Periostin regulates osteogenesis of mesenchymal stem cells from ovariectomized rats through actions on the ILK/Akt/GSK-3β Axis

Figures

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  Figure 1 -. Ovariectomy leads to osteoporosis in rats. (A-D) Representative MicroCT images of distal femurs. (A) and (B) are from rats in the sham group. (C) and (D) are from rats in the OVX group. Scale bar = 1 mm.

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  Figure 2 -. Identification of BMSCs by flow cytometry analysis. Representative images of flow cytometry analysis. Sham-BMSCs were positive for the surface antigens CD29 (97.98%) and CD90 (99.34%) and negative for CD34 (1.92%) and CD45 (0.94%). OVX-BMSCs were positive for the surface antigens CD29 (97.74%) and CD90 (99.37%) and negative for CD34 (0.77%) and CD45 (0.82%).

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  Figure 3 -. BMSCs derived from OVX rats exhibited impaired osteogenesis ability, adipogenesis ability and reduced expression of periostin. (A-B’) Representative images of ALP staining on day 7 after osteogenesis induction. (A, A’) Sham-BMSCs express ALP, while (B, B’) OVX-BMSCs express less ALP. (C) Graph shows the quantification of the relative expression level of periostin on day 7 after osteogenesis induction. Data are presented as the mean ± SD. *** P < 0.001. (D-E’) Representative images of oil red O staining on day 14 after adipogenesis induction. (D, D’) Sham-BMSCs exhibited intracellular lipid droplets. (E, E’) OVX-BMSCs exhibited less lipid droplets. The images are from 1 of 3 independent experiments. (A, B, D, E) Magnification × 40, scale bar = 100 μm. (A’, B’, D’, E’) Magnification × 100, scale bar = 200 μm.

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  Figure 4 -. Overexpression of periostin elevated periostin mRNA and protein levels after osteogenesis induction. (A) The graph shows the quantification of the relative expression levels of periostin in the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction. (B) The expression levels of periostin in the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction were investigated using western blotting; β-actin served as the reference gene. The image is representative of 3 independent experiments. (C) Quantitative western blotting was performed using Image J software. Data are presented as the mean ± SD of 3 independent experiments. ns, not significant. ** P < 0.001; One-way ANOVA.

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  Figure 5 -. Overexpression of periostin promotes ALP production by OVX-BMSCs. (A) Representative images of ALP staining of the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction. The images are from 1 of 3 independent experiments; Magnification × 40, scale bar = 200 μm. (B) Graph shows quantitative analyses of ALP activity of the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction. Data are presented as the mean ± SD of 3 independent experiments. ns, not significant. * P < 0.05, *** P < 0.001; One-way ANOVA.

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  Figure 6 -. Overexpression of periostin increased ARS staining in OVX-BMSCs. (A) Representative images of ARS staining of the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction. The images are from 1 of 3 independent experiments, magnification × 40, scale bar = 200 μm. (B) Graph showing quantitative analyses of ARS of the indicated groups on day 0, day 7, day 14 and day 21 after osteogenesis induction. Data are presented as the mean ± SD of 3 independent experiments. ns, not significant. *** P < 0.001; One-way ANOVA.

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  Figure 7 -. Periostin regulates the expression of ILK, pAkt and GSK3β in OVX-BMSCs. (A) The expression levels of periostin, ILK, GSK3β, pGSK3β, Akt, pAkt in the indicated groups on day 7 after osteogenesis induction were examined by western blotting; β-actin served as the reference gene. (B) Quantitation of western blotting in (A) was performed using Image J software. (C) Graphs showing the relative mRNA expression levels of periostin, OCN, BSP and Runx2 in the indicated groups on day 7 after osteogenesis induction. Data are presented as the mean ± SD of 3 independent experiments. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001; One-way ANOVA.

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  Figure 8 -. Periostin promotes the osteogenesis ability of OVX-BMSCs in part by action on the ILK/Akt/GSK3 axis. (A) Representative images of ALP staining of the indicated groups on day 7 after osteogenesis induction. The images are from 1 of 3 independent experiments, magnification × 40. (B) Representative images of ARS staining of the indicated groups on day 7 after osteogenesis induction, magnification × 40, scale bar = 200 μm.