Experimental Study On Adipose-derived Tem Cells Embedded In Platelet-rich Scaffolds Improving Texture Of Skin Grafts

Background:Skin grafting is a technique that is wildly used in plastic and reconstructive surgery.Comparing to flaps transferring,skin grafting is easily manipulated.However,after transplanted to donor sites,skin grafts lacks blood supply at early stage and revascularization is inevitable.As a consequence,their poor textural durability and associated contracture are less satisfactory than skin flaps.It has been proved that stromal vascular fraction(SVF)suspended with saline can improve the texture for full-thickness skin grafts by promoting angiogenesis.However,during the procedure of the skin graft,the cells in the SVF are easily lost with the leakage of the suspension,resulting in uncertainty about the number of effective stem cells.Thus,it is necessary to find a suitable stem cell carrier to prevent the issue mentioned above.Platelet-rich plasma(PRP)gel is an autologous biological scaffold with a rich source of growth factors.It has a potential to promote stem cells proliferation and differentiation.Adipose derived stem cells(ADSCs)combined with PRP have been proved to promote wound healing and tissue regeneration.Objective:We use ADSCs embedded in PRP gel to assist rat skin grafts.The texture of the skin graft and the indexes of revascularization were detected,so that the validity of the combined use of ADSCs and PRP assisted skin grafting is confirmed,which would provide the reference and theoretical basis for clinical application.Method:1.Adipose-derived stem cells were isolated from adipose tissue of Lewis rats.The stemness was identified by adipogenic and osteogenic induction.2,PRP was prepared by taking the blood from the abdominal aorta of 6-7 weeks male Lewis rats.The number of platelet of PRP were counted.The release of VEGF,bFGF and PDGF in PRP was detected by ELISA kit in vitro.3,After ADSCs were seeded in PRP,PRP was activated and gelling.The microstructure of ADSCs embedded in PRP gel was studied under Scanning electron microscope.4,Animal model was established by removing a 3×3 cm~2 skin area in the midline of the back of male Lewis rats including the panniculus carnosus.Full-thickness skin graft was performed by completely removing the panniculus carnosus tissue.The graft was then reattached to the underlying muscle fascial layer.All rats were randomly divided into 4 groups(18 in each group),group A was control group,group B was PRP treated group,group C was treated with ADSCs,group D was ADSCs+PRP treated group.The contracture ratio,elastic modulus,blood flow perfusion were recorded and statistical analysed on the 14th,30th and 90th day after operation.5,Slide sections were prepared from paraffin embedded skin tissue of grafts from all groups.All sections were stained with haematoxylin and eosin,Masson's trichrome stain and immunohistochemistry staining was used for VEGF and CD31 so as to observe the structural differences,collagen formation and angiogenesis of each group.6,The expression level of angiogenisis factors VEGF,bFGF and PDGF-BB among the four groups was detected by RT-PCR and Western-blot.7,The activation of PI3K/AKT pathway and PLC?/MAPK pathway was detected by RT-PCR.Result:1.The morphology of ADSCs isolated from rat adipose tissue was spindle-shaped,and it had strong self-renew and proliferation ability.It had the potential of adipogenesis and osteogenesis.Its biological characteristics were stable.2,Platelet counting in rat PRP is about 6.19 times of the whole blood.PRP in vitro can continuously release angiogenesis factor VEGF,PDGF-BB and bFGF.3,Scanning electron microscopy showed the porosity of PRP.ADSCs could be seeded evenly in PRP gel and survived well.PRP gel was suitable to be applied under the skin graft.4,Compared to control group,the contracture ratio of the skin in ADSCs+PRP treated group decreased by 46.8%(p<0.005),the softness increased by 44%(p<0.005),the thickness increased by 200%(p<0.005),the subcutaneous collagen fibers were more arranged.5,The blood flow outcome and immunohistochemical results showed enhanced skin graftvascularization.6,The results of RT-PCR demonstrated that in 2weeks,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is7.90 times(P<0.05),3.00 times(P<0.05)and 5.25 times(P<0.05)that of control group.In one month,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is 7.00 times(P<0.01),9.18 times(P<0.01)and 5.00 times(P<0.01)that of control group.In 3 months,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is 22.17 times(P<0.005),50.57 times(P<0.005)and 5.8 times(P<0.01)that of control group.The results of Westernblot demonstrated that in 2 weeks,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is 5.17 times(P<0.05),2.65 times(P<0.05)and 4.30 times(P<0.05)that of control group.In 1 month,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is 1.15 times,8.66 times(P<0.05)and 5.70 times(P<0.05)that of control group.In 3 months,the expression of VEGF,PDGF-BB and bFGF in the ADSCs+PRP group is 1.25 times(P<0.05),1.99 times(P<0.05)and1.75 times(P<0.05)that of control group.7,The expression of AKT and MAPK in ADSCs+PRP group were 1.5 times and 2.32 times(p<0.05)that of control group,as showed by RT-PCR.Conclusion:ADSCs combined with PRP can improve the texture of rat skin graft by increasing the expression of angiogenesis factors and promoting the angiogenesis in rat skin graft.One of the reason of the increase of vascularization of the skin graft is due to activation of PI3K/AKT signaling pathway and PLC?/MAPK pathway,which results in behavioral changes of endothelial cells.ADSCs combined with PRP have a potentiation effect on improving skin grafts by promoting angiogenesis.