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. 2019 Mar;52(2):e12570.
doi: 10.1111/cpr.12570. Epub 2019 Jan 20.

Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF-1α-mediated promotion of angiogenesis in a rat model of stabilized fracture

Yuntong Zhang  1 , Zichen Hao  1   2 , Panfeng Wang  1 , Yan Xia  1 , Jianghong Wu  1 , Demeng Xia  1 , Shuo Fang  3 , Shuogui Xu  1
Affiliations

Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF-1α-mediated promotion of angiogenesis in a rat model of stabilized fracture

Yuntong Zhang et al. Cell Prolif. 2019 Mar.

Abstract

Objectives: Exosomes, as important players in intercellular communication due to their ability to transfer certain molecules to target cells, are believed to take similar effects in promoting bone regeneration with their derived stem cells. Studies have suggested that umbilical cord mesenchymal stem cells (uMSCs) could promote angiogenesis. This study investigated whether exosomes derived from uMSCs (uMSC-Exos) could enhance fracture healing as primary factors by promoting angiogenesis.

Materials and methods: uMSCs were obtained to isolate uMSC-Exos by ultrafiltration, with exosomes from human embryonic kidney 293 cells (HEK293) and phosphate-buffered saline (PBS) being used as control groups. NanoSight, laser light scattering spectrometer, transmission electron microscopy and Western blotting were used to identify exosomes. Next, uMSC-Exos combined with hydrogel were transplanted into the fracture site in a rat model of femoral fracture. Bone healing processes were monitored and evaluated by radiographic methods on days 7, 14, 21 and 31 after surgery; angiogenesis of the fracture sites was assessed by radiographic and histological strategies on post-operative day 14. In vitro, the expression levels of osteogenesis- or angiogenesis-related genes after being cultured with uMSC-Exos were identified by qRT-PCR. The internalization ability of exosomes was determined using the PKH67 assay. Cell cycle analysis, EdU incorporation and immunofluorescence staining, scratch wound assay and tube formation analysis were also used to determine the altered abilities of human umbilical vein endothelial cells (HUVECs) administered with uMSC-Exos in proliferation, migration and angiogenesis. Finally, to further explore the underlying molecular mechanisms, specific RNA inhibitors or siRNAs were used, and the subsequent effects were observed.

Results: uMSC-Exos had a diameter of approximately 100 nm, were spherical, meanwhile expressing CD9, CD63 and CD81. Transplantation of uMSC-Exos markedly enhanced angiogenesis and bone healing processes in a rat model of femoral fracture. In vitro, other than enhancing osteogenic differentiation, uMSC-Exos increased the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). uMSC-Exos were taken up by HUVECs and enhanced their proliferation, migration and tube formation. Finally, by using specific RNA inhibitors or siRNAs, it has been confirmed that HIF-1α played an important role in the uMSC-Exos-induced VEGF expression, pro-angiogenesis and enhanced fracture repair, which may be one of the underlying mechanisms.

Conclusions: These results revealed a novel role of exosomes in uMSC-mediated therapy and suggested that implanted uMSC-Exos may represent a crucial clinical strategy to accelerate fracture healing via the promotion of angiogenesis. HIF-1α played an important role in this process.

Keywords: HIF-1α; angiogenesis; exosomes; fracture healing; umbilical cord mesenchymal stem cell.

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Conflict of interest statement

All authors state that they have no conflicts of interest.

Figures

Figure 1
Figure 1
Characterization of exosomes derived from human umbilical cord‐derived mesenchymal stem cells (uMSCs) and HEK293 cells. A, Representative image of purified exosome particles (left panel) and the particle size distribution in purified uMSC‐Exo (right panel) as determined by NanoSight. The red arrow indicates exosomes. B, Precise particle size distribution of purified uMSC‐Exo and HEK293‐Exo measured by laser light scattering spectrometer. The dashed dot line indicates the peak particle size of purified exosomes. C, Western blot analysis of the exosomes surface markers. D, Morphology of the exosomes observed by TEM
Figure 2
Figure 2
Radiographic and histological analysis of the fracture healing. A, Representative X‐ray images of the fractures on post‐operative day 14. B, Quantitative analysis of the CW on post‐operative days 7 and 21 (left panel). Bone formation in X‐ray images was assessed on post‐operative days 7, 14, 21 and 31 (right panel), using a radiographic score as described in Section 2. n = 6. C, Representative Micro‐CT images of the fractured femur on post‐operative day 14. D, TV and BV of the callus, and BV/TV on post‐operative day 14 was quantified. n = 6. E, BMD on post‐operative day 14 was quantified using Micro‐CT. n = 6. F, Representative Micro‐CT images of the vascular system on post‐operative day 14. G, On day 14 after surgery, vessel volume was quantified on Micro‐CT images. n = 6. H, The fractured callus on post‐operative day 14 stained with anti‐CD31. Representatives were shown, and boxed areas were enlarged on the bottom. Scale bar for original images = 200 mm. I, The number of CD31‐positive vessels (left panel) was counted, and the ratio of vessel area (right panel) was measured n = 6. (*P < 0.05, **P < 0.01, CT, computed tomography; BMD, bone mineral density; BV, bone volume; TV, total volume
Figure 3
Figure 3
The expression levels of selected differentially expressed genes in target cells on 14 day after uMSC‐Exo treatment were quantified by qRT‐PCR. A‐D, Total RNA was extracted, and the expression levels of osteogenesis‐related genes in primary osteoblasts were analysed by qRT‐PCR. n = 3, **P < 0.01. E, F, Total RNA was extracted, and the expression levels of angiogenesis‐related genes in HUVECs were analysed by qRT‐PCR. n = 3, **P < 0.01
Figure 4
Figure 4
Internalization of uMSC‐Exos in HUVECs, and their pro‐angiogenesis effects on the recipient cells. A, Fluorescence microscopy analysis of PKH67‐labelled uMSC‐Exos internalization by HUVECs. The green‐labelled exosomes were visible in the perinuclear region of recipient cells. Scale bar: 50 μm. B, Cell cycle assay of differently treated HUVECs. Representative images were shown in the left. The percentage of G2 population was shown in the right panels. n = 3, **P < 0.01. C, Cells were dispersed, fixed and stained for DAPI and EdU (DAPI and EdU are indicated by blue and red staining, respectively). EdU incorporation for different treatments was visualized using a fluorescence microscope (left panel). Scale bar: 50 μm. The percentage of EdU‐positive (proliferating) cells for each treatment was quantitated using ImageJ software (right panel). n = 3, **P < 0.01. D, The migration ability of HUVECs in different treatment groups was tested by the scratch wound assay. Representative images were shown in the left panels. Scale bars = 250 μm. Quantitative analysis of the migration rates was shown in the right panels. n = 3, *P < 0.05, **P < 0.01. E, uMSC‐Exos stimulated the tube formation ability of HUVECs. Representative images were shown in the left panels. Scale bar: 100 μm. Quantitative analysis was shown in the right panels. n = 3, **P < 0.01
Figure 5
Figure 5
Regulating the expression of HIF‐1α in HUVECs stimulated with uMSC‐Exos‐mediated VEGF expression and angiogenesis ability. A, The inhibitory efficiency of the siRNA targeting HIF‐1α was verified by qRT‐PCR. n = 3, **P < 0.01. B, Western blot analysis showed the inhibition of HIF‐1α decreased the VEGF protein in HUVECs stimulated with uMSC‐Exos. C, qRT‐PCR analysis indicated that inhibition of HIF‐1α decreased the mRNA expression level of VEGF in HUVECs stimulated with uMSC‐Exos. n = 3, **P < 0.01. D, Optical micrographs of tube formation assay of HIF‐1α‐regulated HUVECs stimulated with uMSC‐Exos were shown in the left panels. Scale bar: 100 μm. Quantitative analysis was shown in the right panels. *P < 0.05. E, The migration ability of HIF‐1α‐regulated HUVECs stimulated with uMSC‐Exos was tested by the scratch wound assay. Representative images were shown in the left panels. Scale bars = 250 μm. Quantitative analysis of the migration rates was shown in the right panels. n = 3, *P < 0.05. F, The proliferation ability of HIF‐1α‐regulated HUVECs stimulated with uMSC‐Exos was tested by the EdU test (left panel). Scale bar: 50 μm. The percentage of EdU‐positive (proliferating) cells for each treatment was quantitated using ImageJ software (right panel). n = 3, **P < 0.01
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