Preparation Of Natural Spider Silk Bioinks For 3D Bioprinting Applications

In recent years,spider silk has gradually become a research hotspot in the fields of materials,chemistry and biology.Spider silk has been recognized as a biomaterial because of its high strength,high toughness,low temperature resistance,ultraviolet resistance and biodegradability.Because of the limited access to natural spider silk,most of the current research on spider silk is focused on recombinant spider silk protein(spidroin).But the uncertainty and simplicity of genetic engineering technology make recombinant spider silk not comparable to natural spider silk.The properties of natural spider silk are far beyond that of recombinant spidroin,but the insolubility of natural spider silk still limits its biological application.In this thesis,the influence of spider silk structure on its solubility was analyzed,and the effects of high concentration salt solution,ionic liquids and organic solvents on the solubility of spider silk were discussed in depth.Subsequently,bioink was prepared using natural spider silk as raw material and formic acid as solvent,and then the tissue engineering scaffold was printed using extrusion-based three-dimensional printing technology.The biocompatibility of the scaffolds was also characterized.The details are as follows:(1)Firstly,the influence of spider silk structure on the solubility of spider silk was discussed,and the lipid layer of spider silk outer layer was destroyed by degumming treatment,which greatly enhanced the hydrophilicity of spider silk for subsequent solubility experiments.The results of dissolution experiments showed that spider silk had different solubility in different solvents,among which TBAH(Tetrabutylammonium Hydroxide)(40% aqueous solution)and 98% formic acid had good solubility.Subsequently,the intrinsic reaction of spider silk in different stages of dissolution process was studied,and the mechanism of dissolving spider silk with formic acid was proposed.(2)Self-assembly of spidroin solution is easy to occur at high concentration.In this paper,a high concentration of spidroin solution was prepared by dissolving degummed spider silk with formic acid when exploring spidroin bioink.Subsequently,the gelatinous precipitation obtained by multiple differential centrifugations was used to prepare spidroin bioink.Rheological tests of spider silk gel under different parameters were carried out.The results showed that the prepared bioink had the characteristics of sol-gel transformation and shear thinning.The bioink is placed in a 3D printer and printed layer by layer.The printed matter is placed in deionization to remove formic acid,and then freeze-dried to obtain a scaffold.(3)In order to explore the biocompatibility of spidroin materials,cell compatibility and histocompatibility were used to evaluate them.In the evaluation of cell compatibility,SD rat bone marrow mesenchymal stem cells(BMSCs)were inoculated on the surface of materials/scaffolds,the cell proliferation was evaluated by CCK-8 method,and the cell morphology and adhesion were evaluated by Calcein-AM staining.In the histocompatibility test,subcutaneous embedding of SD rats was carried out with spidroin material/scaffold.HE staining showed that there was no necrosis in the tissues,the cells survived well,and some of them grew into the material.