EPO increases biosynthesis of proteoglycan and upregulates chondrogenic marker genes expression during chondrogenic differentiation.

<p>(A) Alcian blue staining for preoteoglycan synthesis in the chondrogenic micromass culture supplemented with EPO, and with or without EPO block peptide for 7 days. Con, non-treatment control. Magnification: 10×. (B) Quantitation of total sGAG in the micromass culture by dimethylmethylene blue assay. (C) Detection of mRNA expression of chondrogenic marker genes including SOX9, SOX5, SOX6, Col2α1 and aggrecan by real-time PCR in chondrocytes in response to EPO treatment following siRNA mediated EPOR knockdown. (D) Real-time PCR quantification of EPOR mRNA expression in chondrocytes in response to EPO treatment following siRNA mediated EPOR knockdown. Con siRNA, control siRNA. *<i>P</i><0.05; **<i>P</i><0.01, <i>n</i> = 3.</p

EPO improves biomechanical properties during consolidation of bone healing.

<p>Biomechanical parameters including peak load (A), elastic modulus (B), bend strain at maximum (C), and bend strength at maximum (D) were calculated at day 28 post-surgery. Con, non-treatment control. *<i>P</i><0.05; **<i>P</i><0.01, <i>n</i> = 3.</p

Icariin increases chondrocyte proliferation accompanied by upregulation of HIF-1α in alginate-chondrocyte 3D culture system.

<p>(A) Representative images of immunostaining for PCNA in 3D cultured sections from Icariin treated group and control group. Arrows indicate PCNA⁺ chondrocytes. IgG was used as negative control. Scale bar = 50 μm. (B) Quantitation of the percentage of PCNA positive cells in the Icariin treated group and control group. *<i>P</i> < 0.05, n = 3. (C) Representative images of immunostaining for HIF-1α in 3D cultured sections from Icariin treated group and control group. Arrows indicate HIF-1α positive (HIF-1α+) chondrocytes. IgG was used as negative control. Scale bar = 50 μm. (D) Quantitation of the percentage of HIF-1α+ cells in the sections from Icariin treated groups and control groups. *<i>P</i> < 0.05, n = 3.</p

EPO promotes the proliferation of primary chondrocytes.

<p>(A) Examination of mRNA expression of EPOR in chondrocytes by real-time PCR after siRNA mediated knockdown of EPOR. (B) Western blot analysis of EPOR expression in primary chondrocytes with or without siRNA mediated knockdown of EPOR. (C) BrdU incorporation assay in primary chondrocytes with or without siRNA mediated knockdown of EPOR after 48 hours of EPO exposure. Con, non-treatment control. *<i>P</i><0.05; **<i>P</i><0.01; <i>n</i> = 3.</p

Icariin promotes chondrogenesis in alginate-chondrocyte 3D culture system.

<p>(A-C) Representative H&E, Alcian blue and SO histological images for sections from alginate-chondrocyte 3D culture system treated with Icariin (10⁻⁶ M) for 21 days, with none treatment as control. Scale bar = 50 μm. (D) Quantitation of IOD for Alcian blue staining. Icariin treated group compared with control group, **<i>P</i> < 0.01, n = 3. (E) Quantitation of the positive SO staining area. Icariin treated group compared with control group, **<i>P</i> < 0.01, n = 3. (F-I) The alginate-chondrocyte 3D cultures were treated with Icariin (10⁻⁶ M) for 21 days. <i>Sox 9</i>, <i>Aggrecan</i>, <i>Col2α1</i> and <i>Col10α1</i> mRNA expression was quantified by real-time PCR and compared with that of control group with no Icariin treatment. **<i>P</i> < 0.01, n = 3. (J) Representative images of the immunostaining for SOX9 and Col2 in the sections. IgG was used as negative control. Scale bar = 50 μm. (K) Quantitation of SOX9⁺ chondrocytes was presented as percentage of total chondrocytes in the SOX9 stained sections from (J). *<i>P</i> < 0.05, n = 3. (L) Densitometric analysis of Col2 immunostaining in (J) using GraphPad Prism 5 software. *<i>P</i> < 0.05, n = 3.</p

EPO stimulates angiogenesis <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Representative images of endothelial sprouting from metatarsal bones of E17.5 embryos which were treated with different conditions as indicated. The endothelial sprouting was visualized by immunostaining for CD31. VEGF was used as positive control. Con, non-treatment control. Magnification: 2.5×. (B) Quantitation of the endothelial sprouting area for each group. (C) Representative micro-CT 3D reconstruction of vasculature at the fracture site following EPO treatment. The specimens were harvested for micro-CT scanning at day 14 post-surgery. Con, non-treatment control. (D) Quantitation of vascular parameters including the total volume (TV), vessel volume (VV), vessel surface (VS), and vessel thickness (VTh) by micro-CT angiography analysis. ** <i>P</i><0.01, <i>n</i> = 5.</p

EPO enhances cartilaginous callus formation during bone healing.

<p>(A) Safranin O staining of the cartilaginous callus at days 7 and 14 post-surgery. Con, non-treatment control. Magnification: 2.5×. (B) Quantitation of the volume of cartilaginous callus formation at days 7 and 14 post-surgery. *<i>P</i><0.05, <i>n</i> = 5.</p

The expression and localization of EPO and EPOR in the developing bones.

<p>(A) mRNA expression of EPO and EPOR in the developing growth plates of tibiae by RT-PCR at indicated postnatal days. Tissues from kidney (Kid) of new born mice were used as positive control. (B) Immunohistochemistry for EPO and EPOR in growth plates of tibiae from new born mice. Con, non-primary antibody control. Magnification: 40×. (C) Immunohistochemistry for EPO and EPOR in cartilaginous callus of the bone healing in femoral fracture model of mouse. Con, non-primary antibody control. Magnification: 100×.</p

Deletion of HIF-1α eliminates the positive effects of Icariin on chondrocytes.

<p>(A) Western blot analysis for HIF-1α protein expression in MSCs (HIF-1α Floxed) following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10⁻⁶ M) for 12 h. β-actin used as the loading control. (B) Alcian blue staining for preoteoglycan synthesis in MSCs (HIF-1α floxed) following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10⁻⁶ M) for 14 days. (C) Quantitation of the value of integral optical density (IOD) from (B). Compared with Ad-GFP-treated control group, *<i>P</i> < 0.05; n = 3. (D) BrdU incorporation assay for chondrocytes following treatment with Ad-GFP or Ad-Cre and treated with or without Icariin (10⁻⁶ M) for 48 h. Compared with Ad-GFP-treated control group, *<i>P</i> < 0.05, **<i>P</i> < 0.01; n = 3. Compared with Ad-GFP-treated ICA control group, ^##<i>P</i> < 0.01; n = 3. (E, F) Chondrocytes following treatment with Ad-GFP or Ad-Cre were cultured under normal medium in the presence or absence of Icariin (10⁻⁶ M). (E) <i>Sox9</i>, <i>Aggrecan</i> and <i>Col2α1</i> mRNA expression in chondrocytes was detected by real-time PCR. (F) <i>Adamts4</i>, <i>Mmp2</i>, and <i>Mmp9</i> mRNA expression in chondrocytes was detected by real-time PCR. Compared with Ad-GFP-treated control group,*<i>P</i> < 0.05, **<i>P</i> < 0.01; n = 3. Compared with Ad-Cre-treated control group, ^#<i>P</i> < 0.05; ^##<i>P</i> < 0.01; n = 3.</p

Icariin increases chondrocytes proliferation.

<p>(A) MTT assay for cell viability of chondrocytes treated with or without Icariin (0 M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) for 3 days. Treated groups compared with control group, *<i>P</i> < 0.05, **<i>P</i> < 0.01, n = 3. (B) BrdU incorporation assay for chondrocytes treated with or without Icariin (0 M, 10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) for 1 day or 3 days. Treated groups compared with control group, *<i>P</i> < 0.05; ***<i>P</i> < 0.001, n = 3. (C) Colony formation assay for chondroprogenitor cells treated with Icariin (10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M) for 24 h followed by 14 days sub-culture. (D) Quantitation of the colony numbers from (C), *<i>P</i> < 0.05, n = 3.</p