In Vitro Construction Of Capillary-like Network With Human Adipose-derived Stem Cell And Pegylated Fibrin Gel

BackgroundThe tissue engineering products provide an effective alteration treatment for those patients with tissue defects and organ dysfunction.However,due to the barrier to nutrients and oxygen transport,the dimension of implants is restricting to 2 millimeter. Researchers had applied tissue engineering composites such as cardiac muscle and hepatic cells to restore the organ function.Moreover,the autologous tissue including adipose and smooth muscle also had been implanted for reconstruction purposes.The results were positive and also gave a clear conclusion that the dimension of implants must be controlled carefully,otherwise,the transplanting cells would be dysfunction. In these cases,the nutrients and oxygen for implants must be brought by the capillary network.In this condition,researchers focus on the construction of capillary network. Human adipose derived stem cells(hADSCs) possess the multi-directional differentiation ability and were applied as seed cells in the study of cell inducing. Being natural extracelluar matrix,fibrin gel was applied as an ideal scalffold for tissue engineered products,especially for artificial blood vessels construction in vitro. Pegylation is the process of covalent attachment of poly(ethylene glycol) polymer chains to another molecule,normally a drug or therapeutic protein.The technique of pegylation has noticeably developed sine 1970s.To this day,pegylation help to meet the challenges of improving the safety and efficiency of many therapeutics.In this study,we will incubate PEG with the fibrinogen macromolecule to improve biological function and construct capillary-like network with human adipose-derived stem cell and pegylated fibrin gel in vitro.Objective1.To optimize the process of blood vessel cells-like inducing with hADSCs mixed within fibrin gel.To observe the biological characteristics of these seeding cells and scalfford for tissue engineered capillary-like patch construction.2.To optimize the process of incubation of a reactive derivative of PEG with the fibrinogen macromolecule.To observe the alteration of biological characteristics of pegylated fibrin gel.3.To establish and optimize the process of blood vessel cells-like inducing with hADSCs mixed within pegylated fibrin gel.Methods1.The human fat tissue collected by liposuction was digested and filtrated to isolate hADSCs.The biological characteristics of hADSCs was evaluated via morphology of cell growth,cell phenotype(CD133,CD11b,CD34,CD71,CD29,CD45,CD31,CD90,CD11a,CD44,Bcl2å’ŒFlk1) expression and adipose cell-like inducing.2.The hADSCs was induced to differentiate towards blood vessel endothelial cells (BVECs).The condition of inducing was estimated by the expression of BVECs specific mark at protein level(vWF,Flk-1,CD31,CD34) and gene level(CD31,CD34,VE-cadherin and Flk-1) through immunochemistry,immunofluorescence assay and RT-PCR assay.3.The hADSCs(P₃) were cultured within fibrin gel and subsequently,induced towards BVECs.The process was optimized through screening various cell density and concentration of fibrinogen.The biological characteristics of differentiating cells within fibrin gel such as phenotype,growth structure and cell ultrastructure were estimated by optical microscopy,scanning electron microscope and transmission electron microscope.4.PEG agents including sc-PEG-sc and mPEG-sc were added to fibrinogen in different molar ratios and reaction temperature.The pegylation level of fibrinogen was measured by TNBS and SDS-PAGE.The modified biological characteristics of fibrin gel were measured through clotting time,percentage of clottable protein and degradation rate of plasmin and collagenase.The ultrastructure of pegylated fibrin gel was observed through scanning electron microscope.5.The hADSCs(P₃) were cultured within pegylated fibrin gel and subsequently, induced towards BVECs.The biological characteristics of differentiating cells within fibrin gel such as growth curve,phenotype,growth structure and BVEC gene expression(CD31,CD34,VE-cadherin and Flk-1) were measured by optical microscopy and RT-PCR assay.Results1.The hADSCs isolated from human adipose tissue were cultured under Dulbecco's modified Eagle's medium plus 10%fetal bovine serum.After several passaging, the hADSCs presented a kind of fibroblast-like phenotype.Gradually,the shape of hADSCs turned to be irregular shape with increasing intramembranous particles after 8～10 passaging.The cell senescence presented after 15 passaging. The flow cytometry detected hADSCs(P₄) strongly expressed CD29,CD44 and CD90.However,the expression of CD105 and CD71 were weak and CD34 and CD45 were negative.The hADSCs were induced differentiation towards adipose cell and the lipid droplets were observed at 6^th day which were determined by oil red staining.2.Before differentiating to BVECs,the hADSCs presented a fibroblast-like phenotype.At sixth day of inducing,the phenotype turned to be ovoid in shape and to be polygon with increasing proliferation at eighth day.The proliferation of cells reached the summit at 12^th day with high cell density.The cobble-like cells which were typical pattern of endothelial cells were observed as well.The specific mark of BVECs including vWF,Flk-1,CD31 and CD34 were determined at 8th day.The blood vessel endothelial cells specific gene including CD31, CD34 and VE-cadherin were determined in hADSCs which were inducing differentiation after 7 days.Only weak expression of VE-cadherin was observed in hADSCs without inducing.3.Fibrin gel was prepared by adding thrombin into fibrinogen solution.The strength of gel was determined by the concentration of fibrinogen solution.Within fibrin gel,the differentiated hADSCs presented a special growth pattern which was 3-dimenstion.The cells showed a starlike shape after 24hours and formed network structure between adjacent cells at 6^th day.The network structure was obvious at 8^th day and the density reached the summit at 12^th day.With the increasing of cell density(0.5～2.0×10⁵/ml),the density of network increasing accordingly.However,the network density decreased gradually when the concentration of fibrinogen increased from 2.5mg/ml to 10mg/ml.The cells which formed the network structure showed Weible-palade which was endothelial special mark.4.The pegylation level of fibrinogen increased gradually when molar ratios and reacting temperature raised.However,the clotting time,percentage of clottable protein and stability of fibrin gel decreased with the raised pegylation level.For pegylated with mPEG-sc,the resistance to collagenase degradation increased at molar ration 10,and reduced at molar ration ranged from 25 to 100.For sc-PEG-sc,the resistance to protease increased at molar ration ranged from 10 to 50 and reduced at molar ratio 100.For the study of plasmin degradation rate,no increased resistance was observed at all molar ratios for both mPEG-sc and sc-PEG-sc.The pore size of fibrin gel which was prepared by fibrinogen solution at 20mg/ml was smaller than 10mg/ml.For those pegylated fibrin gel,the pore size decreased gradually with the increasing level of pegylation with both mPEG-sc and sc-PEG-sc.Moreover,the density of fibrin decreased significantly in fibrin gel with high pegylation level.Under pegylated molar ratio 50,the pore size of sc-PEG-sc pegylated fibrin gel was smaller than that of mPEG-sc.5.There was no difference for cell proliferation condition between pegylated and no pegylated fibrin gel.For BVEC differentiated study,hADSCs formed network structure successfully within pegylated fibrin gd and expressed specific gene CD31,CD34,VE-cadherin and KDR in sc-PEG-sc group as well as CD31,CD34 and KDR in mPEG-sc respectively. Conclusion1.The hADSCs which was obtained through human liposuction processing could be applied as ideal seeding cells for differenciated blood vessel endothelial cells associated tissue engineering.2.The culturing pattern of hADSCs within fibrin gel could be applied as a method for construction of capillary-like network structure in vitro.3.Pegylation of fibrinogen modified the biological characteristics of fibrin gel and optimized partial function as being scalfford for tissue engineered capillary-like patch.4.The successful formation of capillary-like network structure in pegylated fibrin gel provided us a new method for the study of artificial microvessel construction.