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

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

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

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 inhibits catabolic marker genes expression in chondrocytes.

<p>Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10⁻⁶ M) for 7 (A) or 14 (B) days. <i>Mmp2</i>, <i>Mmp9</i>, <i>Mmp13</i>, <i>Adamts4</i> and <i>Adamts5</i> mRNA expression was detected by real-time PCR in Icariin treated chondrocytes compared with that of the control cells. *<i>P</i> < 0.05; **<i>P</i> < 0.01; n = 3.</p

Icariin enhances chondrogenic marker expression and cartilage matrix synthesis while the effect is limited by knockdown of HIF-1α.

<p>(A) Chondrocytes were processed for micromass culture and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10⁻⁷ M, 10⁻⁶ M, 10⁻⁵ M). The cell masses were stained with Alcian blue after 7 or 14 days culture, respectively. Note that Icariin (10⁻⁶ M) increased proteoglycan synthesis. (B) Quantitation of the value of integral optical density (IOD) from (A). Treated groups compared with control group, *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.01, n = 3. (C, D) ELISA assays for production of aggrecan (C) and hydroxypoline (D) in chondrocytes. Icariin treated groups versus control groups, *<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001, n = 3. (E, F) Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10⁻⁶ M) for 7 (E) or 14 (F) days. <i>Sox9</i>, <i>Col2α1</i> and <i>Aggrecan</i> mRNA expression was detected by real-time PCR in Icariin treated chondrocytes and compared with that in the control cells. *<i>P</i> < 0.05, **<i>P</i> < 0.01; ***<i>P</i> < 0.001, n = 3.</p

Icariin upregulates HIF-1α expression in chondrocytes by inhibiting PHDs activity through competition for iron ions.

<p>(A) The chemical formula of Icariin. (B) Hypoxia response element luciferase reporter assay in C2C12 cells treated with Icariin at indicated concentrations. (C) Western blot analysis for HIF-1α protein expression in primary culture-derived chondrocytes under normoxia or hypoxia or treated with or without Icariin (10⁻⁶ M) for 8 h. β-actin used as the loading control. (D) Detection of HIF-1α nuclear localization in Icariin (10⁻⁶ M)-treated chondrocytes by immunofluorescence staining under confocal microscope. (E, F) Chondrocytes were cultured and induced to differentiate in chondrogenic medium in the presence or absence of Icariin (10⁻⁶ M) for 7 or 14 days. HIF-1α mRNA levels were detected by real-time PCR in Icariin-treated chondrocytes compared with that of the control cells. *<i>P</i> < 0.05, **<i>P</i> < 0.01, n = 3. (G) UV-Vis spectra of the Icariin, FeSO₄ and their mixture (<i>n</i>_Icariin: <i>n</i>_FeSO4 = 3: 1, <i>C</i>_Icariin = 0.5mM) in aqueous solution after incubation at 37°C for 12 h; <i>C</i>_Icariin = 0.5mM; <i>C</i>_FeSO4 = 1mM; The inset shows the visual appearance of each species. (H) Western blot analysis for HIF-1α protein expression in chondrocytes treated with or without Icariin (10⁻⁶ M) and FeSO₄ (100 μM) for 12 h. (I) Western blot analysis for PHDs and HIF-1α protein expression in chondrocytes treated with or without Icariin (10⁻⁶ M) for 12 h. In all Figs, ICA, Icariin.</p

Isolation and purification of CD146⁺ HUCPVCs from human umbilical cord.

<p>A. A dissected human umbilical cord vessel. B. Heterogenous population of HUCPVCs digested from the umbilical cord vessel by collagenase I. Scale bar: 100 µm. C. Homogenous population of HUCPVCs purified with CD146⁺ antibody using MACS system. Scale bar: 50 µm. D. Expression of CD146 in HUCPVCs. Cells were immunostained with anti-CD146 antibody and visualized by fluorescent microscopy. The nucleus was counterstained with DAPI. Scale bar: 50 µm. E. Detection of cell surface markers by flow cytometry analysis. Cells were immunostained with selected cell surface markers CD146, CD44, CD90, CD105, CD34, CD45. The percentage of cell population with positive staining was shown in each figure.</p

Functional engraftment of CD146⁺ HUCPVCs in the new bone regenerates in the subcutaneous transplantation model and the critical-sized bone defect model in SCID mice.

<p>(<b>A</b>) X-ray image of the ectopic bone formation at 6 weeks following CD146⁺ HUCPVCs transplantation subcutaneously in the back of SCID mice (n = 3). (<b>B</b>) H&E staining of the ectopic bone section. (<b>C</b>) Immunohistochemistry staining for human specific mitochondria in the newly formed ectopic bone (<b>D</b>) Mouse IgG isotype control. (<b>E</b>) X-ray image of critical sized bone defect region at 6 weeks following CD146⁺ HUCPVCs transplantation. The newly formed bone is indicated by the white arrow. (<b>F</b>) H&E staining of the new bone regenerate. (<b>G</b>) Immunohistochemistry staining for human specific mitochondria in the new bone regenerate in the critical-sized bone defect model. (<b>H</b>) Mouse IgG isotype control. Red arrow: Osteoblasts; Black arrow: Chondrocytes. All scale bar: 50 µm.</p

Expression profiles of stem cell transcription factors in CD146⁺ HUCPVCs under hypoxia and normoxia.

<p>(A) The table summarizes the fold-change of mRNA expression of stem cell transcription factors in CD146⁺ HUCPVCs in response to hypoxia. (B) Hierarchical clustering analysis on the PCR array data. (C) Illustration of the mRNA fold-change of the top ten upregulated and downregulated transcription factors in CD146⁺ HUCPVCs in response to hypoxia.</p