Fig 2: Immunohistochemical staining of Runx2 and osteocalcin. For Runx2, the (A) control and (B) EPO groups at 4 weeks, and the (C) control and (D) EPO groups at 8 weeks. (E) Quantification of Runx2 expression. For osteocalcin, the (F) control and (G) EPO groups at 4 weeks, and the (H) control and (I) EPO groups at 8 weeks. (J) Quantification of osteocalcin expression levels. Data are presented as the mean ± standard deviation. *P<0.05 vs. the control group. EPO, erythropoietin; Runx2, runt-related transcription factor 2; OD, optical density.

Fig 3: Terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling assays of femoral heads. The (A) control and (B) EPO groups at 4 weeks; the (C) control and (D) EPO groups at 8 weeks. At 4 and 8 weeks following treatment with EPO, less apoptotic cells were present in the trabecular bone and bone marrow cavity of the EPO group compared with the control group. The black arrow indicates an apoptotic cell in the trabecular bone and the red arrow indicates apoptotic cells in the bone marrow cavity. (E) Quantitative analysis of the apoptosis rate in the control and EPO treatment group at 4 and 8 weeks. A significantly higher apoptosis rate was observed in the control group compared with the EPO treatment group at 4 and 8 weeks. Data were presented as the mean ± standard deviation. *P<0.05 vs. the control group. EPO, erythropoietin.

Fig 4: Immunohistochemical staining of VEGF and CD31 in of the femoral heads. For VEGF, the (A) control and (B) EPO groups at 4 weeks, and the (C) control and (D) EPO groups at 8 weeks. (E) Quantification of VEGF expression. For CD31, the (F) control and (G) EPO groups at 4 weeks, and the (H) control and (I) EPO groups at 8 weeks. (J) Quantification of CD31 expression levels. The mean density of VEGF in the EPO treatment group was higher than that in the control group at 4 and 8 weeks. MVD in the EPO treatment group significantly increased compared with the control group at 4 and 8 weeks. Data are presented as the mean ± standard deviation. *P<0.05 vs. the control group. VEGF, vascular endothelial growth factor; CD31, platelet endothelial cell adhesion molecule; EPO, erythropoietin; MVD, microvessel density.

Fig 5: Cytokine dependence of IPE cell expansion. IPE cells were plated at 2.5 × 105 cells/ml in 200 μl IPE base media (without EPO and SCF) and cultured for 6 days with various combinations of EPO and SCF cytokines. Cells were passaged 1:4 every 2 days, and each data point is the average of three technical replicates