Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Accelerate Diabetic Wound Healing via Promoting M2 Macrophage Polarization, Angiogenesis, and Collagen Deposition

Abstract

Some scholars have suggested that the clinical application of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) might represent a novel strategy to improve diabetic wound healing. However, the mechanisms underlying the effects of hucMSCs-exo on wound healing remain poorly understood. This study aimed to identify the mechanism of hucMSCs-exo in treating diabetic wounds. HucMSCs-exo were isolated from human umbilical cord mesenchymal stem cells (hucMSCs) and subcutaneously injected into full-thickness wounds in diabetic rats. Wound healing closure rates and histological analysis were performed. The levels of tumor necrosis factor-α (TNF-α), macrophage mannose receptor (MMR/CD206), platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry. The degree of collagen deposition was examined using Masson's trichrome staining. Gross evaluation of wound healing was carried out from day 0 to 14 post-surgery, and the wound site was harvested for histology on days 3, 7, and 14 post-wounding. HucMSCs-exo transplantation increased diabetic wound healing. In vitro, hucMSCs-exo promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and NIH-3T3 cells. In vivo, hucMSCs-exo reduced wound area and inflammatory infiltration and increased collagen fibers. In addition, wound tissues in the hucMSCs-exo group had higher CD206, CD31, and VEGF expressions and lower TNF-α levels than those in the control group on day 14. Our results demonstrated that hucMSCs-exo facilitated diabetic wound repair by inducing anti-inflammatory macrophages and promoting angiogenesis and collagen deposition.

Keywords: diabetes; exosomes; human-umbilical-cord-derived mesenchymal stem cells; macrophage polarization; wound healing.

Figure 1

Characterization of hucMSCs-exo. (A) TEM micrograph of hucMSCs-exo. The scale bar is 100 nm. (B) Particle size and distribution analysis of hucMSCs-exo by Zetasizer Nano ZS. (C) Western blotting analysis of hucMSCs-exo markers.

Figure 2

Proliferative effects of hucMSCs-exo on HUVECs and NIH-3T3 cells. (A) HUVECs and (B) NIH-3T3 cells were treated with hucMSCs-exo at different concentrations. Untreated hucMSCs-exo was used as the control group. Data represent the mean ± SD (n = 3), * p < 0.05.

Figure 3

Diabetic wound healing induced by hucMSCs-exo in vivo. (A) Representative images of full-thickness dorsal wounds in STZ-induced diabetic rats at days 0, 3, 7, 10, and 14 post-wounding after treatment with phosphate-buffered saline (PBS as a control group) and hucMSCs-exo. (B) Quantification of wound closure at different time points in two groups (n = 5). * p < 0.05 shows a significant difference compared with the control group on the same day.

Figure 4

H&E staining of diabetic wound tissues treated with PBS (control) and hucMSCs-exo on days 3, 7, and 14. Red arrow: blood vessels, green rectangle: characteristic epithelium.

Figure 5

Immunohistochemistry analysis of TNF-α and CD206 expression in diabetic wound tissues on days 3, 7, and 14. Representative images of TNF-α (A) and CD206 (C) staining of wound tissues. The scale bar is 200 µm. Quantitative analysis of the positively stained area percentage of TNF-α (B) and CD206 (D). The data are expressed as mean ± SD (n = 3). * p < 0.05 compared with the control group.

Figure 6

Immunohistochemistry analysis of CD31 and VEGF expression in diabetic wound tissues on days 3, 7, and 14. Representative images of CD31 (A) and VEGF (C) staining of wound tissues. The scale bar is 200 µm. Quantitative analysis of the positively stained area percentage of CD31 (B) and VEGF (D). The data are expressed as mean ± SD (n = 3). * p < 0.05 compared with the control group.

Figure 7

Masson’s trichrome staining in diabetic wound tissues on days 3, 7, and 14. (A) Representative images of Masson’s trichrome staining of wound sections. The scale bar is 200 µm. (B) Quantitative analysis of pigmentation areas (n = 3). * p < 0.05 compared with the control group.