The Optimizing Preparation And Application In Animal Repair Of Recombinant Spider Silk Protein Composite Small Diameter Vascular Scaffold

Vascular diseases have been leading a major threat of human health currently. However, available replacements like autologous vessels harvested from patients for bypass surgery including saphenous vein (SV) and internal mammary artery (IMA) meet their scarcity, while the clinically available artificial conduits such as polyethylene terephthalate (Dacron) and expanded polytetrafluoroethylene (ePTFE) working on the small diameter vascular (inner diameter<6mm) face a huge threat of thrombus formation and intima hyperplasia. The electrospun scaffolds biomimetic extracellular matrix ECM environment with three-dimensional (3D) porous nanofibrous have initiated an potential source of small diameter vascular tissue engineering.Previously, the spider silk protein, PCL and Gt complementary in the material combination of5:85:10has been successfully prepared by electrospinning and finally identified its certain feasibility as scaffold material in small diameter vascular tissue engineering, achieving complementary advantages for better performance in preventing platelet adhesion and promoting endothelial cell proliferation. However, in practical work, bead structure was prone to exist if only using formic acid as solvent, resulting in a decrease on fiber surface area. The preparation of uniform and ultrafine nanofibrous without bead is a structure basis of scaffolds simulating the native ECM with an optimal microenvironment better used in vascular tissue engineering.On the basis of preliminary studies, following an improvement on solvent system, orthogonal design is employed to prepare controlled three-dimensional structure of spider silk protein composite electrospun nanofibers. To go with further study in vivo, we investigate the influence between scaffold structure and biomechanical properties. A particular perspective is of thinking about cell-scaffold interaction that toxic effects of scaffold on endothelial cells can be studied, and in turn the function of endothelial cells on the scaffolds include NO secretion and thrombosis, cytoskeleton organization and cell growth, gelatinase expression and matrix degradation. Finally, the optimizing pNSR16/PCL/Gt scaffold, by way of SD rat abdominal aorta defect model, in vivo behaviour were conducted whether it can keep the physical and chemical characteristics as well as the biological performance, maintain stability and patency, provide the basis for clinical research as an small-diameter vessels substitute.The results can be summarized as follows:1. The orthogonal design was employed to optimize the preparation of pNSR16/PCL/Gt (5:85:10) biomimetic ECM composite nanofibers. The addition of chloroform in solvent system could not only improve the macroscopic morphology of the electrospun membrane but also the microstructure of the electrospun fiber. The fiber diameter and uniform coefficient increased with the higher chloroform into the solvent system. To investigate the influence of five main parameters, including solvent system, solution concentration, voltage, extruding speed and spinning distance, on the diameters and morphologies of electrospun spider silk protein composite nanofibers, optimizing screening tests were performed at three levels. Among the five factors, the spinning distance and solvent system were the most significant factor, and then the spinning solution concentration, voltage, while the effect of extrusion velocity was not significant. By means of range analysis and variance analysis, the optimum-conditions were as follows:solvent system of formic acid/chloroform6/4, spinning solution concentration of10%, voltage of14kV, extruding rate of0.8mL/h and spinning distance of12cm. Being simple and designable, orthogonal test highlights the electrospinning design to prepare controlled three-dimensional structure of spider silk protein composite nanofibers in the vascular tissue engineering, in searching for the optimal balance point between fiber diameter and porosity, formation rate, mechanical strength, degradation rate and organization.2. The biomechanical properties of pNSR16/PCL/Gt after optimal setting were investigated to ensure the transplantation in vivo lately. The thickness of0.1mm-0.3mm conduits were generated by electrospinning with3mm inner diameter and composite nanofibers within50-500nm. According to ISO/DIS7198, the osmosis, bursting strength, suture retention strength and tensile strength were tested, and the influence on mechanical properties with porosity, diameter and wall thickness were explored. The scaffold had excellent mechanical proerties than before, with an appropriate permeability of6.085±0.29mL/(min·cm-2), breaking strength (239±0.1KPa) and elasticity up to125±0.086%strain, being more equal than commercial stents ePTFE and optimized to mimic the nature artery. The burst pressure reached276±7.1kPa as well as an accepted suture retention strength (7.8±1.0N), fully met the clinical needs as well. A good linear relationship with wall thickness was observed on both burst pressure and suture retention strength.3. To evaluate whether the genetic toxicity of scaffold exist on endothelial cells or not, the DNA damage of SDRAECs on the scaffolds were performed by single cell gel electrophoresis assay. Results showed that the morphology and adherent of endothelial cells were normal in the scaffold extracts. The patterns of DNA of these cells were similar to that of in the DMEM culture medium, presenting neat edge, red circular fluorophores, no obvious DNA damage tail, which illustrated a complete volatilization of chloroform via electrospinning, suggesting that pNSR16/PCL/Gt scaffold after optimization with no significant carcinogenicity and genotoxicity laid a safety evaluation basis for further test in vivo.4. On perspective of cell-scaffold interaction three functional characteristics of endothelial cells on the scaffolds were investigated, including NO secretion, cytoskeleton organization and gelatinase expression.1) Nitrate reductase method was used for NO secretion tests. The sequence of NO concentration was:pNSR16/PCL/Gt>pNSR16/PCL>PCL/Gt>PCL group, in which NO concentration on pNSR16/PCL/Gt graft increased with the difference statistically significant (p<0.05) at3,5,7day, when compared to the control group, indicated that pNSR16/PCL/Gt small diameter vascular scaffold could promote SDRAECs release of vasoactive substances NO, being expected to become a "functional" engineered vascular.2) The expression of F-actin protein of SDRAECs on scaffolds with FITC-phalloidin fluorescence was semi-quantitative detected followed by pNSR16/PCL/Gt> pNSR16/PCL> PCL/Gt> PCL group. Apart from PCL, three other groups were able to develop skeleton actin arranged in order, especially the SDRAECs on pNSR16/PCL/Gt graft revealed a highly extended cell morphology, with F-actin constructed active running through the whole cell. Not only an ideal status of cytoskeletal proteins development was shown, there were also so many small tips "contact" between cells. It was visible that the optimized structure of pNSR16/PCL/Gt biomimetic native ECM may provide an optimal microenvironment for the seeded endothelial cells actin function and cell spreading.3) Gelatin zymography studies showed that the MMP-2synthesized and secreted in the SDRAECs on all scaffolds. What’s more, the expression and activity of MMP-2on pNSR16/PCL/Gt had no significant difference (P>0.05) compared with control group, indicating that it could guarantee the normal expression of MMP-2of SDRAECs growth on it at the physiological state.5. To explore the feasibility of pNSR16/PCL/Gt small diameter vascular scaffold in clinical application, In the construction of SD rat abdominal aorta defect model via "two-cuff method", scaffolds showed well operability, no obvious blood leakage. Postoperative rats recovered with normal activity. Blood physiological and biochemical detection showed no effect on animal liver and kidney with implantation. No obvious thrombosis and intimal hyperplasia, the scaffold could still raise host cells in blood environment, with rich synthesis of extracellular matrix, maintained for at least6, months, forming a stable intima and adventitia similar to natural blood vessels. The interim repair effect demonstrated considerable clinical application.To sum up, bionic ECM design from considering the microstructure, mechanical strength, endothelial cell properties have achieved complementary advantages for better performance of scaffold. The practical applications in animal experiment validated that, pNSR16/PCL/Gt small diameter vascular scaffold could be significantly improved when adapted to the bloodstream, allowed to maintain stable structure and patency accompanied by effective natural endothelialization at least6months, which laid a foundation for functional reconstruction of scaffold in later long-term evaluation.