Effects of adipose-derived stem cell released exosomes on wound healing in diabetic mice_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

WANG Jiangwen ,  YI Yangyan , ZHU Yuanzheng , WANG Zhaohui , WU Shu , ZHANG Jing , HU Xuan , NIE Jiaying

Department of Plastic Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;

Corresponding?author：

YI Yangyan, Email: yyy0218@126.com

Keywords：

Adipose-derived stem cells; exosomes; fibroblasts; angiogenesis; wound healing; mouse

DOI：

10.7507/1002-1892.201903058

Video：

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Objective To investigate the effects of adipose-derived stem cell released exosomes (ADSC-Exos) on wound healing in diabetic mice.Methods The ADSCs were isolated from the adipose tissue donated by the patients and cultured by enzymatic digestion. The supernatant of the 3rd generation ADSCs was used to extract Exos (ADSC-Exos). The morphology of ADSC-Exos was observed by transmission electron microscopy. The membrane-labeled proteins (Alix and CD63) were detected by Western blot, and the particle size distribution was detected by nanoparticle tracking analyzer. The fibroblasts were isolated from the skin tissue donated by the patients and cultured by enzymatic digestion. The 5th generation fibroblasts were cultured with PKH26-labeled ADSC-Exos, and observed by confocal fluorescence microscopy. The effects of ADSC-Exos on proliferation and migration of fibroblasts were observed with cell counting kit 8 (CCK-8) and scratch method. Twenty-four 8-week-old Balb/c male mice were used to prepare a diabetic model. A full-thickness skin defect of 8 mm in diameter was prepared on the back. And 0.2 mL of ADSC-Exos and PBS were injected into the dermis of the experimental group (n=12) and the control group (n=12), respectively. On the 1st, 4th, 7th, 11th, 16th, and 21st days, the wound healing was observed and the wound healing rate was calculated. On the 7th, 14th, and 21st days, the histology (HE and Masson) and CD31 immunohistochemical staining were performed to observe the wound structure, collagen fibers, and neovascularization.Results ADSC-Exos were the membranous vesicles with clear edges and uniform size; the particle size was 40-200 nm with an average of 102.1 nm; the membrane-labeled proteins (Alix and CD63) were positive. The composite culture observation showed that ADSC-Exos could enter the fibroblasts and promote the proliferation and migration of fibroblasts. Animal experiments showed that the wound healing of the experimental group was significantly faster than that of the control group, and the wound healing rate was significantly different at each time point (P<0.05). Compared with the control group, the wound healing of the experimental group was better. There were more microvessels in the early healing stage, and more deposited collagen fibers in the late healing stage. There were significant differences in the length of wound on the 7th, 14th, and 21st days, the number of microvessels on the 7th and 14th days, and the rate of deposited collagen fibers on the 14th and 21st days between the two groups (P<0.05).Conclusion ADSC-Exos can promote the wound healing in diabetic mice by promoting angiogenesis and proliferation and migration of fibroblasts and collagen synthesis.

Citation： WANG Jiangwen, YI Yangyan, ZHU Yuanzheng, WANG Zhaohui, WU Shu, ZHANG Jing, HU Xuan, NIE Jiaying. Effects of adipose-derived stem cell released exosomes on wound healing in diabetic mice. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(1): 124-131. doi: 10.7507/1002-1892.201903058 Copy

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2.	Greenhalgh DG. Wound healing and diabetes mellitus. Clin Plast Surg, 2003, 30(1): 37-45.
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4.	Kourembanas S. Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol, 2015, 77: 13-27.
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6.	Zhang B, Wang M, Gong A, et al. HucMSC-Exosome mediated-Wnt4 signaling is required for cutaneous wound healing. Stem Cells, 2015, 33(7): 2158-2168.
7.	Zhang B, Wu X, Zhang X, et al. Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4/β-catenin pathway. Stem Cells Transl Med, 2015, 4(5): 513-522.
8.	Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med, 2015, 13: 49.
9.	Rao RR, Stegemann JP. Cell-based approaches to the engineering of vascularized bone tissue. Cytotherapy, 2013, 15(11): 1309-1322.
10.	Rohringer S, Hofbauer P, Schneider KH, et al. Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells. Angiogenesis, 2014, 17(4): 921-933.
11.	Than UTT, Guanzon D, Leavesley D, et al. Association of extracellular membrane vesicles with cutaneous wound healing. Int J Mol Sci, 2017, 18(5): E956.
12.	張靜, 易陽艷, 陽水發, 等. 脂肪干細胞來源外泌體對人臍靜脈血管內皮細胞增殖、遷移及管樣分化的影響. 中國修復重建外科雜志, 2018, 32(10): 1351-1357.
13.	Hamed S, Bennett CL, Demiot C, et al. Erythropoietin, a novel repurposed drug: an innovative treatment for wound healing in patients with diabetes mellitus. Wound Repair Regen, 2014, 22(1): 23-33.
14.	Martin P. Wound healing—aiming for perfect skin regeneration. Science, 1997, 276(5309): 75-81.
15.	Gadelkarim M, Abushouk AI, Ghanem E, et al. Adipose-derived stem cells: effectiveness and advances in delivery in diabetic wound healing. Biomed Pharmacother, 2018, 107: 625-633.
16.	Trounson A, McDonald C. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell, 2015, 17(1): 11-22.
17.	Gy?ngy?si M, Wojakowski W, Lemarchand P, et al. Meta-analysis of cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data. Circ Res, 2015, 116(8): 1346-1360.
18.	Taverna S, Pucci M, Alessandro R. Extracellular vesicles: small bricks for tissue repair/regeneration. Ann Transl Med, 2017, 5(4): 83.
19.	Hu L, Wang J, Zhou X, et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep, 2016, 6: 32993.
20.	Guo SC, Tao SC, Yin WJ, et al. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics, 2017, 7(1): 81-96.
21.	Geiger A, Walker A, Nissen E. Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice. Biochem Biophys Res Commun, 2015, 467(2): 303-309.
22.	Zhao B, Zhang Y, Han S, et al. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation. J Mol Histol, 2017, 48(2): 121-132.
23.	Liang X, Zhang L, Wang S, et al. Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a. J Cell Sci, 2016, 129(11): 2182-2189.

1. Krug EG. Trends in diabetes: sounding the alarm. Lancet, 2016, 387(10027): 1485-1486.
2. Greenhalgh DG. Wound healing and diabetes mellitus. Clin Plast Surg, 2003, 30(1): 37-45.
3. Hyldig K, Riis S, Pennisi CP, et al. Implications of extracellular matrix production by adipose tissue-derived stem cells for development of wound healing therapies. Int J Mol Sci, 2017, 18(6): E1167.
4. Kourembanas S. Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol, 2015, 77: 13-27.
5. Rani S, Ryan AE, Griffin MD, et al. Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications. Mol Ther, 2015, 23(5): 812-823.
6. Zhang B, Wang M, Gong A, et al. HucMSC-Exosome mediated-Wnt4 signaling is required for cutaneous wound healing. Stem Cells, 2015, 33(7): 2158-2168.
7. Zhang B, Wu X, Zhang X, et al. Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4/β-catenin pathway. Stem Cells Transl Med, 2015, 4(5): 513-522.
8. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med, 2015, 13: 49.
9. Rao RR, Stegemann JP. Cell-based approaches to the engineering of vascularized bone tissue. Cytotherapy, 2013, 15(11): 1309-1322.
10. Rohringer S, Hofbauer P, Schneider KH, et al. Mechanisms of vasculogenesis in 3D fibrin matrices mediated by the interaction of adipose-derived stem cells and endothelial cells. Angiogenesis, 2014, 17(4): 921-933.
11. Than UTT, Guanzon D, Leavesley D, et al. Association of extracellular membrane vesicles with cutaneous wound healing. Int J Mol Sci, 2017, 18(5): E956.
12. 張靜, 易陽艷, 陽水發, 等. 脂肪干細胞來源外泌體對人臍靜脈血管內皮細胞增殖、遷移及管樣分化的影響. 中國修復重建外科雜志, 2018, 32(10): 1351-1357.
13. Hamed S, Bennett CL, Demiot C, et al. Erythropoietin, a novel repurposed drug: an innovative treatment for wound healing in patients with diabetes mellitus. Wound Repair Regen, 2014, 22(1): 23-33.
14. Martin P. Wound healing—aiming for perfect skin regeneration. Science, 1997, 276(5309): 75-81.
15. Gadelkarim M, Abushouk AI, Ghanem E, et al. Adipose-derived stem cells: effectiveness and advances in delivery in diabetic wound healing. Biomed Pharmacother, 2018, 107: 625-633.
16. Trounson A, McDonald C. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell, 2015, 17(1): 11-22.
17. Gy?ngy?si M, Wojakowski W, Lemarchand P, et al. Meta-analysis of cell-based CaRdiac stUdiEs (ACCRUE) in patients with acute myocardial infarction based on individual patient data. Circ Res, 2015, 116(8): 1346-1360.
18. Taverna S, Pucci M, Alessandro R. Extracellular vesicles: small bricks for tissue repair/regeneration. Ann Transl Med, 2017, 5(4): 83.
19. Hu L, Wang J, Zhou X, et al. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep, 2016, 6: 32993.
20. Guo SC, Tao SC, Yin WJ, et al. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics, 2017, 7(1): 81-96.
21. Geiger A, Walker A, Nissen E. Human fibrocyte-derived exosomes accelerate wound healing in genetically diabetic mice. Biochem Biophys Res Commun, 2015, 467(2): 303-309.
22. Zhao B, Zhang Y, Han S, et al. Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation. J Mol Histol, 2017, 48(2): 121-132.
23. Liang X, Zhang L, Wang S, et al. Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a. J Cell Sci, 2016, 129(11): 2182-2189.

Chinese Journal of Reparative and Reconstructive Surgery

Effects of adipose-derived stem cell released exosomes on wound healing in diabetic mice

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