Hypoxia decreases osteogenic differentiation potential but enhances vasculogenic differentiation potential of human adipose stem cells on biphasic calcium phosphate scaffolds in fibrin gels

Background: In bone tissue engineering, biphasic calcium phosphate (BCP) with different hydroxyapatite (HA)/ß-tricalcium phosphate (ß-TCP) ratios can be combined with regeneration-competent stem cells, and bioscaffolds like fibrin that trigger neovascularization. We have shown recently that human adipose stem cells (hASCs) on BCP with a HA/ß-TCP ratio of 20/80 (BCP20/80) incorporated in fibrin gel show enhanced osteogenic and/or vasculogenic differentiation potential compared to BCP with a HA/ß-TCP ratio of 60/40 (BCP60/40) in vitro. A hypoxic microenvironment for the cells might occur in these hASC-seeded BCP-based composites after implantation in vivo. This limits oxygen supply which is essential for successful bone tissue engineering. Objectives: Here we aimed to test the effect of hypoxia on osteogenic and/or vasculogenic differentiation of hASC-seeded BCP60/40 or BCP20/80 scaffolds in fibrin gels as well as fibrin gel degradation. Methods: BCP60/40-based composites and BCP20/80-based composites were cultured under normoxia (20% O₂) or hypoxia (1% O₂) for 12 days, and total DNA content, alkaline phosphatase activity, nitric oxide production, vascular endothelial growth factor production, and fibrin gel degradation were assessed during culture. Results: Hypoxia similarly decreased total DNA content at day 7 (1.3-fold) and 12 (1.2-fold) in both composites. It also decreased alkaline phosphatase activity at day 5 (5.0 and 4.7-fold), day 7 (4.5 and 6-fold), and 12 (7.1 and 6.8-fold) in BCP60/40-based composites and BCP20/80-based composites. Hypoxia similarly increased vascular endothelial growth factor production at day 5 (3.9-fold) and day 7 (2.6-fold) by both BCP-based composites. Hypoxia did not affect nitric oxide production and fibrin gel degradation in both BCP-based composites at all-time points. Conclusion: Hypoxia decreased osteogenic differentiation but enhanced vasculogenic differentiation of cultured BCP-based composites. These in vitro results implicate that cell-seeded BCP-based composites implanted in vivo, might enhance vascular endothelial growth factor production resulting in sprouting and tube formation leading to increased survival of cells in these composites, and stimulation of bone formation.