| Due to its unique and excellent mechanical properties,biocompatibility and biodegradability,spider silk shows great application prospects in many fields such as materials science and biomed-ical medicine,etc.However,because of the cannibalism of most spiders and their strong territorial consciousness as well as low silk productivity,it is not practical to obtain sufficient natural silk proteins for large-scale applications.Nowadays,various recombinant spidroins can be synthesized with bioengineering technologies which provides raw materials for the application and development of spider-silk fibers and spider-silk protein products,and also provides the possibility for the large-scale production and application of the recombinant spider silk fiber.It is still necessary to develop novel recombinant spidroins and explore spinning processes,so as to obtain recombinant silk fibers with high-quality mechanical properties to realize the large-scale production and application of the recombinant spider silk fibers.Achievement of studies on spider silk mainly focuses on natural and recombinant major ampullate silk,while the research on natural and recombinant piriform silk starts relatively late.There are no reports on mechanical properties of natural pyriform silk,and few re-search achievements have been made on the properties of recombinant piriform silk.In this paper,studies were carried on the preparation,modification and mechanical properties of silk fibers spun from recombinant spidroin designed on the basis of piriform spidroin,and then extended to the application study on the properties of nanofibrous membranes electro-spun from blended recombi-nant spidroin.The specific research contents are as follows.Part?.Studies on the preparation of the fibers and their mechanical properties.Piriform silk is one of the main components of spider silk attachment discs.The excellent property of attachment discs also suggests that the pyriform silk may have similar properties.The module of pyriform spi-droin repetitive regions contains QQ and(PX)n motifs,which are new elements to design recombi-nant spidroin for the studies on the relationship between structure and performance.Based on pyri-form spidroin gene of Araneus ventricosus spider,four constructs to express recombinant pyriform spidroins were obtained and used to produce spidroins.The purified spidroins were spun into long fibers with a wet-spinning process.The four kinds of silk fibers had uniform diameter and the cross section exhibited in a circle shape and there were no micro-fibril structures.Thermal gravimetrical analysis results showed that the onset temperature of thermal degradation was around 250?and the four silk fibers possessed similar thermal stability to natural dragline.The results of mechanical properties tests showed that all the fibers exhibited similar and very high extensibility,with maxi-mum strain at breaking more than 60%.The Young's modulus,stress at breaking and toughness increased with the present of non-repetitive units and the increasing number of the repetitive mod-ules.In this study,systematical investigations were performed on the changes of the secondary structure contents of the recombinant spidroin in spinning dope,spun fibers,stretched and broken fibers.The secondary structure of the four recombinant proteins underwent an?-helix to?-sheet transformation during spinning process.The analysis of the secondary structure during the post-spinning stretching process showed that with the increase of the stretching ratio,the content?-sheet decreased gradually,and the?-sheet content recovered after the fiber was broken.In this study,recombinant pyriform spidroins were produced and spun into long fibers with good mechanical properties with a wet-spinning process.This study expanded the studies on the recombinant spidroin and recombinant spider-silk fibers and provided a foundation for further study on these silk fibers.Part?.Study on the modified silk fibers and improvement of mechanical properties of spider-silk fibers.In order to obtain fibers with better mechanical properties,fibers were spun from a re-combinant spidroin with a wet-spinning process in which titanium dioxide and formaldehyde were used for the first time.In terms of mechanical properties,compared with the control spider-silk fibers,the silk fibers treated with titanium dioxide or/and formaldehyde exhibited higher Young's Modulus,stronger stress at break and higher toughness,while decreased extensibility.The fibers treated with the two methods showed the highest toughness up to 249±22 MJ/m~3.Analysis of FTIR showed that,compared with the control,there were difference in the protein secondary structure component between the control fiber and the fibers treated with titanium dioxide or/and formalde-hyde treated silk fibers.The crystal structure analysis results of silk fibers detected by wide angle x-ray diffraction(WAXD)showed that the modified spider-silk fibers had higher crystallinity and different crystalline size.The results of small angle scattering(SAXS)analysis showed that interface thickness in these fibers were different.It was verified in this study that titanium dioxide and for-maldehyde can be used to improve the mechanical performance of the recombinant spider silk fibers.The modification with nanoparticles and organic crosslinking agents was a new strategy to obtain well-performing recombinant spider-silk fibers.This study provided a successful example for the combination of other nanoparticles and the different organic crosslinking agents.Part?.Assessment on the application performance of nanofibrous membrane electro-spun from blended recombinant spidroin.Scaffolds are important for tissue engineering and the present ones fabricated from chemical polymers or native-originated extractor exhibited drawbacks such as insufficient mechanical properties,low hydrophilicity or low degradation rate.Blending with re-combinant spidroin was expected to make up these defects.Recombinant spider silk protein and poly(L-lactide-co-?-caprolactone)were blended in different ratios to electro-spin nanofibrous mem-branes which were characterized with different detective and testing methods.The results of scan-ning electron microscopy(SEM)showed that blended nanofibers had decreased average diameter.Water contact Angle measurement results showed that the surface of pure PLCL nanofibrous mem-brane was hydrophobic and that of blended nanofibrous membrane was hydrophilic.The results of wide-angle-x-ray diffraction(WAXD)showed that the content of amorphous structure increased in the blended nanofibers.Tensile tests results showed that the extensibility of the blended nanofibers decreased with the increased ratio of recombinant spidroin while the strength showed no correlation with the ratio.After preliminary screening,the nanofiber membrane spun from PLCL and protein blending ratio of 75/25 met the requirements of hydrophilicity and mechanical properties of skin regeneration scaffolds.Subsequent hemolysis and cytocompatibility tests preliminarily demon-strated that the blended nanofibrous membrane had good biocompatibility and had the potential to prepare skin regeneration scaffolds.In vitro degradation testing results showed that the degradation rate of blended nanofibers was improved.It was verified that the hydrophilicity,mechanical prop-erties and degradation rate of nanofibrous membrane were improved with blending of 25%recom-binant spidroin.This study provides a scientific reference for further research on the production and application of recombinant pyriform spidroin. |
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