Effect And Mechanism Of CEFFE/Chitosan Chloride Microspheres On Wound Repair

Background:Wound repair is a common problem in clinical.Chitosan chloride microspheres have been widely proved to have good antibacterial properties,but they lack the promoting regeneration ability.Cell-free Fat Extract(CEFFE)is a collection of high-proteins isolated from fat and contains no cells.Like adipose stem cells(ADSCs)and platelet rich plasma(PRP),CEFFE could promote wound regeneration by inhibiting wound inflammation and promoting wound vascularization.It is worth studying whether the modification of chitosan chloride microspheres with CEFFE could enhance their ability to promote wound repair.Methods:1)Performing Scratch,CCK-8 and tube formation assay to identify the functions of CEFFE.Using PCR and immunofluorescence staining technologies to compare the makers expression differences and observe the effect of CEFFE on macrophage polarization.2)Preparation and characterization CEFFE/chitosan chloride microspheres.Testing the antibacterial,promoting HUVEC migration,and tube formation abilities in vitro.And verifying the wound healing property in vivo.Results:1)CEFFE was successfully prepared and showed light red color;Scratch,CCK-8 and tube formation assay results showed that the wound healing distance,cell proliferation rate,branch number(/mm~2)and average tube length of CEFFE group were significantly better than those of PBS group(P<0.05);PCR and immunofluorescence staining results found that M0+CEFFE group highly expressed CD163 and CD206,which were consistent with M2 group.2)CEFFE/chitosan chloride microspheres were successfully prepared.The antibacterial experiment showed that there was no obvious colony formation of S.aureus and E.coli in CEFFE/chitosan chloride microspheres group.The scratch healing distance,cell proliferation rate,number of branch tubes(/mm~2)and average tube length in CEFFE/chitosan chloride microspheres group were higher than those in chitosan chloride group(P<0.05).Animal experiments showed that the wound healing of mice was better.The histological analysis showed that CD31+capillaries density and ki67+proliferating cells were significantly higher,while CD68+inflammatory cells was significantly lower than that in the PBS group(P<0.05).Conclusion:CEFFE can promote HUVEC proliferation,migration,angiogenesis and M2 macrophage polarization.CEFFE modification endows chitosan chloride microspheres with the ability of anti-inflammatory and promoting vascularization and accelerates wound healing.