Exosomes Secreted from Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Prevent Osteonecrosis of the Femoral Head by Promoting Angiogenesis

Abstract

Background: Local ischemia is the main pathological performance in osteonecrosis of the femoral head (ONFH). There is currently no effective therapy to promote angiogenesis in the femoral head. Recent studies revealed that exosomes secreted by induced pluripotent stem cell-derived mesenchymal stem cells (iPS-MSC-Exos) have great therapeutic potential in ischemic tissues, but whether they could promote angiogenesis in ONFH has not been reported, and little is known regarding the underlying mechanism. Methods: iPS-MSC-Exos were intravenously injected to a steroid-induced rat osteonecrosis model. Samples of the femoral head were obtained 3 weeks after all the injections. The effects were assessed by measuring local angiogenesis and bone loss through histological and immunohistochemical (IHC) staining, micro-CT and three-dimensional microangiography. The effects of exosomes on endothelial cells were studied through evaluations of proliferation, migration and tube-forming analyses. The expression levels of angiogenic related PI3K/Akt signaling pathway of endothelial cells were evaluated following stimulation of iPS-MSC-Exos. The promoting effects of exosomes were re-evaluated following blockade of PI3K/Akt. Results: The in vivo study revealed that administration of iPS-MSC-Exos significantly prevented bone loss, and increased microvessel density in the femoral head compared with control group. We found that iPS-MSC-Exos significantly enhanced the proliferation, migration and tube-forming capacities of endothelial cells in vitro. iPS-MSC-Exos could activate PI3K/Akt signaling pathway in endothelial cells. Moreover, the promoting effects of iPS-MSC-Exos were abolished after blockade of PI3K/Akt on endothelial cells. Conclusions: Our findings suggest that transplantation of iPS-MSC-Exos exerts a preventative effect on ONFH by promoting local angiogenesis and preventing bone loss. The promoting effect might be attributed to activation of the PI3K/Akt signaling pathway on endothelial cells. The data provide the first evidence for the potential of iPS-MSC-Exos in treating ONFH.

Keywords: Angiogenesis.; Exosomes; Femoral head; Osteonecrosis; induced pluripotent stem cells (iPSCs).

Figure 1

Characterization of hiPS-MSCs and hiPS-MSC-Exos. (A) The fibroblast-like morphology of hiPS-MSCs shown in light microscopy images. (B) Surface markers of hiPS-MSCs analyzed by flow cytometry. (C) The morphology of hiPS-MSC-Exos shown by transmission electron microscopy. (D) Expression levels of CD9, CD63 and CD81 incorporation into hiPS-MSC-Exos shown by western blotting. (E) Identification of size and concentration of hiPS-MSC-Exos by nanoparticle analysis.

Figure 2

Assessment of trabecular bone structure in the femoral head. (A) H&E staining analysis. More bone trabecular tissue and fewer empty lacunae were observed in the MP+Exo group compared with the MP group. Arrowheads indicate empty lacunae. (B) Quantitative analysis of H&E staining. The number of empty lacunae in the MP+Exo group was significantly less than that in the MP group (P < 0.05). (C) Micro-CT analysis. The subchondral trabeculae appeared much more intact and regular in the MP+Exo group than in the MP group. (D) Quantitative analysis by mirco-CT. The values of BV/TV, bone surface area/bone volume, Tb.Th and Tb.N in the MP+Exo group were significantly higher than in the MP group (P < 0.05).

Figure 3

Assessment of angiogenesis in the femoral head. (A) 3-D microangiography analysis. Greater angiogenesis was observed in the MP+Exo group compared with the MP group. (B) Quantitative analysis of microangiography. Both vessel number and vessel volume in the MP+Exo group were significantly higher than in the MP group. (C) Immunohistochemical analysis. The expression of both VEGFR2 and CD31 was significantly enhanced in the MP+Exo group compared with the MP group.

Figure 4

Functional influence of hiPS-MSC-Exos on HUVECs. (A) Fluorescent microscopy analysis. DiO-labeled hiPS-MSC-Exos (green) were located in the perinuclear region of HUVECs. (B) CCK-8 assay. hiPS-MSC-Exos significantly increased the proliferative ability of HUVECs in a dose-dependent manner. (C) Scratched wound assay. hiPS-MSC-Exos significantly enhanced the migration ability of HUVECs. (D) Quantitative analysis of the migration rate of HUVECs. The value of migration area was significantly greater in the Exo group than in the control group at 12 h. (E) Capillary network formation assay. hiPS-MSC-Exos significantly enhanced tube formation ability in the Exo group compared to the control group in a dose-dependent manner. (F) Quantitative analysis of capillary network formation assay. The values of total branching points, total tube length, cell covered area, and total loops were all significantly higher in the Exo group than in the control group at 4 time point of 4 and 8 h. * represents a statistically-significant difference compared with the control group (P < 0.05). # represents statistically-significant difference compared with the Exos group (1 × 10¹⁰/mL) (P < 0.05).

Figure 5

hiPS-MSC-Exos activate PI3K/Akt in HUVECs. (A) qRT-PCR analysis. Expression levels of PI3K/Akt signaling-related genes were significantly increased under the treatment of hiPS-MSC-Exos. (B) Western blot analysis. Phosphorylation of Akt protein and expression levels of PI3K/Akt signaling-related proteins were increased following treatment with hiPS-MSC-Exos.

Figure 6

The effect of hiPS-MSC-Exos can be abolished by blockade of PI3K with LY294002. (A)(B) qRT-PCR and western blot analysis. hiPSC-MSC-Exos could not increase the expression levels of PI3K/Akt signaling-related molecules after blockade of PI3K with LY294002. (C) CCK-8 assay. The enhanced prolifierative ability conferred by hiPS-MSC-Exos was abolished by blockade of PI3K with LY294002. (D)(E) Scratched wound assay and quantitative analysis. The enhanced migration ability conferred by hiPS-MSC-Exos was abolished by blockade of PI3K with LY294002. (F)(G) Capillary network formation assay and quantitative analysis. The increased tube formation ability conferred by hiPS-MSC-Exos was abolished by blockade of PI3K with LY294002. * represents statistically-significant difference compared with the control group (P < 0.05). # represents statistically-significant difference compared with the Exos+LY294002 group (P < 0.05).