title: Hyperbaric oxygen promotes osteogenic differentiation of bone marrow stromal cells by regulating Wnt3a/β-catenin signaling--an in vitro and in vivo study.
abstract: We hypothesized that the effect of hyperbaric oxygen (HBO) on bone formation is increased via osteogenic differentiation of bone marrow stromal cells (BMSCs), which is regulated by 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 BMSCs. 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. 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 BMSCs, such as alkaline phosphatase activity, type I collagen, osteocalcin, calcium, and the intensity of Alizarin Red staining. HBO dose dependently increased the bone morphogenetic protein (BMP2) and osterix production. We further demonstrated that HBO increased the expression of vacuolar-ATPases, which stimulated Wnt3a secretion from BMSCs. Finally, we showed that the beneficial effects of HBO on bone formation were related to Wnt3a/β-catenin signaling in a rabbit model by histology, mechanical testing, and immunohistochemical assays. Accordingly, we concluded that HBO increased the osteogenic differentiation of BMSCs by regulating Wnt3a secretion and signaling.
authors.affiliation: Institute of Biomedical Sciences, Chang Gung University, Taoyuan, Taiwan; Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Song-Shu
authors.initials: SS
authors.lastname: Lin
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Steve W N
authors.initials: SW
authors.lastname: Ueng
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Chi-Chien
authors.initials: CC
authors.lastname: Niu
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Li-Jen
authors.initials: LJ
authors.lastname: Yuan
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Chuen-Yung
authors.initials: CY
authors.lastname: Yang
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Taoyuan, Taiwan.
authors.forename: Wen-Jer
authors.initials: WJ
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics, Chang Gung Memorial Hospital, Chiayi, Taiwan.
authors.forename: Mel S
authors.initials: MS
authors.lastname: Lee
authors.affiliation: Department of Physiology, Chang Gung University, Taoyuan, Taiwan. Electronic address: jkc508@mail.cgu.edu.tw.
authors.forename: Jan-Kan
authors.initials: JK
authors.lastname: Chen
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D006931:Hyperbaric Oxygenation; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000235:genetics / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects*; D010100:Oxygen / Q000494:pharmacology*; D034622:RNA Interference; D011817:Rabbits; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects*; D060509:Wnt3A Protein / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; C555475:LRP6 protein, human; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C493709:RUNX2 protein, human; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D060509:Wnt3A Protein; D051176:beta Catenin; D015675:Osteocalcin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D010100:Oxygen