Adipose mesenchymal stem cell-derived exosomes stimulated by hydrogen peroxide enhanced skin flap recovery in ischemia-reperfusion injury

Abstract

Background: Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for the treatment of ischemic disease or injury and may be an alternative treatment for cell therapy. This aim of the study was to evaluate whether exosomes derived from adipose mesenchymal stem cell (ADSC) can protect the skin flap during ischemia-reperfusion (I/R) injury and induce neovascularization.

Methods: To investigate the effects of exosomes in the I/R injury of flap transplantation in vivo, flaps were subjected to 6 h of ischemia by ligating the left superficial inferior epigastric vessels (SIEA) followed by blood perfusion. Exosomes derived from normal ADSC (ADSC-exos) and exosomes derived from ADSC preconditioned with H₂O₂ (H₂O₂-ADSC-exos) were injected into the flaps. Then, the blood perfusion unit (BPU) of the flaps was measured by Laser Doppler Perfusion Imaging (LDPI) and microvessel density was determined by the endothelial with cell marker CD31 with Immunohistochemistry (IHC) staining. Inflammatory cell infiltration of the skin flap and apoptosis were detected by hematoxylin & eosin staining (H&E) and the TdT-mediated biotinylated dUTP nick end-labeling (TUNEL) technique.

Results: In vivo, exosomes significantly increased flap survival and capillary density compared to I/R on postoperative day 5, and decreased the inflammatory reaction and apoptosis in the skin flap (P < 0.05). Furthermore, H₂O₂-ADSC-exos had better outcomes compared to normal exosomes (P < 0.05). ADSC-exos could significantly increase human umbilical vein endothelial cell (HUVEC) proliferation (P < 0.05), but no statistic difference was found in exosomes derived from different microenvironments (P > 0.05). HUVEC co-cultured with H₂O₂-ADSC-exos increased the migration ratio and generated more cord-like structures compared to ADSC-exos and the control group (P < 0.05).

Conclusion: ADSC-exos can enhance skin flap survival, promote neovascularization and alleviate the inflammation reaction and apoptosis in the skin flap after I/R injury. The use of a specific microenvironment for in vitro stem cell culture, such as one containing a low concentration of H₂O₂, will facilitate the development of customized exosomes for cell-free therapeutic applications in skin flap transplantation.

Keywords: Adipose-derived stem cells; Exosomes; Hydrogen peroxide; Ischemia-reperfusion injury; Neovascularization; Skin flap transplantation.