Exosomes derived from minor salivary gland mesenchymal stem cells: a promising novel exosome exhibiting pro-angiogenic and wound healing effects similar to those of adipose-derived stem cell exosomes

Abstract

Backgrounds: Minor salivary gland mesenchymal stem cells (MSGMSCs) can be easily extracted and have a broad range of sources. Applying exosomes to wounds is a highly promising method for promoting wound healing. Exosomes derived from different stem cell types have been proven to enhance wound healing, with adipose-derived stem cell (ADSC)-derived exosomes being the most extensively researched. Considering that MSGMSCs have advantages such as easier extraction compared to ADSCs, MSGMSCs should also be a very promising type of stem cell in exosome therapy. However, whether MSGMSC-derived exosomes (MSGMSC-exos) can promote wound healing and how they compare to ADSC-derived exosomes (ADSC-exos) in the wound healing process remain unclear.

Materials: The effects of MSGMSC-exos and ADSC-exos on angiogenesis in wound healing were investigated in vitro using CCK-8, scratch assays, and tube formation assays. Subsequently, the promotion of wound healing by MSGMSC-exos and ADSC-exos was evaluated in vivo using a full-thickness wound defect model in mice. Immunohistochemistry was used to verify the effects of MSGMSC-exos and ADSC-exos on promoting collagen deposition, angiogenesis, and cell proliferation in the wound. Immunofluorescence staining was performed to investigate the role of MSGMSC-exos and ADSC-exos in modulating the inflammatory response in the wound. Furthermore, proteomic sequencing was conducted to investigate the functional similarities and differences between the proteomes of MSGMSC-exos and ADSC-exos, with key protein contents verified by ELISA.

Results: MSGMSC-exos exhibited similar effects as ADSC-exos in promoting the migration, proliferation, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, with a comparable dose-dependent effect. In vivo experiments confirmed that MSGMSC-exos have similar wound healing-promoting functions as ADSC-exos. MSGMSC-exos promoted the neovascularization and maturation of blood vessels in vivo at a level comparable to ADSC-exos. Despite MSGMSC-exos showing less collagen deposition than ADSC-exos, they exhibited stronger anti-scar formation and anti-inflammatory effects. Proteomic analysis revealed that the proteins promoting wound healing in both MSGMSC-exos and ADSC-exos were relatively conserved, with ITGB1 identified as a critical protein for angiogenesis. Further differential analysis revealed that the functions specifically enriched in MSGMSC-exos and ADSC-exos reflected the functions of their source tissue.

Conclusions: Our study confirms that MSGMSC-exos exhibit highly similar wound healing and angiogenesis-promoting functions compared to ADSC-exos, and the proteins involved in promoting wound healing in both are relatively conserved. Moreover, MSGMSC-exos show stronger anti-scar formation and anti-inflammatory effects than ADSC-exos. This suggests that MSGMSCs are a promising stem cell source with broad applications in wound healing treatment.

Keywords: Adipose-derived stem cells; Exosome therapy; Human minor salivary mesenchymal stem cells; Wound healing.

Fig. 1

CCK-8 assay of the effect of ADSC-exos and MSGMSC-exos on HUVECs. (n = 3) A–D Cell viability of HUVECs 24 h, 48 h, 72 h, 96 h after treated with ADSC-exos and MSGMSC-exos respectively. E The line graph that reveals the O.D value of every group at each time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Results of tube formation assay of HUVECs. (n = 3) A Representative images of the formed tubules. (scale bars = 100 μm) B The number tube structures per view field of each group. It represents the tubule that has already formed. C The number of master junctions per view field of each group. It represents the potential to form new tubules. D The line graph of the number of meshes E The line graph of the number of master junctions. *p < 0.05, **p < 0.01

Fig. 3

Cell migration results of HUVECs. (n = 3) A Representative images of cell migration results, scale bars = 500 μm. B The migration rate of each group over time. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C The line graph of the migration rate which can show the growth trend of each group

Fig. 4

Healing process of wounds promoted by MSGMSC-exos and ADSC-exos. A The procedure of in vivo study. B Representative images of wound healing process in Control, GelMa, GelMa + ADSC-exos and GelMa + MSGMSC-exos group. C Wound healing rates at different time points of the four groups. (n = 6) *p < 0.05, **p < 0.01, ***p < 0.001. #p < 0.05, ##p < 0.01 (* stands for MSGMSC-exos group vs GelMa group. # stands for ADSC-exos group vs GelMa group). D Representative photomicrographs of wounds, showing increased neo-vessel formation (Red arrows) in MSGMSC-exos and ADSC-exos groups (scale bars = 5 mm)

Fig. 5

Representative images of H&E staining in four groups. Red arrows stand for the margin of the wound bed. Black lines stand for the nonepithelialized area in the control and GelMa group (scale bar = 1 mm)

Fig. 6

Collagen deposition of wounds promoted by MSGMSC-exos and ADSC-exos A Representative images of Masson staining (scale bars = 1 mm) and immunohistochemical staining of type III collagen and type I collagen (scale bars = 100 μm) in four groups. B Quantitative analysis of overall collagen expression, collagen III and collagen I deposition, and collagen I/ collagen III ratio (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

MSGMSC-exos and ADSC-exos enhance cell proliferation and angiogenesis while suppressing inflammation. A Representative images of Ki67 immunohistochemical (scale bars = 100 μm) and quantification of the number of Ki67 positive cells in the wound area (n = 3). B Representative images of CD31 immunohistochemical staining (scale bars = 250 μm) and microvessel counts analysis in the wound bed (n = 3). C Representative images of α-SMA staining (scale bars = 250 μm) and mature vessels counts analysis (n = 3). D Representative images of immunofluorescence staining of macrophages and regulatory T cells in the wound bed (scale bars = 100 μm). E Quantitative analysis of the immunofluorescence staining (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 8

Bioinformatics analysis of MSGMSC-exos and ADSC-exos. A Venn diagram of MSGMSC-exos and ADSC-exos against ExoCarta. B GO analysis of ADSC-exos C GO analysis of MSGMSC-exos D KEGG analysis of ADSC-exos E KEGG analysis of MSGMSC-exos

Fig. 9

Bioinformatics analysis of the specific proteins detected in each source of exosomes. A The volcanogram of differential proteins for MSGMSC-exos vs ADSC-exos. B Heatmap of MSGMSC-exos vs ADSC-exos C GO and KEGG analysis of specific proteins for ADSC-exos D GO and KEGG analysis of specific proteins for MSGMSC-exos E PPI analysis based on shared pro-angiogenic proteins. F Elisa test of ITGB1 in MSGMSC-exos and ADSC-exos