Human bone marrow mesenchymal stem cell-derived exosomes stimulate cutaneous wound healing mediates through TGF-β/Smad signaling pathway

Abstract

Background: Cutaneous wound healing represents a morphogenetic response to injury and is designed to restore anatomic and physiological function. Human bone marrow mesenchymal stem cell-derived exosomes (hBM-MSC-Ex) are a promising source for cell-free therapy and skin regeneration.

Methods: In this study, we investigated the cell regeneration effects and its underlying mechanism of hBM-MSC-Ex on cutaneous wound healing in rats. In vitro studies, we evaluated the role of hBM-MSC-Ex in the two types of skin cells: human keratinocytes (HaCaT) and human dermal fibroblasts (HDFs) for the proliferation. For in vivo studies, we used a full-thickness skin wound model to evaluate the effects of hBM-MSC-Ex on cutaneous wound healing in vivo.

Results: The results demonstrated that hBM-MSC-Ex promote both two types of skin cells' growth effectively and accelerate the cutaneous wound healing. Interestingly, we found that hBM-MSC-Ex significantly downregulated TGF-β1, Smad2, Smad3, and Smad4 expression, while upregulated TGF-β3 and Smad7 expression in the TGF-β/Smad signaling pathway.

Conclusions: Our findings indicated that hBM-MSC-Ex effectively promote the cutaneous wound healing through inhibiting the TGF-β/Smad signal pathway. The current results provided a new sight for the therapeutic strategy for the treatment of cutaneous wounds.

Keywords: Exosomes; Human bone marrow mesenchymal stem cells; TGF-β/Smad signaling; Wound healing.

Fig. 1

hBM-MSC-Ex promotes proliferation in HaCaT and HDF skin cells. The cell proliferation curve were shown from respective HaCaT and HDF skin cells after treated with hBM-MSC-Ex (a, b). Immunofluorescent staining was performed in HaCaT and HDFs for PCNA-positive cells, and representative images were shown (c). The quantification of PCNA-positive cells (in percentages) in HaCaT and HDFs were determined (d). HaCaT, human keratinocytes; HDFs, human dermal fibroblasts; bar = 100 μm, *p < 0.05, **p < 0.01, n = 3; data are reported as mean ± SD

Fig. 2

hBM-MSC-Ex treatments accelerate cutaneous wound healing process in vivo. The illustration of experimental design and plan of experiment performed in vivo (a). The representative photos shown in the dorsal full-thickness wound area of the rat (b). Quantitative analysis of wound area in the respective treatment groups (c). n = 8/group, scale bar = 5 mm, *p < 0.05, **p < 0.01 compared to the PBS group, ^##p < 0.01 compared to the hBM-MSC group; data are reported as mean ± SD

Fig. 3

hBM-MSC-Ex enhanced the cutaneous wound healing quality. The representative images of H&E staining in the different treatment groups (a). The image quantification of number of cutaneous appendages including hair follicles and sebaceous glands/field (40×) in the healing tissue (b). The blue arrow indicates the presence of sebaceous gland. Immunostaining for α-SMA and VEGF were shown respective images at 16 days after treatment (a). The percentage quantification of α-SMA and VEGF positive area (c, d). The purple arrow indicates the α-SMA-positive cells, and red arrow indicates VEGF positive cells. Scale bar = 1 mm, *p < 0.05, **p < 0.01, ***p < 0.001, n = 8/group, data are reported as mean ± SD

Fig. 4

hBM-MSC-Ex regulate the TGF-β/Smad signal pathway. Representative Western blot of key TGF-β/Smad signaling-related protein levels in skin tissue treated with hBM-MSC-Ex (a). The quantification of relative mRNA expression levels of major TGF-β/Smad signaling-related gene in skin healed tissue treated with hBM-MSC-Ex (b). *p < 0.05, **p < 0.01, n = 3, data are reported as mean ± SD

Fig. 5

Illustration of hBM-MSC-Ex stimulates cutaneous wound healing by regulating the TGF-β/Smad signal pathway. hBM-MSC-Ex inhibited TGF-β1 and activated TGF-β3 expression; TGF-β isoforms and activins stimulate intracellular signaling via Smad-2/3 transcription factors; phosphorylated Smad-2 and Smad-3 bind to Smad-4 leading to the transcription and expression of α-SMA; inhibitory Smad7 are activated by the binding of the TGF-β super family to the cell surface receptors