Study Of Engineer Small Diameter Tissue Engineering Vascular Grafts Immobilized With SDF-1α For Regeneration And Transplantation Of Blood Vessels

Bypass procedure is a common treatment for vascular diseases. To overcome the unavailability of autologous grafts, tissue-engineered blood vessels have been created in vitro by using cells with or without scaffolds. However, these approaches are time consuming and are only appropriate for customized and non-urgent therapies. The use of synthetic grafts is limited to large diameter grafts because of the acute thrombotic clogging and the lack of endothelialization to maintain long-term patency. Small-diameter synthetic vascular grafts have high failure rate due to thrombogenic responses. Tissue-engineered blood vessels have been created in vitro by using cells with or without scaffolds, but the long construction time limits the applications. Advanced biomaterials and scaffolds for tissue engineering place high demands on materials and exceed the passive biocompatibility requirements previously considered acceptable for biomedical implants. Here, we present a one-step preparation of fully synthetic, bioactive and degradable extracellular matrix-mimetic small diameter scaffolds by electrospinning, using poly-L-lactide (PLLA) and poly-caprolactone (PCL) as the matrix polymer. Here we present an in situ tissue engineering approach that recruits two types of vascular progenitor cells for the regeneration of blood vessels.PART ONE:Using electrostatic spinning PLLA/PCL small-diameter grafts and its characteristics researchObjectiveBy chosing the right material and using electrostatic spinning technology to creat tissue-engineered vascular grsfts of fully bioactive.MethodsMicrofiber scaffolds were fabricated by using poly(L-lactic acid)(PLLA) and polycaprolactone (PCL). The polymer blends (e.g.,19%PLLA and5%PCL; w/v) and pure PLLA were dissolved in1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP), and electrospun into grafts with minor modifications. The mandrel collector was a rotating stainless steel rod (1.0mm diameter). The structure of the scaffolds was characterized by using a scanning electron microscope. The mechanical testing were performed between the two materials’grafts.Results1. Using19%PLLA/5%PCL solution electricspun get the grafts, the fiber average diameter was982.3nm.2. Average inner diameter of TEVG was1.000±0.013mm, average outer diameter of1.302±0.021mm, average thickness was150.1±10.5μm.3. PLLA/PCL grafts’ average Yang’s modulus was5.19±0.7Mpa,19%PLLA and autologous vascular were12.85±0.11Mpa and9.66±0.13Mpa, has the remarkable difference P<0.05.ConclusionScanning electron microscopy (SEM) images showed that the electrospun grafts had a porous structure of microfibers. The addition of low molecular weight of PCL made the scaffolds more flexible, and increased the conjugation sites (carboxylic groups) on the microfibers. The maximum stress of PLLA/PCL grafts significantly better than PLLA, but worse than autologous vascular.PART TWO:Study of heparin-SDF-la immobilization and release on TEVG before implant and test the mechanical properties of grafts post implant.ObjectiveConjugate heparin and SDF-1α on TEVG by covalent bonding and immobilization, and test the stability and in vitro release of these two materials. Implanted TEVG with inner diameter lmm to SD rats carotid artery by end to end intestinal anastomosis. Study the blood vessels patency and test the mechanical properties of biological active graft.MethodsTthe density of reactive carboxylic groups on the microfibers was increased by briefly treating the scaffolds with0.01N NaOH. Di-NH2-PEG molecules were then covalently attached to the carboxylic groups on the microfibers by using EDC and Sulfo-NHS. Heparin was conjugated to the free amines on the di-NH2-PEG molecules via EDC and sulfo-NHS. Following heparin conjugation, SDF-la in PBS were incubated with the scaffolds for its binding to heparin and its immobilization on the scaffolds. Immunofluorescent staining was performed by using a SDF-la antibody. To test the stability and in vitro release of bioactive SDF-1α and heparin from microfibrous scaffolds, an ELISA kit was used to determine the concentration of SDF-1α, Toluidine Blue was used for heparin. Implant TEVG to SD rat carotid common artery by end to end intestinal anastomosis. Test the patency of very time points using doppler ultrasound. Mechanical test was performed for different time point grafts.ResultsAverage of heparin content on each graft was15.31±2.31μg/mm3test by Toluidine Blue. Average of SDF-1α content on each graft was140.5±4.1ng/mm3. The starting solution concentration and SDF-la content on graft have equal ratio. Necropsy showed that patency after-operation was100%.89%(8of9) of heparin-treated grafts, and89%of (8of9) heparin-SDF-la-treated grafts were patent at1week after implantation better than that of untreated grafts P<0.05. Patency of heparin-SDF-1α-treated grafts was obviously better than the other two after1week P<0.05. Mechanical tests demonstrated that the elastic modulus of the grafts of all three groups increased significantly over time after implantation. At4weeks, heparin-SDF-la-treated grafts had higher elastic modulus than that of untreated group and heparin-treated group, and similar to native artery, P<0.05.Conclusion19%PLLA/5%PCL TEVG has great compatible with heparin, and heparin can efficiently bind with SDF-1α by immobilization, we made TEVG with biological activity, the starting content of SDF-1α can be500ng/ml. Heparin-SDF-1α group of TEVG recent and long-term patency rate were significantly higher than that of heparin group and control group. The elastic intensity (elastic modulus) of heparin-SDF-1α group of4weeks was close to that of autologous blood vessels, after12weeks the other two groups were also close to that of native artery.PART THREE:Study endothelialization on the luminal surface of the graftsObjectiveFocus on biological activity tissue engineering grafts efficiency and mechanism of endothelialization by in vivo and in vitro experiment.Methods1. In vitro experiment:Bovine endothelial cells culture, add different concentration of growing the SDF-la solution to M199, found endothelial cell migration ability.2. Investigated different time points TEVG, immunohistochemical method analysis the inner surface for the rate and the endothelialization and its mechanism.ResultsThe control group, heparin solution with concentration of15μg/ml, and SDF-la solution with concentration of respectively20ng/ml,50ng/ml,150ng/ml was add to transwell, calculated ECs number after migration were3.5±0.58as control,7.9±1.09as haperin,18.3±1.73,24.7±2.05,31.1±2.46as different concentration of SDF-la, which was significant difference (P<0.05).1week SDF-la treated TEVG inside surface attract much more cells (1570±413, n=3, P<0.05), and can be observed a lot CD34+/CD133+/CD31-EPCs recruited on the luminal surface. Ratio of endothelialization for4weeks SDF-1α TEVG was higher than the other two groups, while CD31positive suggest the mature of ECs.ConclusionIn vitro study shows SDF-1α can effectively attract endothelial cells, proved SDF-1α induce vascular endothelial cells to migration2. Heparin-SDF-la treated TEVG can be effective recruited endothelial progenitor cells (EPCs), within4weeks that the endothelialization the whole luminal surface was well.PART FOUR:Research on outer layer of the vascular graftsObjectiveStudy biological activity tissue engineering vascular grafts’ability of recruiting smooth muscle precursor cells and differentiation into mature smooth muscle cell of the outer layer.Methods1. In vitro study:Vascular grafts for in vitro cell isolation were harvested at3days post-surgery and identified cells, and Real-time PCR detected each time point smooth muscle cells markers of alpha-SMA/CNN1to the corresponding gene expression2. In vivo study:Immunofluorescence test all time points of the outer layer tissue with markers of alpha-SMA, MHC, CNN1, CXCR4,CXCR7,Sox10and Sox17, calculated the ratio of positive cells, Verhoeff’ elastin stainning detected elastin and collagen of the outer layer of TEVG at4weeks.Results1. In vitro study:cell culture found SDF-la group have α-SMA positive cells compare with the other two groups. α-SMA+/CNN1-/7MHC-cells were found at1week, at4weeks a-SMA+/CNN1+/MHC+cells were found, Real-time PCR results show that a-SMA/CNN1/MHC mRNA increase gradually, statistical analysis with significant difference (P<0.01)2. In vivo study:at1week SDF-la group ratio of a-SMA positive cells is87.2±5.1%(n=3), much higher than that of heparin group64.2±4.2%(n=3) and the control group46.2±3.7(n=3), have significant difference (P<0.05). At4weeks Heparin-SDF-la treated grafts showed more and denser collagen-I and CNN1deposition surrounding the vascular grafts, Verhoeff’s elastin staining suggest Heparin-SDF-1α-treated grafts have more elastic and collagen than the others.Conclusion1. In vitro studies found a Sox10+Sox17+/CD34-smooth muscle progenitor cells (SMPCs) to characteristics as stem like cells.2. In vivo experiments Heparin-SDF-la treated grafts increased the recruitment of SMPCs, and stimulate SMPCs differentiate to smooth muscle cell as well as available for proliferation and related protein products.