Experimental Study Of Promotion Effect Of ADSC-exo On Fibroblast Proliferation Derived From Granulation Tissues And The Repairing Effect Of Skin Defect From Nude Mice

Objective:1.The traditional treatment is ineffective for the chronic wound or tissue defect inthe later stage of orthopaedic patients.This experiment aims to find a treatmentmethod from autologous cells or tissues to accelerate the wound healing andachieve the goal of closing the chronic wound quickly.2.In this study,adipose-derived mesenchymal stem cell-derived exosomes wereextracted to intervene in granulation-derived fibroblasts,and to treat fullthickness skin defects in nude mice,trying to find a way to promote healing andreduce scar formation.Methods:1.Adipose tissue discarded from operation was taken for isolation and culture ofADSC.Immunofluorescence staining was used to detect the expression ofCD105,CD90 and CD45 on cell surface.The cells were induced differentiationinto adipogenic,osteogenic and cartilage cells.2.ADSC-exo was separated by ultrafiltration and the morphology was observed bytransmission electron microscopy.Nanosights were used to detect the distributionand size of exosomes,and Western blot was used to detect the expression ofCD81 and CD63.Granulation tissue fibroblasts were isolated and identified.3.Cell scratch test was used to detect whether ADSC-exo could increase themigration ability of fibroblasts;Transwell medium was used to detect theproliferation of ADSC-exo and ADSC at different time points;Flow cytometrywas used to detect the apoptotic effect of ADSC-exo on fibroblasts.4.ADSC-exo was used to treat full-thickness skin tissue defect in nude mice.Thegeneral condition and healing rate during healing were observed.HE staining andMasson staining in ADSC-exo group were compared with those in control group.5.The expression of proinflammatory factors TNF-??IL-6 and anti-inflammatoryfactors IL-10? IL-13 were detected by Elisa method,the expression of collagentype I and III in wounds was detected by qRT-PCR.Western blot was used todetect the expression of ?-SMA,p-Smad2/3 and TGF-?1 protein at 3,4 and 5weeks of wound healing,and qRT-PCR was used to detect the expression of?-SMA and TGF-?1 protein at 3,4 and 5 weeks of wound healing.Western blotand qRT-PCR showed that the contents of ?-SMA,p-Smad2/3 and TGF-?1 inADSC-exo group were significantly lower than those in control group,suggesting that the degree of wound fibrosis in ADSC-exo group wassignificantly lower.Results:1.Immunofluorescence staining showed high expression of CD105 and CD90 onthe surface of ADSC,but no expression of CD45.The separated cells coulddifferentiate into adipogenic,osteogenic and cartilage cells,which confirmed thatthe separated cells were ADSC.The isolated granulation tissue fibroblasts werepositive by vimentin immunochemical staining after culture and passage in vitro,and the overall proliferation ability was weak.2.The vesicle structure and lipid bilayer membrane of ADSC-exo were observedunder electron microscopy.The peak diameter of ADSC-exo gathered at 55 nmand distributed between 40 and 100 nm by Nanosights.The expression of CD81and CD63 was positive by Western blot.3.Cell scratch test showed that the area of scratch migration in ADSC-exo group at24h(p < 0.05)and 48h(p < 0.01)was significantly higher than that in blankcontrol group.Transwell co-culture of fibroblasts showed that the proliferation ofADSC-exo on fibroblasts at 72 h and 96 h was significantly higher than that ofcontrol group(p < 0.01).ADSC-exo could significantly reduce the apoptosis offibroblasts(p < 0.01).4.The scar of ADSC-exo group was significantly lighter than that of control group,and the scar texture was soft.The wound healing rate of ADSC-exo group wassignificantly higher than that of control group at the 2nd,3rd,4th and 5th weeks(p < 0.05).HE staining and Masson staining showed that collagen arrangement inADSC-exo group was relatively neat and inflammatory media infiltration wasrelatively less than that in control group.The expression of TNF-? in woundtissue decreased gradually,and there was a significant difference betweenADSC-exo group and control group(p < 0.05).The levels of anti-inflammatoryfactors IL-10 and IL-13 increased significantly in ADSC-exo group at the 2ndand 3rd weeks,and the difference was statistically significant.5.The ratio of collagen type I to collagen type III in the wounds of ADSC-exogroup and control group gradually tended to the ratio of collagen content innormal skin tissue with wound healing.Western blot and qRT-PCR showed thatthe contents of ?-SMA,p-Smad2/3 and TGF-?1 in ADSC-exo group weresignificantly lower than those in control group,suggesting that the degree ofwound fibrosis in ADSC-exo group was significantly lower.Conclusion:1.The proliferation of isolated ADSC was stronger than that of fibroblasts derivedfrom granulation tissue of chronic wounds.ADSC-exo can significantly increasethe proliferation and migration of fibroblasts,and reduce the apoptosis offibroblasts.2.ADSC-exo treatment can significantly improve the healing speed offull-thickness skin defects in nude mice and reduce scar formation.ADSC-exotherapy can regulate the inflammatory response of wounds and reduce theproduction of inflammatory mediators.3.ADSC-exo may accelerate wound healing and reduce scar formation byup-regulating the expression of collagen gene in wound tissue.ADSC-exo mayinhibit the transdifferentiation of fibroblasts into myofibroblasts by inhibiting theactivation of TGF-?1/Smad2/3 signaling pathway.