Background: Hypoxia induces mesenchymal stem cells (MSCs) proliferation but results in a population with impaired osteogenic differentiation potential. Bone healing of tibial lengthening is enhanced by hyperbaric oxygen therapy. However, little is known about the effects of HBO on the Wnt signaling pathway in MSCs.
Materials and methods: MSCs were cultured in complete medium and the osteogenic groups were cultured in osteogenic induction medium. Control cells were maintained in 5% CO2/95% air throughout the experiment. The hyperoxic cells were exposed to 100% O2 for 25 min and then to 5% CO2/95% air for 5 min at 2.5 ATA (atmospheres absolute) in a hyperbaric chamber. Cell proliferation was quantified using the WST-1 cell proliferation reagent. The mRNA and protein levels of Wnt3a, sclerostin, β-catenin, GSK-3β, Runx2, as well as alkaline phosphatase activity and calcium deposition were analyzed after HBO treatment. We investigated whether HBO affects the translocation of β-catenin between nucleus and cytosol by western blot. Alizarin Red staining was tested in osteogenetic differentiation MSCs after HBO treatment.
Results: Our data showed that HBO increased cell proliferation in osteogenically differentiated MSCs. The mRNA and protein levels of Wnt3a, β-catenin, and Runx2 were upregulated while sclerostin and GSK-3β were downregulated after HBO treatment. The relative density ratio (phosho-protein/protein) for GSK-3β was up-regulated while β-catenin was down-regulated after HBO treatment. Our western blot analysis showed increased levels of translocated β-catenin that stimulated the expression of target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of osteogenic markers of MSCs, such as alkaline phosphatase activity, osteocalcin, calcium, and the intensity of Alizarin Red staining.
Conclusion: Hyperbaric oxygen promotes osteogenic differentiation of MSCs by regulating sclerostin expression and Wnt/β-catenin signaling.
17 May 2014 - 20 May 2014