Study Of Promoting Angiogenesis In Adipose Tissue Engineering Using Adipose-derived Stem Cell Transfected With VEGF₁₆₅ Gene

BACKGROUND:Tissue engineering is a newly emerging subject that provides a new method to repair or regenerate construction and function of tissue defects using principle of cytobiology and engineering.Recently,tissue engineering has been being constructed as a newly emerging biomedical technology to repair or regenerate a body defect by combining cells of high proliferation and differentiation potential with an artificial matrix of cells scaffold and growth factor,including bone,skin,cartilage,vascular, and adipose tissues.However,the only products of tissue engineering that have been used in a clinical setting are of a relatively circumscribed dimension,supplied by diffusion. Because diffusion can supply cells only over a distance of 150μm,this nutrition alone is insufficient for three-dimensional constructs of larger size.The nutrient supply to cells at the center of the graft is inadequate,leading to cell necrosis.To overcome this limitation in the application of cultured engineering tissue,research must focus on accelerating vascularization of the transplanted cells.Especially,fat tissue represents a highly vascularized tissue and the substitution of soft tissue by autologous transplantation of cultivated ASCs requires a large cell volume.Nutrition of these composites by diffusion alone is insufficient.A volume-persistent culture of adipose tissue can be successful only via early vascularization of the composite in order to avoid considerable cell death,especially in the central graft area. VEGF is a kind of the most important angiopoiesis regulatory factor in reconstructing blood circulation as specificity mitogen of vascular endothelial cell, and it is important in process of neovascularization.It is a optima development potentiality therapy mode that transplanting cell imported with VEGF₁₆₅ gene to reconstruct blood circulation in vivo.Studies for combined application of cellular transplant and angiogenic factors in small animal model have been reported recently to enhance the effects of neovascularization.Until now,the research about transplanting human adipose tissue-derived stem cell transfected with VEGF₁₆₅ gene in the adipose tissue engineering for increasing neovascularization and the survival has no report.In this study,we applied this method to the adipose tissue engineering and investigate to see whether autologous ASCs encoding by VEGF₁₆₅ gene are capable of improving neovascularization in the adipose tissue engineering,with potential clinical application for the adipose tissue engineering.OBJECTIVES:1.To find a new method which can be used for isolation and cultivation of ASCs from liposuction,and the ASCs ability differentiated into lipocytes and osteoblast with the foundation of ASCs is the seed cell of adipose tissue engineering。2.To investigate the transfection efficiency and affect of recombinant adenovirus in human adipose tissue-derived stem cell,and to find a better method to label ASCs.3.To establish the foundation of advancing vascularized capability of adipose tissue engineering,by detect the gene expression of ASCs transfected with vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus.4.To assess the possibility of constructing adipose tissue via the attachment of ASCs to typeâ… collagen scaffold.5.To investigate the feasibility of transplanting human adipose tissue-derived stem cell(ASCs) transfected with VEGF₁₆₅ gene to the adipose tissue engineering for increasing neovascularization and the survival.METHODS:1.ASCs Isolation and cultivation and identification ASCs were isolated and cultivated from the liquid portion of liposuction aspirates by directly centrifugate for primary culture and observe changes in morphology and function of cells.The cells are used for experiments after the generation four.Flow cytometry was used to detect the molecular expression which is associated with the surface of the stem cells;making cells in vitro to adipogenic, osteogenic,cartilagenic differentiation.Oil Red O staining and Alizarin red staining and Alcian blue staining to identify the success of differentiation.2.Transfection and marker of EGFP in human adipose-derived stem cellsThe ASCs was infected with adenovirus expressing the EGFP gene by different multiphcity of infection(MOI=0,10,20,30,50,100).The expression of EGFP was detected by fluorscent microsecope.The cytotoxicity of adenovirus to ASCs was evaluated by cell proliferation cell vitality and cell differentiation.3.Expression of adipose-derived stem cell transferred by VEGF₁₆₅ geneHuman adipose tissue were isolated and cultured the cells that the ASCs of human was infected with adenovirus expressing VEGF₁₆₅ gene according to multiphcity of infection equaling to 50.Gene expression of transtected cells was detected by immunofluorescent staining and ELISA analysis in vitro.4.Construction of tissue-engineered adipose using typeâ… collagen in nude mouseHuman adipose tissue were isolated and cultured the cells from fluid portions of liposuction aspirates.We evaluated the use of a combination of adipose tissue derived adult stem cells and collagen sponge for adipose tissue engineering.Adipogenesis was examined in nude mice imbeded subcutaneously with collagen sponge(â… ),ASCs attached collagen sponge(â…¡),or adipogenic differentiation of ASCs attached collagen (â…¢) cultured in medium for 3 days.After 8 weeks,newly formed adipose tissue was observed by fluorescent microscope,histology and Oil red O.5.Transplanting ASCs transfected with VEGF₁₆₅ in the adipose tissue engineering for increasing neovascularizationASCs was isolated from fluid portions of human liposuction aspirates.After transfection by VEGF₁₆₅ gene,then adipogenesis was examined in nude mice imbeded subcutaneously with adipogenic ASCs with differentiation attached collagen sponge(groupâ…£),differentiation and indifferentiation with EGFP gene transfection attached collagen sponge(groupâ…¤),differentiation and indifferentiation with VEGF₁₆₅ gene transfection attached collagen sponge(groupâ…¥) cultured in medium for 3 days.Its were transplanted to the adipose tissue engineering at the same of a nude mouse.The capillary density of transplanted the engineered adipose tissue was detected by blood vessel counting.RESULTS:1.There was a large amount of ASCs in the liquid portion,the cells growed appearance as fibroblasts-like,but it has strong tendency for high proliferation and multiple differentiation.With the effect of adipogenic and osteogenic and cartilagenic differentiation medium,it is proved to be able to differentiate into mature adipocyte and bone cells and chondrocyte.Adipogenic differentiation of ASCs was assessed by Oil Red O staining after 2 weeks and significant fraction of the cells contained multiple,intracellular lipid-filled droplets that accumulated Oil Red-O.After 2 weeks' osteogenic induction,cells were positively stained by alizarin red.After 2 weeks' cartilagenic induction,cells were positively stained by Alcian blue.These kind of cell (ASCs) is also proved to be CD29and CD44 positive expression which are one of the main proof for stem cells.2.Adipose-derived stem cells were successfully infected by adenovirus.EGFP was initially expressed in the adipose tissue-derived stem cell of all transfected groups 24h following infected by Ad.EGFP.The level increased with the incease of MOI and reached peak on 5d.Transfection efficiency of the Expression were 0%(0); 10.3%(10);26.6%(20);47.6%(30);94.7%(50);96.8%(100),respectively.During the period of the culture,cell proliferation,cell vitality and cell differentiation in the transfected and untransfected groups had no difference on 1,3,5,7,9,11d.3.ASCs were successfully infected by vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus.The expression of human VEGF₁₆₅ in the transfected human ASCs was demonstrated by immunofluorescent and ELISA analysis.The level increased with the incease of time and reached peak on 7d.4.ASCs were successfully cultured in collagen sponge.collagen represent good compatibility and adhesion with ASCs without a little toxicity;newly constructed tissue are found in the experiment.After 8 weeks,newly formed adipose tissue was observed in groupsâ…¡andâ…¢,but groupâ… was fully reabsorbed.The wet weigh of the groupsâ…¢was significantly higher than those of the groupsâ…¡(P＜0.05).HE or Oil red O staining of newly formed tissue showed that there was substantially more tissue regeneration and adipogenic effect in groupâ…¢than in groupâ…¡.EGFP expressed in the newly formed adipose tissue was observed by fluorescent microscope.5.Two weeks after transfection,transplanted ASCs survived and were incorporated into the capillary networks in the engineered adipose tissue.The wet weight of transplanted tissue of VEGF₁₆₅ gene transfection group was 22.95±0.79mg, significantly higher than that of the groupâ…£(20.85±1.35mg,P＜0.01),but groupâ…¤(22.52±0.75mg,P＞0.05).The vessels counting of the newly formed adipose tissue for each group were:6.50±2.07/HPF,9.56±2.73/HPF,12.00±2.45/HPF,respectively.The capillary density of transplanted tissue of VEGF₁₆₅ gene transfection group was significantly higher than those of the groupâ…£and groupâ…¤(P＜0.05).Twelve weeks after transfection,transplanted ASCs survived and were incorporated into the capillary networks in the engineered adipose tissue.The survival volume of transplanted tissue of VEGF₁₆₅ gene transfection group was 22.80±0.72mg, significantly higher than that of the groupâ…¤(21.20±1.00mg,P＜0.05) and groupâ…£(13.53±1.16mg P＜0.01).The vessels counting of the newly formed adipose tissue for each group were:11.11±1.94/HPF,15.55±2.77/HPF,18.39±3.73/HPF,respectively. The capillary density of transplanted tissue of VEGF₁₆₅ gene transfection group was significantly higher than those of the groupâ…£and groupâ…¤(P＜0.01).These kind of cell(ASCs) is also proved to be CD31 positive expression in vivo,and to take part in angiopoiesis.DISCUSSIONS:One of the major challenges facing the emerging field of regenerative medicine is finding a reliable source of cells for tissue repair and regeneration.In experiment, Human ASCs form fluid portions of human liposuction aspirates in vitro meet many of the requirements required of the 'ideal' cell for tissue engineering.These stem cells can be easily obtained,easily purified,and are readily expanded in culture,using a relatively noninvasive method.Stem cells offer a potentially unlimited source of cells for tissue engineering;thus,research in using stem cells to produce adipose tissue has become increasingly popular.Another of the major challenges facing the emerging field of regenerative medicine is finding a reliable source of scaffold for tissue repair and regeneration.A lot of scaffolds were applied to tissue engineering,but there were much disadvantage. In the experiment,ASCs were successfully cultured in collagen protein.Collagen protein represents good compatibility and adhesion with ASCs without little toxicity; newly constructed tissue are found.The collagen protein was positived about application of engineering adipose tissue.At the same time,one of the major challenges facing the emerging field of repair mechanism of engineering adipose tissue is finding a better method to label ASCs.It is important for us to find a better method to label ASCs.In the experiment, transduction of EGFP gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label ASCs.In addition,On molecular level,angiopoiesis was a aggregation of a series of molecular event.Vascular growth factor was the most superlative and multiplicity in angiopoiesis,of the total,VEGF was the most effective in the initial stage.VEGF₁₆₅ is predominant and powerthl secrete modality.In general,applied way of VEGF was divided to direct application and indirect application.In the direct application,there were much disadvantage of expensive price,and hard to maintain effective concentration in vivo,and inability self-adjustment.If the factor was inefficiently to maintain formation of new vasoganglion,it is not to prevent the retrogradation and collapse of vasoformation.However,it is very good for indirect application to overcome disadvantage of direct application.In the experiment,transduction of VEGF₁₆₅ gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro.It may be used to increase vascularized capability of ASCs in the study of adipose tissue engineering.Eventually,in experimental result,ASCs from fluid portions of human liposuction aspirates in vitro were successfully infected by vascular endothelial growth factor 165(VEGF₁₆₅) mediated by adenovirus,and can increase the engineered adipose tissue neovascularization and the survival,and bring into full play effectiveness of genic remedial angiopoiesis.Meanwhile,the kind of treatment is safe, because we do not find pathological changes of vascular tumor by the detection of specimen and histology.In vivo,ASCs can be differentiated the endothelial cell to construct blood vessel by immunofluorescent staining,which is possible for us to increase application of the adipose tissue engineering in clinical.CONCLUSIONS:1.ASCs was isolated by directly centrifugate and filtrate the fluid portions of autologous liposuction aspirates of human without enzymatic digestion.The way of Isolation and cultivation is convenient and easier to carry out.possessed the capabilities of pluripotential differentiation.The ASCs can rapidly in vitro,and reached the number for implantation in short time.Thus,the use of ASCs as the seed cell of to engineer adipose tissue seems to be more inspiring.2.Transduction of EGFP gene to ASCs by adenovirus is safe and high efficient, and the target protein can be expressed in cell in vitro.EGFP transfection is a better method to gene therapy and label ASCs.3.Transduction of VEGF₁₆₅ gene to ASCs by adenovirus is safe and high efficient,and the target protein can be expressed in cell and secreted to surrounding at a high level in vitro.It may be used to increase vascularized capability of ASCs in the study of adipose tissue engineering.4.This study provides significant evidence that ASCs obtained from fluid portions of liposuction aspirates and collagen can be used in adipose tissue engineering.5.ASCs form fluid portions of human liposuction aspirates in vitro can increase the engineered adipose tissue neovascularization and the survival,and the ability of promoting neovascularization of ASCs transfected with VEGF₁₆₅ gene is more potent than ASCs alone.In vivo,ASCs can be differentiated the endothelial cell to construct blood vessel,which is possible for us to increase application of the adipose tissue engineering in clinical.