JOURNAL ARTICLE

[Comparative study between hypoxia and hypoxia mimetic agents on osteogenesis of bone marrow mesenchymal stem cells in mouse]

Lei Zhang, Yuekun Gong, Xueling Zhao, Houjun Zhou
Chinese Journal of Reparative and Reconstructive Surgery 2016 July 8, 30 (7): 903-908
29786329

OBJECTIVE: ?To compare the effects on the osteogenesis of bone marrow mesenchymal stem cells (BMSCs) between hypoxia and hypoxia mimetic agents dimethyloxalylglycine (DMOG) under normal oxygen condition.

METHODS: ?BMSCs were isolated and cultured from healthy 3-4 weeks old Kunming mouse. Cell phenotype of CD29, CD44, CD90, and CD34 was assayed with flow cytometry; after osteogenic, adipogenic, and chondrogenic induction, alizarin red staining, oil red O staining, and toluidine blue staining were performed. The passage 3 BMSCs were cultured under normal oxygen in control group (group A), under 1%O2 in hypoxia group (group B), and under normal oxygen and 0.5 mmol/L DMOG in DMOG intervention group (group C). BMSCs proliferation was estimated by methyl thiazolyl tetrazolium assay at 1, 2, 3, and 4 days. Alkaline phophatase (ALP) expression was determined at 7 and 14 days after osteogenic induction. Western blot was employed for detecting hypoxia inducible factor-1α (HIF-1α) at 24 hours. Real time fluorescence quantitative PCR was employed for detecting the mRNA expression of runt-related transcription factor 2 (RUNX2) and Osterix at 3 and 7 days. Alizarin red staining was applied to assess the deposition of calcium tubercle at 21 days.

RESULTS: ?The BMSCs presented CD29(+), CD44(+), CD90(+), and CD34(-); and results of the alizarin red staining, oil red O staining, and toluidine blue staining were positive after osteogenic, adipogenic, and chondrogenic induction. No significant difference in BMSCs proliferation was observed among 3 groups at 1 day (P>0.05); compared with group A, BMSCs proliferation was inhibited in group C at 2, 3, and 4 days, but no significant difference was observed (P>0.05); compared with group A, BMSCs proliferation was significantly promoted in group B (P<0.05). At each time point, compared with group A, the ALP expression, HIF-1α protein relative expression, and mRNA relative expressions of RUNX2 and Osterix were significantly up-regulated in groups B and C (P<0.05); compared with group B, the ALP expression, the RUNX2 and Osterix mRNA relative expression were significantly up-regulated in group C (P<0.05); compared with group C, the HIF-1α protein relative expression was significantly up-regulated in group B (P<0.05). The alizarin red staining showed little red staining materials in group A, some red staining materials in group B, and a large number of red staining materials in group C.

CONCLUSIONS: ?Hypoxia can promote BMSCs proliferation, DMOG can not influence the BMSCs proliferation; both hypoxia and DMOG can improve osteogenic differentiation of BMSCs, and DMOG is better than hypoxia in improving the BMSCs osteogenesis.

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