Construction Of Biomimetic Vascular Network Applied To A Tissue-Engineered Liver Scaffold With Computer Assisted

Objective:To study the isolation and cultivation of mesenchymal stem cell (MSCs) from the mice bone marrow in vitro. To study the efficiency and functional changes of MSCs differentiated into endothelial cells.Methods:Separate the mice femora and tibia, wash the bone marrow with DMEM-LG medium (lOu heparin/ml), and collect the bone marrow into a10ml centrifuge tube. MSCs from bone marrow were extracted、cultured and purification by Percoll density gradient centrifugation and adherence-sieve method. Mesenchymal stem cells growth curve detected with MTT method. The identification of MSCs were detected with flow cytometry by CD2、CD90、CD105、CD34、CD45monoclonal antibody. MSCs induced to differentiated adipose cells by insulin and dexamethasone. After induction2weeks, identified the adipose cells by red oil dyeing assay. MSCs differentiated into endothelial cells(ECs) by adding vascular endothelial growth factor(VEGF), Identification the induced ECs with the immunofluoresence and immunohistochemistry assays after culture for10days.Results:(1) MSCs were cultured by Percoll density gradient centrifugation and adherence-sieve method. Adherent cells were round after cultured12hours and spindle-like cells were formed and radiated towards periphery in72hours. After cell cultured for10days, a high density of cell layer were formed, and the extremes of cells start regularly arranged in a beam shape.(2) The flow cytometry positive rate of CD29、CD90、CD105were all higher than90%. CD34、CD45positive rate were both less than8%.(3) After2weeks induction, adipose cells were formed, the lipid droplets were detected. The lipid droplets were dyed with red oil.(4) After induction1weeks with VEGF, there are scattered pebbles shapes cells in the culture dish. After induction2weeks, forming a high density of cell layer, most of cells in a round or oval shape, cells presented a typical "stepping stones" shape. The positive rate of CD34and KDR of differentiated cells were89.24±6.37%and85.19±4.52%。 FITC-UEA-1and DiI-acLDL double dye rate was89.83±4.49%. The induced ECs could form a tube-like structure when cultured on the matrigel after2weeks.Conclusion:Bone marrow-derived MCSs could isolated and cultivated with the methods of density gradient centrifugation combined adherence-sieve, could differentiated into adipose cells and ECs with inducible factors in vitro. Objective:To synthesis a biodegradable copolymer poly(EC-CL) at room temperature. To prepare poly(EC-CL) nanofibrous membranes by electrospinnning process.Methods:Ring-opening copolymerization of ethylene carbonate (EC) with s-caprolactone (CL) was carried out using neodymium tris(2,6-di-tert-butyl-4-methylphenolate) as a single component catalyst and toluene as solvent. NMR was performed to detect the chemical structure, FTER. Fourier total reflection infrared spectral analysis of the change of the polymer group characteristics. HFIP was used as the solvent, the nanofibrous scaffolds were electrospinned with different concentration of Poly(EC-CL) with different ratio of EC:CL,(1:9,1:6,1:4mol:mol). The morphology and diameter of the fibers were observed by scanning electron microscope(SEM). Mechanical properties test detection the rigidity of the membrane tensile strength, young’s modulus and elongation at break.Results:The FTIP spectrum and NMR assays confirmed that poly(EC-CL) was as a random copolymer. When Poly(EC-CL) concentration was5%, the ratio of EC/CL from1:9rise to1:4, fiber diameter from212±52nm rise to522±68nm. When Poly(EC-CL) concentration was10%, the ratio of EC/CL from1:9rise to1:4, fiber diameter from539±79nm rise to956±142nm. When Poly(EC-CL) concentration was15%, the fiber diameter of EC/CL1:9was867±93nm. EC/CL(1:6,1:4) could not form an effective electrospun scaffolds. Mechanical properties test found that as EC content increased, polymer tensile strength, yield strength and young’s modulus gradually declined, and elongation at break with the EC content increases increased.Conclusion:The diameter of scaffolds fibers increased with the increasing concentration of Poly(EC-CL) solution and the ratio of EC/EC of Poly(EC-CL). Poly (EC-CL) is a kind of good mechanical properties and biodegradable controlled polymer materials. Objective: To prepare a novel degradable nanofibrous scaffolds with Poly(EC-CL) and vascular endothelial growth factor as raw materials by electrospinning method, and to tested the biological compatibility and mechanical properties of the blended scaffolds.Methods:VEGF was dissolved in500ul M199(Gibco, USA) medium, and Poly(EC-CL) dissolved in1,1,1,2,2,2,-hexafluoro-2-propanol(HFIP, Yumei Co.Ltd, China) at a concentration of10%(w/v) for electrospinning. We blended both solutions at a ratio of lug/g100ng/g, lOng/g, Ong/g,(VEGF/poly(EC-CL), wt%/wt%) respectively, the electrospinning process was performed at15kV voltage,15cm distance between the needle tip and a rotating mandrel. The flow rate was2mL/h. Scanning electron microscopy (SEM) was used to measure the fiber diameter distribution. The composition of the composite fibrous membranes were analyzed by immunofluorescence. cell proliferation test, Live/Dead cells kit assay, cell adhesion test, LDH release test, indirect contact with hemolytic test, subcutaneous implant test nanofibrous scaffolds’s compatibility and blood compatibility. Poly (EC-CL) electric spinning membrane differentiated MSCs test d the ability of VEGF contained in the nano scaffolds in vitro.Results:The average diameters of these scaffolds were estimated to be440±55nm. We increased the percentage of VEGF/poly(EC-CL) from10ng/g to lug/g(w%:w%). The diameters of the scaffolds were not changed with the increase of VEGF content, and there were no significant difference between the pure poly(EC-CL) and blended VEGF/poly(EC-CL)(p>0.05). Cell proliferation test, Live/Dead cells test kit, cell adhesion tests confirmed that Poly (EC-CL)/VEGF films possess excellent cell compatibility. VEGF/Poly (EC-CL)(100ng/g μ;1g/g) group was higher than the cells on Poly (EC-CL) and VEGF/Poly (EC-CL)(10ng/g)(P<0.05). The hemolysis rate of hemolysis test group all<6%, subcutaneous implant test showed no inflammation around. When VEGF content>100ng/g, can effective induction MSC into endothelial cell.Conclusion:Poly (EC-CL)/VEGF copolymer has good biological compatibility and blood compatibility, can be used as biomedical implant material. When VEGF of Poly(EC-CL) content>100ng/g, can effective induction MSC to endothelial cell differentiation. Methods:Using64row CT to collect normal liver blood vessels image, then transform the CT images into4cm x4cm vascular fault plane coordinate chart, according to liver vessels different section plane coordinates, laser prepare biomimetic vascular network on the electrospun Poly(EC-CL) scaffolds. The SEM tested the bionic vascular network. each of the liver tissue engineering scaffolds will punch in the order attached together using the matrigel adhesive. Using GFP-adenovirus marked ECs, then implanted ECs into bionic blood vessels in the bionic network. Cultured the bionic liver vessel network at37oC5%CO2incubator,after3weeks culture, tested the formation of blood vessels with fluorescence microscopeResults:after laser drilling, scanning electron microscopy (sem) shows the bionic vascular edge smooth. After3weeks culture, immune fluorescent detected visible wall endothelial layer formation.Conclusion:helped to promote metabolism functional tissue engineering viscera research progress.