The Application Of Polyether-ester Block Copolymer In Vascular Tissue Engineering

Objective To investigate the effect of surface hydrophobicity of different polyether-ester block copolymers [poly ( ethylene glycol-terephthalate) / poly (butylene terephthalate), PEGT/PBT] on the growth of cells derived from vascular tissue and achieve the preliminary selection of polyether-ester copolymer expected to be used as scaffolds in vascular tissue engineering. To investigate further the adhesion behavior of endothelial cells and explore the adhesive structures related to integrin. To prepare the porous scaffolds of PEGT/PBT and get the first-hand application evaluation. And to study the effect of different methods of disinfection and sterilization on the cytocompatibility of the polyether-ester copolymer. Moreover, to explore the potential property changes of PEGT/PBT after the high-pressure steam sterilization.Methods The canine aorta smooth muscle cells, canine external jugular vein endothelial cells, and human umbilical vein endothelial cells were harvested and cultured. These cells were identified by immunocytochemistry. And the human dermal fibroblasts were provided and used without further identification. The polyether-ester copolymer with different compositions were synthesized and prepared as 1000-T20, 1000PEGT70/PBT30 and 600PEGT70/PBT30. The water up-take and static contact angle of these copolymer were determined. And the growth offibroblasts, SMCs and ECs were compared among them. Then the 600PEGT70/PBT30 were divided into three groups: fibronectin-coating group, type I collagen-coating group and untreated group. The adhesion and proliferation of HUVECs were investigated as a function of culture time and adhesion structures labeled by vinculin and paxillin were immuno-staining. The porous scaffolds were prepared by the solvent casting / particulate leaching, and vortexing modeling / particulate leaching technique. After the SMCs were cultured on the porous scaffolds by static seeding, dynamic rotation seeding or pressure-filtration seeding for a scheduled time, the samples were harvested and examined by scanning electron microscope and histological section. The cytocompatibility of polyether-ester after 7 radiation sterilization, ethylene epoxide sterilization, high-pressure steam sterilization, ultraviolet radiation sterilization or 70% alcohol disinfection, were evaluated by cytotoxicity test and cell culture. The 600PEGT70/PBT30 after high-pressure steam sterilization was characterized by ]H NMR, ATR-FTIR, DSC, and SEM, and the intrinsic viscosity, static and dynamic contact angle of the copolymer were measured. The cytocompatibility after high-pressure steam sterilization and after treatment combined with ultraviolet radiation sterilization or 70% alcohol disinfection, were compared.Results The water-uptake of three copolymers increased as the sequence 600PEGT70/PBT30, 1000-T20, 1000PEGT70/PBT30, and contact angle decreased as the same sequence, indicating 1000PEGT70/PBT30 more hydrophilic. However, canine SMCs and ECs grown poorly on 1000PEGT70/PBT30 membrane after low density seeding, with a significant difference (PO.05) compared to 1000-T20 and 600PEGT70/PBT30. Thefibroblasts and HUVECs on 1000PEGT70/PBT30 proliferated much less than on 1000-T20 and 600PEGT70/PBT30, and had a significant difference(P<0.05) compared to 600PEGT70/PBT30. The adhesion percent of HUVECs reached 45.0%±6.5% at 20th min after seeding, and the adhesion percent increased to 85.3±7.9% at 6th h . The immuno-staining of vinculin at 20th min showed the linear dot-like distribution at the edge of lamellipodium, indicating the formation of focal complexes. At 2nd h, the staining of vinculin became numerous elongated, oval bar emerged in the cell periphery, indicating the formation of focal adhesion. The treatment with fibronectin or collagen increased the proliferation of HUVECs on 600PEGT70/PBT30 significantly. On Fn-coating membrane, HUVECs formed focal adhesion at 20th min after seeding, and the adhesion percent increased much more at 20th min and at 6th h respectively. The SMCs could growth and proliferate to form multilayers on the porous membrane made from 600 PEGT70/PBT30. But on tubular porous scaffolds, although the different seeding technique were used, the results of cell seeding and proliferation were poor. The examination showed the internal and external surfaces of tubular porous scaffolds much less open than the interior, the surfaces of pores uneven, and interconnect channels between pores much narrow. The cytotoxicity test showed that after various disinfection and sterilization treatments, the cytotoxicity was grade 0-1. But the seeded cells, including L929, fibroblasts, SMCs and ECs, couldn't adhere to and grow on the copolymers after sterilization by -y^ray radiation sterilization, ethylene epoxide sterilization and high-pressure steam sterilization. Increasing the cell density at seeding or treating membrane with gelatin or collagen, didn't change the results. However, the cell growth on copolymers wasn't influenced by the ultraviolet sterilization and 70%alcohol disinfection. Compared to the 600PEGT70/PBT30 before sterilization, the H NMR characterization after high-pressure steam sterilization didn't show the change of PEGT/PBT composition and EG repeating unit, the ATR-FTIR examination didn't show the emergence of new absorption peak, and the intrinsic viscosity measurement didn't show the difference. On the other hand, DSC measurement showed an increase of 3% in crystallinity. And the measured static contact angle decreased to 34.5°±0.9°,having a significant difference to 38.9°±0.7°of untreated membrane (P<0.05). The advancing angle 8a and receding angle 8r of dynamic contact angle showed the same decrease. Surprising, the L929 cells could grow on high-pressure steam sterilized 600PEGT70/PBT30 after sterilized by ultraviolet or disfected by 70% alcohol again.Conclusion Contrasting to more hydrophilic 1000PEGT70/PBT30, moderately hydrophilic 1000-T20 and 600PEGT70/PBT30 supported vascular cells growth well. HUVECs could form numerous focal complexes and focal adhesion at the early stage after seeding on 600PEGT70/PBT30. In porous membrane made from 600PEGT70/PBT30, SMCs could adhere and grow to form multilayers. These were the first evidences that indicated moderately hydrophilic 600PEGT70/PBT30 had appropriate vascular cytocompatibility and showed the possible application of PEGT/PBT in vascular tissue engineering. The deficiencies of tubular porous scaffold made from 600 PEGT70/PBT30 were related to the nature of particulate leaching technique adopted. The other techniques, such as thermally induced phase separation/freezing and electronspinning deserved to applying. The cells couldn't grow on copolymers after y-ray radiation, ethylene epoxide sterilization and high-pressure steam sterilization, which wasn't due to thecytotoxicity effect. The 600PEGT70/PBT30 didn't degrade after high -pressure steam sterilization, and it was most likely the motility of PEGT chains made the surface more hydrophilic and unsuitable for cell growth.