Preparation And Characteristics Of Autologous Acellular Matrix Vascular Grafts

Part ⅠMechanism of the formation of autologous biological conduitObjective:To explore the mechanism of autologous biological conduit formation.Methods:1.Teflon tubes were implanted into the subcutaneous tissue of the abdominal wall of male SD rats,and they were removed on the 3rd,7th,and 28 th day after operation.The implants were removed and the outer layer tubular connective tissues were detached from the Teflon tube mandrel by using microsurgical techniques.Then the tissues were embedded and frozen sections followed by immunofluorescence staining and fluorescence microscope observation.Six rats were included at each time point.2.Teflon mandrels were implanted subcutaneously in Vav1-cre/R26R-EYFP mice.After 7 days,the Teflon mandrels were harvested and the outer layer tubular connective tissues were detached from the Teflon tube mandrel by using microsurgical techniques.Then the tissues were embedded and frozen sections.The histological analysis of the fibrotic conduits around the mandrels were performed by immunostaining of CD45,CD68,CCR7,CD206,CD31,FSP1,and α-SMA to trace the fate of inflammatory cells.Results:1.Various cells were found to contribute to the formation of autologous biological conduits in vivo.Fibroblasts(FSP1+)and vascular smooth muscle cells(α-SMA+)were found to be involved in the formation of autologous biological conduits wall at 4 weeks after operation.Immune response was found to play an important role in autologous biological conduit formation: macrophages,T cells and B cells were recruited as early as day 3.The phenotype of macrophages was determined by immunostaining the CD68(surface markers of pan-macrophages),CCR7(markers of M1 macrophages)or CD163(markers of M2 macrophages).CCR7+pro-inflmmatory macrophages reached highest level at day 3,mostly found at the interface of the autologous biological conduits and the mandrels,and then gradually reduced after at day 7.The number of CD163+ anti-inflammatory macrophages reached the peak around at day 7,illustrating the transformation of macrophage from M1 to M2.CD3+ T cells,CD4+ T helper cells or Fox P3+ regulatory T cells also showed a peak level at day 7.Few B cells were found in capsule tissues.2.Inflammatory cells could trans-differentiate into fibroblast-like cells: most EYFP+ cells were positive for CD45 and CD68 but not CD31,suggesting that macrophages were a major population of EYFP+ cells.Both CCR7+ and CD206+(anti-inflammatory)macrophages were found in the fibrotic capsules,and most EYFP+ cells were CD206+ and weakly positive for CCR7,suggesting a potential conversion of macrophage phenotype.After 7 days,many EYFP+ cells expressed FSP1,and a fraction of cells next to the surface of the mandrel expressed α-SMA,implying that inflammatory cells could trans-differentiate into fibroblast-like cells.Conclusions:1.Fibroblasts and macrophages and T cells participated in and played an important role in the formation of autologous biological conduits wall.2.By tracing the lineage of cells derived from inflammatory cells,it was found that macrophage phenotypic transformation and the transdifferentiation of inflammatory cells to fibroblast-like cells participated in the formation of autologous biological conduits wall.Part Ⅱ Mechanism of stenosis in the rat carotid artery implantation model with autologous biological conduitsObjective:To investigate the main cells source of autologous biological conduit vascular graft stenosis in the rat carotid artery implantation model.Methods:The Teflon tubes were implanted into GFP+ SD rat abdomen subcutaneously,and the external wall tissue of Teflon tubes were stripped by the microsurgical technique to make autologous biological conduits as vascular grafts at 4 weeks after operation,which were implanted into the left common carotid arteries of SD rats.The vascular grafts were explanted at 4 weeks after surgery(n=7).The samples were stained with HE and DAPI respectively,and the staining results were observed.Then the number of neointima GFP+ cells were calculated.Results:Severe stenosis wereobserved from the samples of all autologous biological conduit vascular grafts.The results of HE staining showed neointima formation in vascular grafts,and 85.9±6.6% of neointimal cells were GFP+ cells.Conclusions:The cell source of the neointimais mainly from autologous biological conduit wall,which is due to the stenosis.Part Ⅲ Preparation and characteristics of autologous acellular matrix vascular graftsObjective:To explore the preparation methods and characteristics of autologous acellular matrix vascular graft.Methods:The Teflon tubes were implanted subcutaneously into the abdomen of male SD rats and explanted at 2,3,4,and 5 weeks after operation to make autologous biological conduits,which were washed thoroughly in 1M Na Cl,and then treated with the zwitterionic 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate-(CHAPS)detergent solution(8m M CHAPS,1M Na Cl,25 m M EDTA)on a stir plate at 37℃ for 6-8 hours.The acellular samples were embedded and sliced,then the histological analysis of the samples were performed by Masson’s trichrome,Verhoeff’s stainings and Western blotting.The inner and outer surface of the conduit cavity was observed by electron microscope,and the mechanical properties of the samples were measured before and after decellularization at all time points.Results:1.Histological analysis showed that the main collagen fibers were preserved after decellularization of the autologous biological conduit vascular grafts,and high porosity on the inner and outer surfaces were observed by electron microscope.No elastin were observed in the autologous biological conduit vascular grafts.At the same time,this decellularization process completely removed the cellular structure of the graft wall.2.By comparing the pre-and post-decellularization mechanical properties of grafts at different time points,we observed a significant increase in the Young’s modulus and ultimate tensile strength(UTS)of grafts at 2,3,4 weeks.And no significant difference of the Young’s modulus and UTS were observed at 4 and 5weeks.Conclusions:The zwitterionic detergent solution can effectively remove the cells of autologous biological conduits without decreasing the mechanical properties.