| Vascular diseases have become a major threat of human health, usually use blood vessel transplantation surgery for treatment. Autologous blood vessels and artificial blood vessels are the main graft. However, autologous blood vessels own limited sources despite of its pleased biocompatibility and non-immunity. The artificial blood vessels perform well in the large diameter vascular injury repair but fail in the small diameter vascular with intimal hyperplasia or thrombosis. As a result, it also can’t meet the clinical requirements. The small diameter blood vascular grafts have become a hot topic of research in the field of vascular tissue engineering.The phase separation structure endow polyurethane (PU) with good mechanical properties and biocompatibility for biomedical materials.Gelatin has a similar structure with collagen of the extracellular matrix. As the product of collagen by a mild fracture, gelatin shows an incomparable water-soluble ability with rich hydrophilic amino acids that its application is also extensive. Spider silk protein with good biocompatibility and biodegradability have become a potential material of tissue engineering. On the basis of the laboratory preliminary studies, we continue to prepare a double-layer vascular by electrospinning. The inner layer is composed of blend fibers of gelatin and spider silk protein while the polyurethane outer layer on the outside. The physical and chemical characteristics as well as the biological performance of scaffold are evaluated in this test. Results are as follows:1. The electrospun device was successfully modified with the loading position of the voltage and rotation of receiving rod. The reduced voltage significantely improved the operational safety. Formic acid as solvents of gelatin and spider silk protein, dimethylformamide and acetone as a mixed solvent of the polyurethane, were used to prepare double tubular scaffold formed with Gt/pNSR16inner layer and PU outer layer by electrospinning. The best electrospun parameters of polyurethane including:the solvent ratio VDMF:VACE=1:1, the concentration of spinning solution is5%(w/v), the voltage of18kV, the receiver distance of20cm and the extrusion speed of0.5mL/h. 2. The physical and chemical characteristics of the scaffolds. Polyurethane is a hydrophobic material, The water uptake test results confirmed that gelatin can significantly improve the hydrophilicity of scaffold. The porosity of88.7±1.5%meet the requirements of cell infiltration. The mechanical test results showed that the composite scaffold with lower permeability (6.8±0.2mL/(min·cm-2)), the bursting strength of276±7.1KPa and the suture strength of4.9±0.8N, wich all met the requirements of operation. The fracture stress and fracture strain of the scaffolds are superior than that of natural blood vessels and commercialized ePTFE stent. The degradation rate in PBS seemed slower than in multi-enzyme system.3. The evaluation of the blood coMPatibility of scaffolds. Hemolysis test, recalcification clotting time test, dynamic clotting time test and platelet adhesion test showed that the polyurethane scaffold has a good blood coMPatibility. The hemolysis rate of1.22%is in full compliance with the ISO requirement of less than5%. The recalcification clotting time of379s, a longer dynamic coagulation time experienced as well as the less platelet adhesion on the stent surface were observed. In addition, the adhered platelet didn’t change its appearance, showing disc-shaped with no pseudopodia.4. Tissue adherence method was used to isolate vascular endothelial cells. The cell morphology was viewed with the Inverted phase contrast microscope. The cell morphology was polygonal or fusiform. The cells at passage2were identified by immunoreactivity with factor vWF antigen. The cytoplasm emerged a green fluorescence and the nuclei shown a blue fluorescence by DAPI. The results confirmed that the isolated cells were endothelial cells.5. The cell compatibility of the scaffolds. Cytotoxicity test confirmed that the performance of scaffolds is stability, non-toxic to cells at room temperature with toxicity grade0to1. The PU and composite scaffolds can support ECs adhesion, proliferation and morphology and function maintenance. In conclusion, the composite scaffold with RGD spider silk protein and gelatin is more conducive to cell proliferation... |
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