Summary: | 博士 === 長庚大學 === 生物醫學研究所 === 102 === Hyperbaric oxygenation (HBO) was shown to increase bone healing in a rabbit model. We hypothesized that the effect of HBO on bone formation is increased by osteogenic differentiation of the bone marrow mesenchymal stem cells (MSCs) through Wnt3a/β-catenin signaling. Our in vitro data showed that HBO increased cell proliferation, Wnt3a production, LRP6 phosphorylation, and cyclin D1 expression in osteogenically differentiated MSCs. The mRNA and protein levels of Wnt3a, β-catenin, and Runx2 were upregulated while those of GSK-3β were downregulated after HBO treatment. The relative density ratio (phospho-protein/protein) of Akt and GSK-3β was both up-regulated while that of β-catenin was down-regulated after HBO treatment. We next investigated whether HBO affects the accumulation of β-catenin. Western blot analysis showed increased levels of nucleus translocated β-catenin and the expression levels of its target genes after HBO treatment. HBO increased TCF-dependent transcription, Runx2 promoter/Luc gene activity, and the expression of MSC osteogenic markers, such as alkaline phosphatase, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the production of bone morphogenetic protein (BMP2) and osterix. We further showed that HBO increased the expression of Wntless and vacuolar-ATPases, and in turn, stimulated transportation and secretion of Wnt3a in MSCs. The involvement of Wnt3a/β-catenin in promoted bone formation was demonstrated in vivo in a rabbit model by histology, mechanical testing, and immunohistochemical assays.
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