| Silk fibroin is natural protein obtained from silkworm silk, consists 18 kinds of amino acid and has excellent physicochemical characters and superior biocompatibility. There has been a long standing history of use for silk fiber in clinical suture, and this application opened the door which led to biomedicine for it. Recently, the research and application of silk fibroin in biomedicine has gotten more and more attention, such as suture, controlled release of medicine, support materials for immoblization, contact lenses, manual vein, artificial ligament and muscle tendon, wound dressing, cell culture matrix. But pure silk fibroin membrane is brittle after insolubilization, which restrict silk fibroin from comprehensive application in biomedicine. Now, many authors have processed different kinds of silk fibroin materials, such as silk fibroin fiber, membrane, gel and sponge, in various physical and chemical methods that were used to improve the properties of silk fibroin materials. But with the improvement of tissue engineering and the need for porous scaffold, the preparation of silk fibroin scaffolds have been studied more.There are many methods to prepare porous materials, such as freeze drying, particle leaching, gas foaming, thermally induced phase separation and rapid prototyping manufacturing. As so far, porous silk fibroin scaffolds have been obtained by freeze drying, salt leaching and gas foaming. The scaffolds obtained from these methods were characterized as having a good porous structure and mechanical properties for tissue engineering. But the preparation of silk fibroin porous scaffolds, using ice particles to generate pores, usually need freeze drying to make silk fibroin molding, so the preparation was always complicated.A new sample method without freeze drying to prepare silk fibroin scaffolds was used in this study. Porous silk fibroin scaffold (PFGS) was obtained by freezing the mixed solution of silk fibroin and polyepoxy compound at -20℃, followed by washing and freeze drying. Cross-linked silk fibroin scaffold (CFGS) was obtained by putting the mixed solution of silk fibroin and polyepoxy compound under 60℃, followed by treatment of washing and freezing. From the results of Infra red (IR) spectra, Differential Scanning Calorimeter (DSC) curves, amino acid analysis and dissolution, it was found that epoxide and silk fibroin didn't act in PFGS, but they cross-linked in CFGS. The SEM images indicated that the two kinds of silk fibroin scaffolds had different porous structure. The surface of PFGS was a coat with many tiny pores, and interior structure was round and highly interconnected pores, with the size of more than 150μm. Meanwhile, there was no pores in the surface of CFGS which was more smooth, and the pores inside were tubular with the size of more than 150μm too. Repeating freeze-thaw treatment increased the amount of tiny pores in the surface of PFGS, and made the surface of CFGS uneven, while freeze-thaw treatment made the interior porous structure more complicated with new pores generated by the break of pore walls. The porosities of two kinds of scaffolds were both above 90% measured by liquid displacement.The cytotoxicity of silk fibroin scaffolds was tested by detecting the effect of the extracted liquid to the cell relative proliferation rate of L-929 mouse fibroblasts. The result indicated that they all displayed high relative proliferation rate, and the cytotoxicity grade was 1 grade, being in the allowed range of medical application. The cell compatibility of silk fibroin scaffolds was further studied by culturing fibroblasts on the materials directly. The results indicated that attachment ratio and growth of fibroblasts in PFGS were better than CFGS, while both of them were lower than pure silk fibroin membrane. The fibroblasts on the surface of PFGS started to proliferate from the 5th days, and fibroblasts can grow well on it. On the other hand, fibroblasts can't attach and grow well on the surface of CFGS. All of thses differences of fibroblasts on material surface were due to the surface properties, like hydrophilicity, physical morphology and the change of chemical groups. Although there were no excellent attachment ratio and growth of fibroblasts on the surfaces of scaffolds, fibroblasts can grow well inside both of the scaffolds. We speculated the reason was that the interior porous structure was different from the surface. In this study, the generation of PFGS was based on the thermally induced phase separation of trinary solution, while the generation of CFGS was based on the freezing of cross-linked gel, and both of the two kinds of silk fibroin scaffolds had optimal porous structure. The study indicated that different silk fibroin scaffolds with different porous structure could be obtained through the mutual action of polyepoxy compound and silk fibroin at different temperature. This afforded new thought and method for preparation of silk fibroin porous scaffolds. In addition, the study indicated that the surface properties of scaffold materials had a big effect on the attachment and growth of fibroblasts, the function of scaffold. This provided reference for the preparation of silk fibroin scaffolds in future, such as the choice of methods, the surface modification and the choice of chemical cross-link reagent.
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