Preliminary Study Of Tissue Engineered Blood Vessels Contructed By Using Mensenchymal Stem Cells And Vessels Acellar Matrix

Part one Cultivation of the Mesenchumal stem cellsAimTo explore the method about the isolation and culture of human bone marrow mensenchymal stem cells (BM-MSCs) which derived from human bone marrow in vitro, and to evaluate their biological properties, so as to provide the preparation for the establishment of tissue engineered blood vessels (TEBV).MethodsBone marrow samples were collected from healthy donors. 5ml bone marrow aspired from the iliac crest and equivalent in DMEM 5ml aliquot was layered over a ficoll solution and centifuged at 1200rmp for 30 minutes at 4°C, bone marrow mononuclear cells (BM-MNC) at the interface were recovered, and washed twice in DMEM. Detect vigor of the cells with typanblau. All the cells seeded into flasks containing DMEM, 10% fetal calf serm (FCS). BM-MSCs culture at 37°C in 5% CO₂. Non-adherent cells were removed after 24 hours but not changing medium.The medium was changed half subsequent every 3 days. Twelve later the culture reached 90% confluency. BM-MSCs were reconered using 0.25% Trypsin- 0.02% EDTA and seeded at flask as passagel (P1) cells. The morphology of cells and growth of BM-MSCs were studied with inverted phase contrast microscope, the biological feature of BM-MSCs and cryogentic storaged cells were studied. Draw the growth curve and calculate the adherence rate of the No.1-3 generation cultured cells. Cells were trypsinized, washed with PBS, fixed with paraformaldehyde and incubated with antibodies against CD34 to No.3 generation cells. Analysis was performed with a flow cytmoeter.ResultsAfter dyeinged by typanblau, calculation indicate that the survival rate of cells is 95%. Nearly all BM-MSCs adhered after 24h plating. Non-adherent cells removed with changes in meium. The adherent elonged with spindle-shaped, triangular or polygonal morphology. They grew in clusters later and reached confluent after 12d. Passaged cells attached several hours after plating. They gradually stretched to spindle-shaped or polygonal. When reached confluence after 6d, they became uniformly shaped and densely arranged. The resuscitative cell have powerful proliferative ability and the same growth feature as the same kind of passge cells, which has 95% survive rate. After having draw growth curve of No.1.5.10 generation cells, we find human-MSCs growth feature, the cell number doesn't change after No.1-2 day when cells are in incubation period. Then the cells reach to logarithm growth period at No.3 day, reach to peak period at No.5 day and then to platform period. Adherence rate show no difference betweent No.1-3 generation cultured cells CD34 antigen is negative expressed at the human-MSCs, but hemopoietic stem cells are positive. That is to say cultured cells are nonhemopoietic stem cell.ConclusionsThe method is established for culuring human MSCs and studying their biological feature in vitro, which provide a new source for the tissue engineering.Part two Study on cell extraction from human aortasAimTo investigate a method to remove cellular components from human aortas, resulting scaffolds of acellular tissue matrix (ACTM) for tissue engneering of vessel.MethodsThe active aorta conduit preserved in liquid nitrogen were selected as experimental material and decellularized by sodium dodecyl sulplate (SDS) containing low osmotic Tris buffer, DNase and RNase. First put the sample into low osmotic Tris buffer (0.1 %EDTA, aprotinin10KIU/ml), after 14 hours remove it to low osmotic Tis buffer (0.1%EDTA, aprotinin10KIU/ml, 0.1% SDS), cultivated at 37°C and culture it for 24 houres. Then put it into iso-osmia Tris buffer (DNase200μg/ml, RNase200μg/ml PH7.6). Then the acellular tissue matrix (ACTM) was obtained. The morphological integrity of acellular scaffold was investigated by means of inverted phased contrast microscope and scanning electron microscope. The shrinkage temperature, thickness and water content were compared between active aortic wall and acellular wall.The differential significance is judged by P< 0.05.ResultsLight and electron microscopy confirmed that all the cellular constituents were removed without ultrastructural damage to fibrous component. Water content in decellular aortic wall was increased significant(P>0.05), thickness and shrinkage temperature of the decellularized human homograft aortic had no significant differences(P< 0.05).ConclusionHuman aortic can be almost completely acelluarized by SDS containing low osmotic Tris buffer procedure without imparing the mechanical propertiy of the tissue. This kind of acellular tissue could be used to develop tissue engineering for homograft aortic conduit substitute.Part three Preliminary study of mesenchymal stem plant on intrsvascular stentAim To study the possibility of seeding human marrow stem cells on decellularized human aortas.MethodBone marrow samples were collected from healthy donors. Marrow stem cells were isolated using Ficoll gradient, cultured and passaged. Using SDS, DNase and RNase to remove cellular components from human aortas.put the brackets into fetal bovine serum 12 houres, that can increase the adherence of the brackets. Cells were seeded on the decellularized aortas one times. After 7 days the samples were observed by light and electron microscopy.ResultsThe light and electron microscopy showed that large amount of cells growing on the acellular tissue matrix.ConclusionIt is possible to seed MSC on decelluarized human aortas to create a viable tissue-engineering aortas.