Exosomes Derived from Bone Marrow Mesenchymal Stem Cells Alleviate Ischemia-Reperfusion Injury and Promote Survival of Skin Flaps in Rats

Abstract

Free tissue flap transplantation is a classic and important method for the clinical repair of tissue defects. However, ischemia-reperfusion (IR) injury can affect the success rate of skin flap transplantation. We used a free abdomen flap rat model to explore the protective effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs-exosomes) against the IR injury of the skin flap. Exosomes were injected through the tail vein and the flaps were observed and obtained on day 7. We observed that BMSCs-exosomes significantly reduced the necrotic lesions of the skin flap. Furthermore, BMSCs-exosomes relieved oxidative stress and reduced the levels of inflammatory factors. Apoptosis was evaluated via the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay and Western blot analysis of Bax, Bcl-2. Simultaneously, BMSCs-exosomes promoted the formation of new blood vessels in the IR flap, as confirmed by the increased expression level of VEGFA and the fluorescence co-staining of CD31 and PCNA. Additionally, BMSCs-exosomes considerably increased proliferation and migration of human umbilical vein endothelial cells and promoted angiogenesis in vitro. BMSCs-exosomes could be a promising cell-free therapeutic candidate to reduce IR injury and promote the survival of skin flaps.

Keywords: angiogenesis; bone marrow mesenchymal stem cells; exosomes; ischemia-reperfusion injury; skin flap.

Figure 1

Identification and internalization of BMSCs-exo in vitro. (A) Representative images of BMSCs-exo morphology obtained via transmission electron microscopy (yellow arrow, scale bar, 100 nm); (B) Size distribution of BMSCs-exo, as determined by NanoSight; (C) Identification of exosomes via detection of Alix, CD9, and CD63 using Western blotting; (D) DiI-labeled exosomes were detected in HUVECs by confocal fluorescence microscopy (Scale bar, 50 μm); (E) In vivo tracking of DiR-labeled BMSCs-exo.

Figure 2

The effects of BMSCs-exo on the ischemia-reperfusion injury flaps’ survival rate and histological lesions. (A) Digital photograph of flaps from day 1 to day 7 after surgery; (B) The tissue side of flaps on day 7; (C) Necrosis rates (necrosis area/flap area) of the IR+PBS and IR+Exo groups, *** p < 0.001; (D,E) H&E and Masson staining of tissue sections of the three groups; (F) Histological score of the three groups, *** p < 0.001; (G) Collagen volume fraction (collagen area/total area) of the three groups, ** p < 0.01.

Figure 3

BMSCs-exo promoted tissue regeneration and angiogenesis. (A) Co-staining of CD31 (green) and PCNA (red) of the three groups (scale bar, 50 μm); (B,C) Number of vessels and relative expression of PCNA, *** p < 0.001; (D) The expression of VEGFA in the three groups detected by immunohistochemistry (scale bar, 50 μm); (E) Positive area of VEGFA, ** p < 0.01; (F) Western blot analysis of VEGFA, CD31, and PCNA expression of the three groups; (G) Quantitative analysis of the expression levels of VEGFA, CD31, and PCNA, * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 4

BMSCs-exo reduced the apoptosis of skin flap. (A) TUNEL staining (red) of tissue sections of the three groups (scale bar, 50 μm); (B) Number of TUNEL positive cells, *** p < 0.001; (C) Western blot analysis of Bax and Bcl-2 expression in the three groups; (D) Quantitative analysis of the expression levels of Bax and Bcl-2, * p < 0.05 and * p < 0.01; (E,F) Evaluation of SOD activities and MDA contents of the three groups, ** p < 0.01; (G,H) Expression levels of TNF-a and IL-1β were measured by ELISA analysis, *** p < 0.001.

Figure 5

The proliferation, migration, and angiogenesis ability promotion effects of BMSCs-exo on HUVECs in vitro. (A) CCK8 test for different concentrations of BMSCs-exo on the HUVECs’ proliferation. Compared to the PBS group, BMSCs-exosomes significantly increased endothelial cell proliferation at 24 h after undergoing hypoxia reoxygenation injury, **** p < 0.0001); (B) The images of wound healing assay in HUVECs with/without BMSCs-exo for 12 h and 24 h (Scale bar, 100 μm); (C) The wound closure rates of HUVECs are presented as migration area/original area, ** p < 0.01, *** p < 0.001 and **** p < 0.0001). (D) For capillary network formation assay in vitro, HUVECs were incubated in Matrigel with/without BMSCs-exo. (E,F) Quantification of capillary network formation of HUVECs, **** p < 0.0001.