Periostin Mediates Oestrogen‐Induced Osteogenic Differentiation of Bone Marrow Stromal Cells in Ovariectomised Rats

Figures

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  Figure 1. Effect of 17β-oestradiol (17β-E2) on bones in ovariectomised rats. (a) Timeline of the experiment. (b) Three-dimensional computed tomography images of the distal femurs from representative rats of different ages and with different interventions. (c) BV/TV, CtTh, and BMD of the distal femur of 16- and 20-week-old rats determined via micro-CT. (d) Longitudinal BV/TV, CtTh, and BMD of the distal femur of OVX-rats determined via micro-CT (n = 6/group). Bars represent the means ± SD. ∗P < 0.05, ∗∗P < 0.01. BV/TV: bone volume fraction; CtTh: cortical thickness; BMD: bone mineral density.

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  Figure 2. Bone formation and POSTN expression are increased by 17β-oestradiol (17β-E2) in the endosteum of ovariectomised rats. (a) Timeline of the experiment. (b) MAR, MS, and BFR, as determined via calcein (green) and xylenol orange (red) staining, are shown in the photomicrographs (scale bar: 20 μm) in longitudinal undecalcified sections of femurs from 18-week-old female rats. (c) Immunohistochemical staining of POSTN in longitudinal sections of the proximal femur of the rats 6 weeks after 17β-E2 treatment shows increased POSTN expression at the endosteum (Es). Magnification: ×10. In the photomicrographs, POSTN is indicated by the arrows (n = 6/group). Bars represent the means ± SD. ∗P < 0.05. NS: not significant; MAR: mineral apposition rate; MS/BS: mineralised surface area; BFR: bone formation rate.

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  Figure 3. POSTN protein expression in response to 17β-oestradiol (17β-E2) was increased in vitro. (a) Immunofluorescence of POSTN (red) localization in BMSCs. Magnification: ×40. POSTN IOD quantification using Image-Pro Plus 6.0. (b) Postn, Wnt3a, and β-catenin mRNA levels shown, as measured via RT-PCR analysis after 48 h of 17β-E2 treatment. (c) RUNX2 and ALP mRNA expressions observed in OVX+E2-BMSCs are higher than in OVX-BMSCs. Bars represent the means ± SD; n = 3, ∗P < 0.05, ∗∗P < 0.01. BMSCs: bone marrow-derived mesenchymal stem cells; IOD: integrated optical density.

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  Figure 4. Overexpression of POSTN in OVX-BMSCs increased the expression of POSTN, Wnt3a, β-catenin, and osteogenic differentiation. (a) Higher expression of POSTN, Wnt3a, and β-catenin was observed via western blot in the Lev-POSTN than in the Lev-GFP group; there was no significant difference relative to the sham group. (b) RT-PCR showing partly restored mRNA levels of Postn, RUNX2, and ALP of OVX-BMSCs following POSTN overexpression. (c) Overexpression of POSTN partially restored ALP activity of OVX-BMSCs. Bars represent the mean ± SD; n = 3, ∗∗P < 0.01. NS: not significant.

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  Figure 5. Inhibition of the expression of POSTN, Wnt3a, and β-catenin and osteogenic differentiation in OVX-BMSCs with or without 17β-E2 using Postn-siRNA. (a) Expression of POSTN, Wnt3a, and β-catenin was lower in OVX-BMSCs with Postn-siRNA compared with the negative control siRNA and control groups. (b) The mRNA levels of POSTN, Wnt3a, and β-catenin were lower in OVX-BMSCs with Postn-siRNA compared with the negative control siRNA and control groups. (c) Treatment of OVX-BMSCs with 10−9 M 17β-E2 did not reverse the levels of POSTN, Wnt3a, and β-catenin in the Postn-siRNA group. (d) The mRNA levels of RUNX2 and ALP in OVX+E2-BMSCs with Postn-siRNA were lower than those in the negative control siRNA and control groups. (e) Postn knockdown significantly decreased the mineralised node formation. Bars represent the means ± SD; n = 3, ∗∗P < 0.01.