| Skin wound repair is an important topic in Burns and Reconstructive Surgery.Delayed wound healing brings serious burden to patients,families and society.hUMSC,which plays an active role in tissue reconstruction and wound healing,are a kind of pluripotent stem cells with potent self-renewal and multi-differentiation ability.hUMSC exosomes are hUMSCderived nanoparticle(30-150 nm).Containing complex proteins and RNA,mesenchymal stem cell exosomes participate in intercellular communication and immunomodulatory,they also play an important role of inhibitory oxidative stress,anti-inflammatory,anti-fibrosis,and enhanced angiogenesis.Exosomes are the key to the effectiveness of hUMSC.However,due to the limited purification technology,the extraction process of exosomes is timeconsuming,high cost,low purity so far.Therefore,we proposed to prepare a kind of engineering nano-particle called exosome mimetics(EMs),which could simulate hUMSC exosomes and the double-layer membrane structure of stem cell exosomes by physical film extrusion,so as to retain the functional contents of stem cells.In order to simulate microenvironment characteristics of full-thickness skin injury,hUMSC-EMs were loaded on Gel MA,and the EMs loaded-Gel MA was applied for skin injury to investigate the application effect of hUMSC-EMs on wound healing and tissue reconstruction.Although the benefits of hUMSC-derived EMs addressed issues in wound healing,the proteome cargo of EMs has not been comprehensively characterized and may potentially have an effect on recipient tissues and cells.We investigated the proteome cargo of EMs and compared it to that of natural exosomes secreted from the same cell line.This should mean something to expand and provide theoretical basis for the potential clinical application of hUMSC EMs loaded-hydrogel in wound healing and tissue reconstruction.Part 1 the preparation of hUMSC engineered-exosome mimeticsObjective: Isolate,purify and identify hUMSC engineered-EMs and hUMSC-derived exosomes,providing the foundation for the following experiment.Methods: After hUMSCs was cultured into the optimal period(3 ～ 8 passages),the culture supernatant was collected,and hUMSC exosomes were separated and purified by supercentrifugation.hUMSC engineered EMs suspension was prepared from hUMSC by physical transmembrane extrusion technology.hUMSC-EMs suspension was separated and purified by supercentrifugation.After extraction,transmission electron microscopy,nanoparticle tracking analysis and western blot were used to identify and observe the morphology,particle size and surface specific markers of hUMSC-EMs and hUMSC exosomes.Results: Except for a little difference,the morphology and particle size distribution of hUMSC-derived exosomes and EMs were similar.However,their specific surface markers were significantly different,which might be related to different biogenesis.Conclusions: Both hUMSC-derived exosomes and EMs have demonstrated their nanoscale structures and favorable biocompatibilities.From the aspect of morphology and biocompatibility,EMs could well simulate exosomes.Part 2 The application of hUMSC engineered exosome mimetic loaded-hydrogels for wound healing and tissue reconstructionObjective: To investigate the application of hUMSC engineered EM loaded-hydrogels for wound healing and tissue reconstruction.Methods: Human dermal fibroblast(HDF-a)were cultured to stable state and they were divided into control group,hUMSC exosome group,hUMSC engineered EMs group.Ed U(5-ethynyl-2 ‘-deoxyuridine)incorporation assay,CCK-8(Cell Counting Kit-8)assay and scratch experiment were used to explore the cell proliferation and migration promotingability of hUMSC engineered EMs.Using full-thickness skin injury model of mice,which were divided into control group,hUMSC exosomes group,hUMSC EMs group,hUMSC exosome loaded Gel MA group and hUMSC EMs loaded Gel MA group,we recorded and collected the skin tissue at 3,7 and 14 days after surgery.The wound size was measured,and the skin tissue sections were prepared for HE(hematoxylin-eosin)staining and immunohistochemical staining,and the expression of tissue repair related signal proteins,for example,ki67,were measured.Results: In vitro experiment results showed that both exosomes and EMs could promote proliferation and migration of fibroblasts,and EMs even had better proliferation or migration effect at some concentrations.For experiments in vivo,through the evaluation and analysis of histomorphology and immunohistochemistry,we found that the Gel MA loaded with EMs had significantly wound healing effect.Conclusions: Exosome mimetics not only have the same effect as exosomes,but have an even better effect on wound repair under some circumstance.There is enormous clinical transformation potential for exosome mimetics.Part 3 The mechanism of wound healing and tissue reconstruction by hUMSC exosome mimeticsObjective: Further explore the mechanism of hUMSC engineered EMs on wound repair and tissue reconstruction.Methods: hUMSC exosome and hUMSC engineering EM proteins were determined and relative quantified by label-free proteomic quantitative technique.Quantitative and identified protein information was subjected to a series of quality control to ensure that the quality of the results met the standards.After retrieval in the database,the subcellular structure localization,Gene Ontology(GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathways were evaluated in detail.Results: Proteomic profiles showed that,compared to exosomes,the upregulation differential proteins enriched in EMs were expressively abundant.From the perspective of subcellular structure,the effect of EMs for wound healing may be related to the function of mitochondria.GO and KEGG analysis showed that,Notch,Wnt/β-catenin,PI3K/Akt,JAK/STAT3,Ras/ERK and other classical signaling pathways may also be involved.Conclusions: EMs promote wound healing is associated with multiple biological processes,such as,mitochondrial energy metabolism,Notch signaling,etc. |
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