Background: There is a critical need for management of vascularization in bone tissue engineering. The purpose of this study was to use Hypoxia-Inducible Factor-1α (HIF-1α) transduced Bone Marrow Mesenchymal Stem Cells(BMSCs) to fabricate prevascularized osteogenic cell sheets In vitro and explore HIF-1α promoted osteogenic-angiogenic coupling response of BMSCs cell sheets.

Methods: HIF-1α was over-expressed by using a lentiviral vector which encoded Green Fluorescent Protein(GFP) and transduced steadily in Wistar rats BMSCs to form HIF-1α/BMSCs. While empty lentiviral transduced BMSCs(Empty/BMSCs) were as a negative control group and BMSCs without transduced were as a blank control group. Fluorescence microscope was used to detect GFP expressions, meanwhile Real-time quantitative and western blot were performed to assess the expression level of HIF-1α. Next, HIF-1α/BMSCs were cultured to form Osteogenic Cell Sheets (OCTs) with a density of 1 × 105/cm2 under osteogenesis medium. Then, Alkaline Phosphatase staining (ALP) at day 14 and Alizarin-red staining at day 21 were performed to check the characteristic of osteogenesis. Empty/BMSCs formed OCTs served as control. Simultaneously, HIF-1α/BMSCs were induced to differentiate into endothelial-like cells(i-ECs) for 14 days, and the conversion rates were performed by Flow Cytometry assay. At the same time, we used Transwell assay to detect whether HIF-1α could migrate i-ECs In vitro. Finally, i-ECs were seeded onto OCTs with a density of 5 × 104/cm2 to fabricate Prevascularized- Osteogenic Cell Sheets (P-OCTs). In order to detect the role of HIF-1α involved in osteogenic-angiogenic coupling response of P-OCTs, Immunofluorescent staining for CD31 was performed at 1,3,7,14 days to check i-ECs migration and networks formation, and western blot of Osteopontin (OPN) and Ssteocalcin (OCN) at 1,7,14 days to check bone formation. Meanwhile, Empty/BMSCs formed P-OCTs were as control.

Conclusion: BMSCs were transduced by Lenti-HIF-1α with optimal multiplicity of infection was 30(MOI = 30) and the GFP expression over 90%. At the same time, qPCR and western blot confirmed a high HIF-1α expression in HIF-1α/BMSCs group, which had a statistic significance among BMSCs group and Empty/BMSCs group (p < 0.05). Next, Flow Cytometric analysis results showed the conversion rate of HIF-1α/BMSCs to i-ECs was 92.43% in experimental group (p < 0.05), which indicated BMSCs transduced by HIF-1α had a great superiority to differentiate into endothelial cells under experimental conditions. Transwell assay showed that HIF-1α could recruit i-ECs in vitro, with an average of over 400 i-ECs migrating per field of view in HIF-1α/BMSCs group. Meanwhile no migrating i-ECs in Empty/BMSCs group (p < 0.05). On the other hand, ALP at day 14 and Alizarin-red staining at day 21 of OCTs in HIF-1α/BMSCs group showed an obviously osteogenic differentiation characteristic with more deep stained calcium nodules deposits than Empty/BMSCs group (p < 0.05). Finally, we fabricated P-OCTs and detected angiogenesis by Immunofluorescent staining for CD31 at day 1,3,7,14, which showed i-ECs migrated reticulated fast and formed a large number of lumens and networks in HIF-1α/BMSCs group. While i-ECs migrated slowly and the lumens and networks formation was limited in Empty/BMSCs group (p < 0.05). At the same time, the expressions of OCN, OPN at day 1,7,14 showed that HIF-1α could promote osteogenic response in P-OCTs significantly in vitro (p < 0.05). All in all, the over-expressed HIF-1α of BMSCs cell sheets strategy can provide a new promising method for bone engineering, which could promote osteogenic-angiogenic coupling response In vitro.