Mobilization of Transplanted Bone Marrow Mesenchymal Stem Cells by Erythropoietin Facilitates the Reconstruction of Segmental Bone Defect

Abstract

Reconstruction of segmental bone defects poses a tremendous challenge for both orthopedic clinicians and scientists, since bone rehabilitation is requisite substantially and may be beyond the capacity of self-healing. Bone marrow mesenchymal stem cells (BMSCs) have been identified as an optimal progenitor cell source to facilitate bone repair since they have a higher ability for proliferation and are more easily accessible than mature osteoblastic cells. In spite of the potential of BMSCs in regeneration medicine, particularly for bone reconstruction, noteworthy limitations still remain in previous application of BMSCs, including the amount of cells that could be recruited, the compromised bone migration of grafted cells, reduced proliferation and osteoblastic differentiation ability, and likely tumorigenesis. Our current work demonstrates that BMSCs transplanted through the caudal vein can be mobilized by erythropoietin (EPO) to the bone defect area and participate in regeneration of new bone. Based on the histological analysis and micro-CT findings of this study, EPO can dramatically promote the effects on the osteogenesis and angiogenesis efficiency of BMSCs in vivo. Animals that underwent EPO+BMSC administration demonstrated a remarkable increase in new bone formation, tissue structure organization, new vessel density, callus formation, and bone mineral density (BMD) compared with the BMSCs alone and control groups. At the biomechanical level, we demonstrated that combing transplantation of EPO and BMSCs enhances bone defect reconstruction by increasing the strength of the diaphysis, making it less fragile. Therefore, combination therapy using EPO infusion and BMSC transplantation may be a new therapeutic strategy for the reconstruction of segmental bone defect.

Figure 1

The characteristics of BMSCs. The passage 3 BMSC immunofluorescent labelings were positive for CD29 (a), CD44 (b), CD90 (c), and CD271 (d) and negative for CD11b (e) and CD45 (f). (g) BMSCs manifested typical spindle or polygonous shape and adhered to the plastic culture plate. (h) Positive oil red O staining on day 14 revealed the differentiation of passage 3 BMSCs into adipocytes. (i) ALP staining of passage 3 BMSCs indicated formation of calcification nodes on day 21. Scale bar is 100 μm for (a, c, g, h, and i) and 50 μm for (b, d, e, and f).

Figure 2

Western blot and Transwell migration assay. (a) Western blot assay revealed that the expression levels of CXCR4 increased with the increment of EPO centration. (b) The CXCR4/GAPDH ratio was the lowest without EPO intervention and the highest at the maximum concentration of EPO. (c) EPO-induced migration of BMSCs was confirmed by inverted phase contrast microscopy. (d) The OD value in the EPO group was significantly higher than that of the control groups, while no significant difference was detected between the EPO+AMD3100 group and the control group. The data are plotted as mean ± SD. N.S=no significant difference.

Figure 3

Immunofluorescent microscopy. (a) Immunofluorescence staining of GFP+ cells in four groups at each time point. BMSCs: bone marrow mesenchymal stem cells. Scale bar is 100 μm. (b) Quantitative analysis of the number of GFP+ cells. The data are plotted as mean ± SD. N.S=no significant difference.

Figure 4

Histological examination of newly regenerated bone at 4 and 8 weeks after operation. Representative H&E images (scale bar is 200 μm). New bone areas were stained in pink red.

Figure 5

Representative Masson's trichrome staining images at 4 and 8 weeks after operation (scale bar is 200 μm).

Figure 6

Histomorphometry analysis for bone defective model at 4 and 8 weeks. (a) Percentage of new bone area. (b) New blood vessel density. The data are plotted as mean ± SD. N.S = no significant difference.

Figure 7

Three-point flexural test results at 4 and 8 weeks of postoperation. (a) The bending stiffness of tibia in each group. (b) The ultimate loading of tibia in each group. The data are plotted as mean ± SD. N.S = no significant difference.

Figure 8

Micro-CT scans were used to evaluate in vivo new bone formation. (a, b) Representative 2D cross-sectional images and 3D reconstructed images at 8 weeks of postinjury. (c, d) Quantification of newly formed bone volume and bone mineral density by micro-CT. N = 6 for each population. The data are plotted as mean ± SD. N.S = no significant difference.