Improvement Of The Viability Of Human Autologous Fat Transplantation By Using VEGF-transfected Adipose-derived Stem Cells

BackgroundAutologous fat transplantation(fat grafting or injection) for the correction of soft tissue defects was first described 100 years ago by Neuber.It is currently a technique that is favored by plastic surgeons as it is considered to be easy and associated with little donor site morbidity.However,the clinical outcomes of this technique are limited by resorption of the transplanted fat and calcification due to fat necrosis.Histological studies have suggested that revascularization of autologous fat transplants only occurs after 48 h.This delay appears to critically impair the survival of the fat cells,which undergo degenerative changes,including the destruction of their nucleus and cell membranes.After this cell destruction,fatty cysts develop and the fat gradually becomes absorbed.Thus,early and abundant neovasculization seems to be the key to the survival of free fat transplants,which in turn leads to good transplant outcomes.Recent advances in vascular biology have suggested possible targets whose therapeutic manipulation may increase ischemic tissue survival.One of these targets is vascular endothelial growth factor(VEGF).The ability of exogenous VEGF(and other agents) to stimulate the development of new blood vessels has been investigated. It has been shown that when VEGF is applied topically or injected intravenously,it enhances revascularization of ischemic tissues such as skin flaps.However,the effect of such exogenous cytokine administration is limited by the number of endothelial cells in the transplant.Thus,an alternative strategy is to increase the number of endothelial cells in the transplant.Previous studies have identified a putative stem cell population within the adipose stromal compartment.This so-called adipose-derived stem cells(ASCs) population can be isolated from adipose tissue.Like bone marrow-derived mesenchymal stem cells(MSCs),ASCs can differentiate toward the osteogenic,chondrogenic,adipogenic,myogenic,neurogenic and angiogenic lineages. However,ASCs have several advantages over MSCs in that they are easier to obtain, cause less donor site morbidity,and are available in large numbers upon harvesting.ObjectiveHere,we evaluated whether mixing ASCs with fat tissues prior to transplantation would enhance neovascularization and increase the survival of fat transplants.We also asked whether this effect would be augmented if the ASCs were transduced with the human VEGF gene.Methods1.Isolation and identification of ASCsASCs were isolated from the fatty portion of the liposuction aspirates by using enzyme digestion method and observe changes in morphology and function of cells.The cells are used for experiments after the generation three.We make these cells to adipogenic,osteogenic and chondrogenic differentiation in vitro,then Oil Red O staining and Alizarin red staining and Alcian blue staining to identify the success of differentiation in order to identify the success of multi-directional differentiation.2.DiI staining of ASCsASCs was labelled with fluorescent dyes DiI in vitro,then we use inverted microscope and fluorescence microscopy to observe changes of ASCs.Almost 100% ASCs show red fluorescence.3.Adenoviral-VEGF₁₆₅ infect ASCs and determination of VEGF expression in vitroASCs of human was infected with adenovirus expressing VEGF₁₆₅ gene according to multiplicity of infection equaling to 50.Gene expression of transtected cells was detected by immunofluorescent staining and ELISA analysis in vitro.4.Animal model and groupsEighteen nude mice served as free fat transplantation models.Human fat tissue mixed with DiI-labeled VEGF-transfected ASCs(Group A),mixed with DiI-labeled ASCs(Group B),with insulin(Group C),with Medium(Group D).Four groups were injected subcutaneously into the four random points on the back of eighteen nude mice.5.Examination of Transplants(1) Evaulate the survival of harvested fat at 6 months after transplantation.(2) Histopathological examination of transplants.(3) Determination of neovascularization by measuring capillary density.(4) Determination of the origin of the endothelial cells in neovascular capillaries.Results1.The original cultured ADSCs looks like to fibroblasts,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic,osteogenic and chondrogenic differentiation medium,it is proved to be able to differentiate into mature adipocyte,osteoblast and chondrocyte,respectively. The differentiation can be proved by Oil Red O,Alizarin red and Alcian positive staining respectively.2.Almost all ASCs is labelled with DiI,a kind of fluorescent dye,It is proved not to destroy cells and interfere with cell proliferation.3.ASCs were transduced with the human VEGF gene with a multiplicity of infection(MOI) of 50 pfu/cell.When immunofluorescence staining for VEGF was performed 3 days later,over 90%of the ASCs were stained positively.In contrast,the untransfected ASCs exhibited little VEGF expression.4.(1) The wet weight for Groups A-D were(190.91±29.51) mg,(165.90±32.47) mg,(98.45±13.36) mg,(71.69±15.59) mg,respectively.(2) Group D exhibited excessive fibrosis and fat necrosis while theGroup C showed obvious fibrosis,numerous mixed cell infiltrations,and fat necrosis(left bottom image).In contrast,the Group A and Group B consisted predominantly of surviving mature adipose tissue and had significantly lower levels of fat necrosis and fibrosis.(3)Capillary density was much higher in the VEGF-transfected ASC transplants than in the other three transplants.The transplants mixed with untransduced ASCs also had a reasonably good capillary density.In contrast,few capillaries could be detected in the Group C and D transplants.(4) DiI-labelled ASCs were demonstrated by the presence of red fluorescing cells.Staining of the transplant sections with a FITC-labeled mAb against the endothelial cell marker CD31 revealed the neovascular capillary endothelial cells(green fluorescence).Merging of picture indicates that some endothelial and fat cells are corresbonding with ASCs.Only miscellaneous light can be seen in the Insulin and Control group.DiscussionAutologous fat transplantation is useful for filling soft tissue and contour defects and has the advantage over other transplant techniques in that the transplant material is relatively cheap,easy to harvest,available in large quantities,and autogenic,which eliminates the potential risks associated with allogenic fillers and implants.Fat tissue is a large and diffuse tissue with high metabolic activity.Histological studies have suggested that revascularization of autologous fat transplants only occurs after 48 h. This delay appears to critically impair the survival of the fat cells,which undergo degenerative changes,including the destruction of their nucleus and cell membranes. After this cell destruction,fatty cysts develop and the fat gradually becomes absorbed.Thus,early and abundant neovasculization seems to be the key to the survival of free fat transplants,which in turn leads to good transplant outcomes.Cell therapy is a technology in which autologous,allogeneic or xenogeneic somatic living cells are transplanted into patients for therapeutic,diagnostic or preventive purposes.After recent developments in stem cell research,cell therapy has been extended to assist the revascularization of transplanted tissue or promote wound healing.At present,it has been shown that MSCs,ASCs and endothelial progenitor cells can all promote the revascularization of ischemic tissue.Compared to the other sources,ASCs are ideal in many aspects:they are easily harvested and handled,and they multiply readily,non-invasively,and effectively.Furthermore,ASCs have been shown to have angiogenic characteristics and to differentiate into vascular endothelial cells in experimental settings.These observations together led us to examine here whether mixing fat transplants with ASCs would improve their survival.We found that mixing the fat tissues with autologous ASCs before free fat transplantation significantly increased their capillary density and viability.First,we found that some of the CD31-positive endothelial cells in the fat tissues that were DiI-positive,which indicates that these cells had differentiated from the ASCs.Second,we detected adipogenic differentiation of the DiI-labeled ASCs in the surviving fat tissue,which indicates that some of the adipocytes in the tissue had been derived from the exogenous ASCs.Thus,the differentiation of some ASCs into mature adipocytes that partly constitute the fat transplant may compensate for any early loss of transplanted adiopcytes.Recent advances in vascular biology have suggested possible targets for therapeutic intervention that increases ischemic tissue survival.VEGF has been confirmed to be a particularly relevant target.VEGF can induce angiogenesis and endothelial cell proliferation and stimulates vascular permeability.Given that the half-life of VEGF is short,we used ASCs as carriers for releasing VEGF.Our results indicate that VEGF-transduced ASCs can secrete more VEGF and promote capillary density and fat viability more efficiently than untransduced ASCs.This strongly supports the notion that VEGF can improve the neovascularization and viability of autologous transplanted fat tissue.ConclusionASCs can differentiated into vascular endothelial cells and adipocytes.It contributed to angiogenesis in free transplanted fat tissue.ASCs can increase the survival rate and decrease the rate of fibrosis and steatonecrosis of free transplanted fat tissue.These findings suggest that ASCs assisted transplantation may be an ideal cell therapy.VEGF-transduction of the ASCs augments this effect.