Study On The Role And Mechanism Of Hypoxia-induced UCMSCs-derived Exosomes In GelMA Hydrogel To Promote Wound Healing

Objective: Stem cell therapy has brought fresh concepts for trauma rehabilitation in recent years.Umbilical cord Mesenchymal Stem Cells(UCMSCs),particularly their Exosome(Exo),have been shown to stimulate wound healing via paracrine pathways.Furthermore,changes in the milieu in which Mesenchymal Stem Cells(MSCs),are found can cause changes in exosomal proteins and mi RNAs,boosting the therapeutic effect.Most MSCs exist in a hypoxic environment in vivo;however,in vitro,MSCs are typically cultured in an environment with normal oxygen content.As a result,we predicted that hypoxiainduced UCMSC-derived exosome(H-Exo)could boost the biological role of repairrelated cells and improve wound healing.To investigate the effects of hypoxia,we compared the effects of H-Exo and normoxia-induced UCMSC-derived exosome(N-Exo)on trauma healing-associated cells.The effect of methacryloyl gelatin(Gel MA)hydrogel loaded with H-Exo on wound healing was studied in order to develop a new technique for wound treatment.Methods: First,the effects of H-Exo on human skin fibroblast cells(HSFs)migration were evaluated using a cell scratch test,and the effects of H-Exo on endothelial cells(ECs)tube formation ability were evaluated using a tubule formation assay.Then,using differential mi RNAs analysis,target gene prediction,and gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis,high-throughput sequencing of HExo and N-Exo mi RNAs was undertaken to investigate the various processes by which HExo improves wound healing.Ultimately,a total skin defect model was established in nude mice,which were randomly separated into four groups: control,Gel MA,H-Exo,and Gel MA/H-Exo,each of which was treated differently and photographed on days 3,5,7,and 14.Image J and Graph Pad Prism software were used to calculate the trauma healing rate,and laser Doppler flowmetry was used to detect trauma perfusion.Hematoxylin and eosin staining was used to examine trauma healing,and immunofluorescence staining for von Willebrand factor(v WF)and alpha-smooth muscle actin(α-SMA)was used to measure trauma angiogenesis.Results: First,scratch results revealed that H-Exo promoted HSFs migration substantially better than the Control and N-Exo groups.The results of tube formation experiments revealed that the tube length and node number of tube formation at the 8th hour were significantly higher in the H-Exo group than in the Control and N-Exo groups.Furthermore,mi RNA sequencing data revealed a total of 910 distinct mi RNAs in the two exosomes.There were 328 differential mi RNAs with a P value <0.05,including 147 up-regulated mi RNAs and 181 down-regulated mi RNAs.Using Target Scan and mi Randa software,the number of projected target genes of the differential mi RNAs was 1048575.GO and KEGG enrichment analysis of the predicted target genes revealed that angiogenesis was enriched in the biological process of GO analysis,and the target genes were considerably enriched in the PI3K-AKT signaling pathway and the HIF-1 signaling pathway associated to angiogenesis in KEGG analysis.Lastly,in vivo findings revealed that the Gel MA/H-Exo group of mice with complete skin defects had richer angiogenesis and perfusion,better wound repair regeneration,and stronger immunofluorescence signals in wound healing than the other groups.Conclusion: 1.H-Exo is better capable of promoting HSFs migration and ECs tubule formation.2.H-Exo may promote wound healing via the angiogenesis-promoting mechanism.3.Gel MA hydrogels containing H-Exo can considerably stimulate angiogenesis and have a promising application in wound healing.application prospects.