| Cardiovascular disease has become the major threaten to human health. The death rate reached 40%. In order to effectively prevent cardiovascular disease, more and more surgical transplantations were used to replace the blood vessels of the diseased sites. At present, large and medium caliber vascular grafts have achieved good results. The long-term patency is acceptable. However, the transplantation based on small diameter artificial blood vessel is still unsatisfied. There are many factors that cause the graft failure of small diameter artificial blood vessel. The comprehensive performance mismatch between the biomaterials and host vessel is the major cause. How to prepare small diameter artificial vascular materials with excellent properties is difficult in the research of small diameter artificial blood vessels.In addition, most of the biocompatibility experiment is tested under static state. There are few reports under dynamic culture about interaction between cells and materials. Although the material has good biocompatibility under static test, it is easy to fail when grafted.Based on these difficulties of small diameter artificial blood vessel, silk was selected. We aimed to prepare medium molecular weight regenerated silk fibroin through degumming and dissolving processes, which showed excellent biocompatibility. By layer-by-layer self-assembly, the silk fibroin fabrics are modified by regenerated silk fibroin and alginate in order to improve the biocompabibility of silk fabric.This study includes 4 parts as followed:(1) Preparing medium molecular weight regenerated silk fibroin. The medium molecular weight silk fibroin has suitable solubility and film forming properties. It is good for selfassembly. The 0.5 wt% sodium carbonate solution and 8 M urea aqueous solution were used for degumming. Three kinds of method, mass loss rate, picrocarmine staining and scanning electron microscopy observation, were used to evaluate the effect of degumming. And 9.3 M Li Br aqueous solution and calcium chloride-ethanol-water(molar ratio 1:2:8) solution were used to dissolve the silk fibroin fibers to obtain silk fibroin. The molecular weight and distribution of the regenerated silk fibroin was observed by SDS-PAGE. Finally, the process of degumming and dissolving was selected to generate medium molecular weight silk fibroin.(2) Surface modification on silk fibroin fabrics by layer-by-layer self-assembly. And the physical properties and biocompatibility were evaluated. The sericin can not be removed completely only by degumming. The residual sericin may cause inflammatory in the host. We used the medium molecular weight regenerated silk fibroin and alginate as polyelectrolytes for layer-by-layer self-assembly to cover the sericin. In order to fixing the silk fibroin deposited on the fabric, 75 % ethonal was used to induce structure changing from random style to ?-sheet. At the same time, we observed the surface morphology, mechanical properties, stability and biocompability.(3) Establishing the dynamic culture system for vascular materials, and analysing the interaction between cells and materials under shear stress. Most of the dynamic culture devices are used for tissue engineering, which are not suitable for artificial vascular materials. We designed a convenient dynamic culture device for artificial vascular materials. It could simulate the blood stream flow through the surface. The shear stress could be adjusted by changing the inlet velocity.(4) Fabricating the tubular small diameter vascular based on the layer-by-layer self-assembly on silk fabrics, and evaluating the dynamic compliance. A tubular silk artificial blood vessel with a diameter of 6 mm was fabricated. Through the heat treatment method, the threedimensional tubular was obtained. After that, degumming and layer-by-layer self-assembly were operated to improve the biocompability of silk vascular. Based on the Test Instrument Biodynamic test system, the dynamic compliance of modified silk tubular artificial blood vessels, porcine carotid arteries and other artificial blood vessels were compared. After a few of experiments, we got the results as followed:(1) In this project, 0.5 wt% sodium carbonate degumming 1 hour, 9.3M lithium bromide dissolving silk fibroin under 95? to obtain the regenerated silk fibroin solution with medium molecular weight. The molecular weight distributes between 20 to 100 k Da.(2) Based on the theory of layer-by-layer self-assembly, we used regenerated silk fibroin solution and sodium alginate solution as the polyelectrolytes. Adjusting the p H of the solution, we made the solutions with different zeta potential. Sodium alginate was used as a polyanion polyelectrolyte. The zeta potential value was-35.75 ± 0.81 m V when p H was adjusted at 8. Regenerated silk fibroin solution was used as polycationic polyelectrolyte. The value of zeta potential was 10.80 ± 0.64 m V at p H=2. And regenerated silk fibroin solution was used as polyanion polyelectrolyte when assembling the outermost layer. At that time, the zeta potential was-9.23 ± 0.46 m V at p H=8. Under these conditions, the polyelectrolyte solution could generally stable and suitable for self-assembly.(3) Layer-by-layer self assembly could improve the surface roughness of the material. And the thickness increased with the modification. At the beginning of the self-assembly, the surface was rough. A lots of humps distribute on the surface of the silk fibroin fabrics. When assembled 1.5 layer, the thickness of deposition was 23 nm. With the increasing of the assembled layers, more and more depositions absorbed on the surface. The topography becomes fine and smooth. The humps become small. The thickness increased rapiedly. When assembled 9.5 layers, the surface was smooth. The thickness reached to 215 nm.(4) Layer-by-layer self assembly could improve the stability and mechanical properties. After assembled, the silk fibroin fabrics were fixed by the 75 % ethanol. The characteristic peaks shifted at amide ?, amide ? and amide ?, which means the structure of the fibroin changed from random conformation to ?-sheet conformation. At the same time, the decomposition temperature also improved from 314.9? to 322.3? observed by TGA. After 24 hours flushing, the depositions could withstand flushing fluid and remain stable. Bursting strength is significantly improved with the increase of the assembly layers. The breaking strength reaches 15.28 ± 0.16 N / mm2 after 9.5 layers assemble, and the value of commercial polytetrafluoroethylene vascular is 15.68 ± 0.8 N/mm2.(5) Layer-by-layer self assembly could improve the cytocompatibility and hemocompatibility. The endothelial cells were non-proliferation and did not adhere on the materials. This means the unmodified silk fibroin fabrics showed unfriendly for cells. And the platelets were apt to adhere on the materials and activated. It is easy to form thrombus when used. Using layer-by-layer technology, the surface of the silk fibroin fabrics were covered by silk fibroin and alginate. The biocompatibility improved significantly. With the increase of assembled layers, the cells proliferated well. And as assembled to 5.5 layers, more and more cells could be observed on the surface. The shape of cells like spindle. When assembled to 9.5 layers after a period of culture, cells covered the surface of the materials completely. At the same time, with the increase of the assembled layers, the platelets were significantly reduced, and the hemolysis was also improved. When assembled to 3.5 layers or more, the hemolysis rate decreases below 0.45 % which conforms to standards ASTM F756-00. When assembled to 5.5 layers, no platelets adhere on the materials.(6) Different inlet velocity created different shear stress by the fluid simulation analysis. Under low flow rate, the stream was in free flowing state. It was turbulent flow. As the velocity increased, the fluid in the central region of the dynamic incubator becomes uniform. We focused on the middle area for cell culture. Based on the results of tissue engineering dynamic culture, the shear stress increased gradually. There were four values: 3 dyn/cm2, 6 dyn/cm2, 9 dyn/cm2, 12 dyn/cm2. Finally it reached to the normal carotid shear stress. When the fibers and flows had a parallel, the cells would spread along the flow direaction and be elongated. When the fibers and flows had a vertical, the cells would be round. In the warp and weft yarns interlacing point, the cells gathered. An endothelial cell layer was formed.(7) The dynamic compliance of the tubular silk fibroin artificial blood vessel can not reach the hosted vessel. In the testing system, we set the wave type as sine, and the frequency was 1Hz. The pressure were 50-90 mm Hg, 80-120 mm Hg, 110-150 mm Hg and 140-180 mm Hg. Four types of blood vessels, including the pig's carotid artery, the commercial woven artificial blood vessel, the corrugated knitting artificial blood vessel and the e-PTFE artificial blood vessel, were selected as the reference samples. The dynamic compliance of the pig's carotid artery reached 7.21%/100 mm Hg, which was obviously superior to the dynamic compliance of the artificial blood vessels(below 2.52%/100 mm Hg). Comparing three kinds of artificial blood vessel, the dynamic compliance of corrugated knitting artificial blood vessel was better than others. Due to the high modulus and tex, the dynamic compliance of silk fibroin artificial blood vessel was inferior to the commercial woven artificial blood vessel. At the same time, the dynamic compliance decreased releated to the deposition of silk fibroin film after surface modification.In summary, there was no impact to the physical properties after modification on the silk fibron fabrics by medium molecular weight silk fibroin. And the cytocompatibility and hemocompatibility were improved observably. The materials were suitable for cell adhesion and proliferation both in static and dynamic culture. However, the dynamic compliance of silk fibroin artificial blood vessel was inferior to host, which need to be improved in future study. The results laid a foundation for the research of small diameter artificial blood vessel. |
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