheads, inset in n). ANOVA with Bonferroni post hoc analysis was carried out for statistical analysis in (f). Error bars represent SEM. **p < 0.01. ad adenoviral, GFP green fluorescent protein