The Effect Of Adipose Derived Stem Cells On Full-thickness Skin Grafts In SD Rats

Background:Full-thickness skin graft(FTSG)is one of the common methods to treat with acute or chronic wounds in plastic surgery.As consisted of the epidermis and the whole dermis,the effect of FTSG is better than the other two kinds of skin grafts(split-thickness skin graft and epidermis graft).However,with the increase of skin thickness,the grafted skin seems difficult to survive.As a type of MSCs,ADSCs have the ability of multilineage differentiation and secretion of several growth factors such as VEGF,EGF and FGF.During the process of isolating ADSCs,SVF cell suspension,whose effect is even considered greater than that of ADSCs in some aspects,should be obtained first and both of them are supposed to promote the establishment of blood supply in the wound,which plays an important role in the healing process of skin graft.So that SVF cells and ADSCs are considered to have the ability to improve the survival of FTSG theoretically.Objective:1.To clear the multi-differentiation and paracrine functions of ADSCs obtained by enzymatic digestion;2.To verify the effect of allogeneic ADSCs and autologous SVF cells on full-thickness skin graft transplantation and compare the difference of them;3.To adjust the methods of using allogeneic ADSCs and to verify its effect on FTSG.Methods:1.The groin fat pads of SD rat were gained,minced and digested with collagenase and centrifuged to obtain SVF cell suspension.The suspension was inoculated in the culture dish and cultured in the incubator.Different concentrations of ADSCs were inoculated on 24-well plates,and the serum-free medium was collected for ELISA to detect their paracrine ability.The adipogenic,osteogenic and chondrogenic abilities of the cells were tested by relative induction mediumly respectively.2.24 SD rats were divided into three groups and beneath the graft,autologous SVF suspension(group A),allograft ADSCs(group B)and PBS buffer(group C)were injected respectively.3d,7d and 14d after the operation,the replanted skins were observed and the rates of skin necrosis were evaluated.Meanwhile,HE and Masson staining,as well as VEGF and CD31 immunofluorescence were detected.3.To enhance the effect of allogenic ADSCs,two methods were applied,including intradermal and subcutaneous injection of ADSCs and PBS buffer(group a and group b)7days preoperatively and the usage of 3%sodium hyaluronate(ADSCs+HA=group c and PBS+HA=group d).The effects of both methods were evaluated by animal experiments.Results:1.With collagenase digestion and centrifugation,SVF cell suspension could be obtained successfully,and relatively homogeneous ADSCs could be obtained after culture,which could be induced to differentiate into adipocytes,chondrocytes and osteocyte under the induction of relative culture medium.Furthermore,it was verified that it had a strong paracrine function and the order of 10~6 seemed the best;2.The skin necrosis rates at the 3 observation points of group A were 25.29±4.54%,28.18±4.76%and 32.17±6.16%respectively,which were better than those of group B(46.42±3.95%,51.86±5.53%and 59.68±5.70%),and those of group C(51.91±5.04%,62.63±5.33%and 71.51±12.70%),with significant statistical significance.The skin necrosis rate of group B and group C was statistically significant on day 7,but there was no significant difference in day 3 and day 14.3.3days after surgery,the skin necrosis rates of group a and group b were 37.87±3.01%and 49.86±2.86%respectively,with statistical significance.Compared with the allogeneic ADSCs injection subcutaneously,the results were also better.However,the skin necrosis rate was still higher than that of autologous SVF cells.7days after surgery,the skin necrosis rates of group a and group b were 44.12±2.82%and 57.45±3.74%respectively,with statistical significance.The skin necrosis rate was still higher than that of autologous SVF despite no significant statistical difference between the two groups.But14 days after operation,the skin necrosis rates of group a and group b were 45.51±3.33%and 60.58±2.39,which were statistically different.Even if it was without statistical significance,compared with subcutaneous injection of allogeneic ADSCs,the skin necrosis rates of group a were slightly superior while that was still not dominant compared with autologous SVF cells.Conclusion:1.With enzymatic digestion and centrifugation,SVF cells were extracted from rat groin fat and cultured to obtain ADSCs cells,which were induced by lipogenic,osteogenic and chondrogenic differentiation culture medium.In addition,ADSCs have strong paracrine ability and can secrete a variety of growth factors including VEGF,EGF,FGF and GM-CSF,and the effect is the best at the order of 10~6.2.Compared with PBS buffer solution,the subcutaneous use of autologous SVF cells and allogeneic ADSCs can improve the effect of FTSG,but the effect of autologous SVF cells is better;3.Injection of allogeneic ADSCs before FTSG can improve the survival of skin graft to a certain extent,but this situation does not have obvious advantages over immediate use of allogeneic ADSCs,and it is still not as effective as autologous SVF cells.However,the addition of HA in ADSCs cell suspension did not improve the effect of ADSCs on full-thickness skin graft transplantation.