Hypoxia improved vasculogenesis in distraction osteogenesis through Mesenchymal-Epithelial transition (MET), Wnt/β-catenin signaling pathway, and autophagy

Introduction: The osteogenesis rate of distraction osteogenesis is 4-6 times faster than that of infants, far beyond fracture healing. However, the osteogenesis mechanism of DO is complicated and inconclusive owing to two significant elements: mechanical tension which is well explored and trauma caused by bone fracture. Vasculogenesis and EPCs are critical for successful bone regeneration during DO. Thus, this study aimed to explore the effects of hypoxia caused by trauma or CoCl2 on the vasculogenesis of DO and EPCs. Material and methods: Mandibular DO and BF models were generated using 6 beagle dogs with a distraction rate of 1 mm per day for 7 days or acute lengthening for 7 mm. The vasculogenesis in DO-gap or BF-gap were assessed via histological analyses, qRT-PCR and immunofluorescence staining. Dog bone marrow EPCs were isolated and cultured with or without 0.1 mM CoCl2. The effect of hypoxia caused by CoCl2 were subsequently valuated via in vitro assays including Cell Counting Kit-8, transwell assay, qRT-PCR, western blot, and immunofluorescence staining. Results: Histological analyses, qRT-PCR and immunofluorescence staining revealed that vasculogenesis markedly accelerated in DO-gap compared with BF-gap, and the DO-gap displayed more positive to CD133, CD34, HIF-1 alpha, E-cadherin, beclin1, beta-catenin, VEGF, bFGF, and less positive to ZEB1 than BF-gap. In addition, in vitro analyses revealed CoCl2 treatment enhanced EPCs proliferation and migration, and the levels of HIF-1 alpha, E-cadherin, beta-catenin, beclin1, VEGF, bFGF of EPCs were increased, but the level of ZEB1 was decreased. Conclusion: Our studies showed that hypoxia promoted vasculogenesis in DO and EPCs, and the mechanism may involve autophagy, Wnt/beta-catenin signaling pathway, and Mesenchymal-Epithelial transition (MET).