Promoting Effect Of Adipose-derived Stem Cells Loaded Chitin Nanofiber Hydrogel On Wound Healing And Its Mechanism

With the development of regenerative medicine,adipose mesenchymal stem cells provide a new possible for the treatment of diseases such as diabetes wound,bone defect,but there are some limitations in application.Chitin is a kind of multifunctional material with good biological activity,which can be used to prepare stem cell scaffolds and provide a new method for local application of stem cells.However,the biological characteristics of chitin nanofiber and the effect of chitin nanofiber on adipose mesenchymal stem cells are not clear.The role and mechanism of adipose mesenchymal stem cell-chitin nanofiber hydrogel in acute and chronic wounds remain to be further studied.In this study,we prepared chitin nanofiber biological scaffold and clarified its biological characteristics.Chitin nanofiber hydrogel was used to culture adipose mesenchymal stem cells and its effect on adipose mesenchymal stem cells was investigated.Furthermore,the effects of adipose mesenchymal stem cell-chitin nanofiber hydrogel in acute and chronic wounds were clarified.1.Preparation and characterization of β-chitin nanofiber stem cell scaffolds(1)Preparation of β-chitin nanofiber suspension: Chitin nanofiber was successfully prepared from Squid cartilage by acid extraction and alkali purification,which had a certain fluidity.The structure of nanofiber was observed under scanning electron microscope,and the β chitin was proved by the characteristic peaks of infrared ray and X-ray..(2)Preparation of chitin nanofiber stem cell scaffolds with different morphology: Chitin nanosuspension can react with cell culture medium to form a jelly-like gel.Chitin nanosuspension was prepared into gel scaffold and thin film scaffold by gelation reaction and extraction.Scanning electron microscopy showed that the gel had porous structure before and after formation.Rheological examination showed that the strength of chitin nanofiber hydrogel material increased after the reaction with cell culture medium.There is no obvious change when chitin nanofiber membrane was treated in cell culture medium,and its structure scanned by electron microscopy showed compact.(3)Effect of different chitin nanofibers forms on wound healing: Compared with model group,chitin nanofiber hydrogel can accelerate the wound healing of full-thickness skin defect in rats,while there was no significant difference between the chitin nanofiber membrane group and the model group.(4)Biological evaluation of chitin nanofiber hydrogel: The survival rate of L929 and adipose stem cell was more than 90% after 12 h,24h and 48 h exposed to chitin nanofiber extraction solution,which showed no obvious cytotoxicity and could be used for cell culture and nutrition delivery.Sensitization evaluation results showed that guinea pig skin had no allergic reaction to the material.In vivo degradation experiment results showed that chitin nanofiber hydrogel was gradually wrapped by tissue and shrunk over time,and basically degraded after 1 month.ELISA results showed that the expression levels of interleukin-1α,interleukin-4 and transforming growth factor-β(TGF-β)in the surrounding skin tissue did not change significantly after the chitin nanofiber hydrogel was placed under the skin.These results indicate that the chitin nanofiber suspension can react with the medium for gelation,and the formed hydrogel has good porous structure,mechanical strength and biological properties,which can be further used in the study of adipose mesenchymal stem cells culture and wound healing.2.Effects of β-chitin nanofiber hydrogel on adipose mesenchymal stem cells(1)Morphological observation of adipose mesenchymal stem cells: The spherical growth of adipose stem cell in chitin nanofiber hydrogel was observed by ordinary optical microscopy,and green fluorescent cell spheres were seen when GFP labeled adipose stem cell were observed under fluorescence microscope,which further confirmed the above experimental results.The results of scanning electron microscopy showed that adipose mesenchymal stem cells were evenly distributed as cell spheres in chitin nanofiber hydrogel.(2)Proliferation,migration and exosome secretion of adipose mesenchymal stem cells: CCK8 showed that the adipose mesenchymal stem cells could grow in the gel and were still alive at 15 days.Transwell experiments showed that cells migrated from the upper compartment to the lower compartment.Transmission electron microscopy,NTA and Western blot were used to detect the secretion of exosomes in the supernatant of adipose stem cell cultured from chitin nanofiber hydrogel.(3)Transcriptome sequencing and wound healing related factors detection: GO analysis of transcriptome sequencing results enriched the changes of cell differentiation,cell cycle,cell adhesion,positive regulation of cell population proliferation,membrane genes and protein-binding genes,which provided reference for material optimization and stem cell modification.KEGG enrichment further proved the gene changes such as platelet derived growth factor D(PDGFD),Insulin-like growth factor 1(IGF1)and TGF-β1.Western blot results showed that the expressions of PDGFD,IGF1 and TGF-β1 were increased after chitin nanofiber hydrogel culture,which were consistent with transcriptome sequencing results.These results indicate that adipose mesenchymal stem cells from chitin nanofiber hydrogels can effectively proliferate,survive and secrete exosomes.Chitin nanofiber hydrogels may affect the expression of genes and proteins related to wound healing in adipose mesenchymal stem cells.3.Effect and mechanism of adipose mesenchymal stem cell-chitin nanofiber hydrogel on acute wound healing(1)Effect of chitin nanofiber hydrogel on promoting acute wound healing:Adipose mesenchymal stem cells-chitin nanofiber hydrogel intervention significantly improved the wound healing rate,accelerated the epithelialization of wound tissue,promoted collagen formation,and increased the expression of angiogenesis related proteins VEGFR and α-SMA in rats.The promoting effect of adipose mesenchymal stem cells-chitin nanofiber hydrogel on wound healing was better than that of chitin nanofiber hydrogel or adipose mesenchymal stem cells.(2)Effect of adipose mesenchymal stem cells-chitin nanofiber hydrogel on key proteins of wound healing through exosomes: Previous studies have shown that stem cells exert biological activities mainly through cell differentiation and exosome secretion.To further elucidate the mechanism of adipose mesenchymal stem cells-chitin nanofiber hydrogel promoting wound healing through exosomes,the effects of adipose mesenchymal stem cells exosomes-chitin nanofiber hydrogel on acute wound healing in rats were observed and analyzed by transcriptome and proteomics.The complement factor D(CFD)and aldolase A(Aldoa)proteins were changed after adipose mesenchymal stem cells exosomes-chitin nanofiber hydrogel treatment,which were verified by western blot.Furthermore,the expressions of CFD and Aldoa proteins in new wound tissue were also increased after adipose mesenchymal stem cells-chitin nanofiber hydrogel intervention.(3)Adipose mesenchymal stem cell exosomes promote fibroblast proliferation by regulating Aldoa protein: Aldoa is associated with cell proliferation,and the effect of adipose mesenchymal stem cell exosomes on cell proliferation was studied in vitro.Western blot results showed that adipose mesenchymal stem cell exosomes promoted the proliferation of L929 mouse fibroblasts and the expression of CFD and Aldoa proteins.After the Aldoa protein was inhibited,the proliferation of L929 mouse fibroblasts promoted by adipose mesenchymal stem cell exosomes was weakened.These results suggest that adipose mesenchymal stem cell-chitin nanofiber hydrogel can regulate CFD and Aldoa protein expression to accelerate skin fibroblast proliferation and promote acute wound healing in rats by adipose mesenchymal stem cell exosomes.4.Effect and mechanism of adipose mesenchymal stem cells-chitin nanofiber hydrogel on wound healing in diabetic mice(1)Effect of adipose mesenchymal stem cells-chitin nanofiber hydrogel on wound healing in diabetic mice: To further explore the promoting healing effect of adipose stem cells-chitin nanofiber hydrogel on chronic wounds,we established full-thickness defect wound model in db/db diabetic mice.After wound intervention by adipose stem cells-chitin nanofiber hydrogel,the wound healing rate was significantly increased,the scar decreased,the granulation tissue became nearly normal,the formation of epithelialization was accelerated,the collagen deposition was increased,and the expressions of cytokeratin and Ki67 proteins were also significantly increased.(2)Adipose mesenchymal stem cells-chitin nanofiber hydrogel promoted wound healing by angiogenesis and extracellular matrix remodeling: In addition to cell proliferation,angiogenesis and extracellular matrix remodeling play an important role in diabetic wound healing.ELISA results showed that adipose mesenchymal stem cells-chitin nanofiber hydrogel could promote the expression of VEGF,VEGFR,MMP-9 and TIMP-1 proteins in mouse wound tissue.Because Aldoa can activate HIF-1α by feedback,the expressions of Aldoa and HIF-1αwere detected on the results of promoting acute wound healing.Adipose mesenchymal stem cells-chitin nanofiber hydrogel increased the expression of Aldoa and hypoxia inducible factor-1(HIF-1α)in newborn tissue.The expression levels of HIF-1α,vascular endothelial growth factor(VEGF),VEGFR,matrix metalloproteinase-9(MMP-9)and tissue inhibitor of matrix metalloproteinase-1(TIMP-1)were decreased after local cutaneous interference with HIF-1α inhibitors.The wound healing rate was slower than that without interference.(3)Adipose mesenchymal stem cell exosomes promote cells migration by regulating HIF-1α/MMP9: HIF-1α can affect the expression of MMP-9 and play an important role in cell migration.Therefore,the role of adipose mesenchymal stem cell exosomes promoting fibroblasts migration was further studied.Cell scratch and transwell assay results showed that the migration of L929 mouse fibroblasts increased after intervention with adipose mesenchymal stem cell exosomes.These were attenuated by interfering with HIF-1α.These results suggest that adipose mesenchymal stem cells-chitin nanofiber hydrogel can promote diabetic wound healing in mice.The effect may be related to HIF-1α/MMP9 activation,which can promote cell migration during wound angiogenesis and extracellular matrix remodeling.In summary,adipose mesenchymal stem cells-β-chitin nanofiber hydrogels were prepared,and the role and mechanism of hydrogels in acute and chronic wound healing were discussed.These play a foundation for adipose mesenchymal stem cells culture and solving the problem of acute and chronic wound treatment.