The Study Of Preparation On Regenerated Bombyx Mori Silk Fibroin With Controllable Molecular Weight

As the main components, silk fibroin has been widely used inbiomedical for the excellent mechanical properties, biocompatibility anddegradability. The silkworm silk fibroin used in biomedical is primarily intwo forms. One form is silk fibroin fibers, which is the degummed silk.The other form is regenerated silk fibroin, which obtain from silk fibroinfibers through dissolution, dialysis and drying. There are four steps fromnatural silk to regenerated silk fibroin. They are degumming, dissolution,dialysis and drying. Now there are many ways to degum natural silk, suchas enzymatic degumming, sodium degumming, urea degumming, high-temperature and high-pressure water degumming and so on. There are alsomany method to dissolve silk fibroin fibers, for instance lithium bromidesolution, lithium thiocyanate solution and ethanol-water-calcium chloridesolution. Hence different research groups have different process to prepare regenerated silk fibroin. However, degumming and dissolution have asignificant effect on molecular weight and its distribution, which affect thestructures and properties of regenerated silk fibroin-based biomaterials.In this study, we study the processes of preparing regenerated silkfibroin from natural silk and focus on the effect of degumming anddissolution on molecular weight and its distribution. We aim to get processof preparing regenerated silk fibroin of controllable molecular. There arethree parts in this study.The first part is the effect of degumming on molecular weight and itsdistribution of regenerated silk fibroin. The silkworm cocoons aredegummed using conventional0.5%sodium carbonate solution and8Murea solution at different temperatures and different time. The results of thedegumming rates of different methods, dyed using picric acid carmine andSEM show that the silk can be degummed completely using0.5%sodiumcarbonate solution and8M urea solution at90℃for3h.The silk fibroinfibers degummed using0.5%sodium carbonate solution and8M ureasolution at90℃for3h are dissolved in9.3M lithium bromide solution.The result of SDS-PAGE show the molecular weight distribution ofregenerated silk fibroin solution degummed using0.5%sodium carbonatesolution is below50kDa,while it is below116kDa degumming using8Murea solution at90℃for3h.Therefor,8M urea solution have a minor effecton silk fibroin molecular weight. The second part is the effect of dissolution on molecular weight andits distribution of regenerated silk fibroin. The silk fibroin fibersdegummed using8M urea solution at90℃for3h are dissolved in9.3Mlithium bromide solution and the molar ratio of1:2:8of calcium chloride-ethanol-water ternary solution. The result of SDS-PAGE shows theregenerated silk fibroin solution dissolving silk fibroin fibers in9.3Mlithium bromide solution have less effect on the molecular weight and itsdistribution than calcium chloride-ethanol-water ternary solution. Thesilk fibroin fibers degummed using8M urea solution dissolve in9.3Mlithium bromide solution at25℃、60℃and95℃.The result of SDS-PAGEand HPLC shows that different temperature have different effect onmolecular weight and its distribution. With increasing temperature, themolecular weight becomes smaller.The third part is study of structure and properties of differentmolecular regenerated silk fibroin hydrogel. Choosing8M urea solution todegum and9.3M lithium bromide solution to dissolve at25℃、60℃and95℃and getting different molecular regenerated silk fibroin solution.Three-dimensional porous scaffolds are prepared by freeze-dryingdifferent molecular regenerated silk fibroin solution. Through SEM,compression test, FTIR and XRD, the mechanical properties, surfacemorphology, the secondary structure and crystallinity are analyzed andcompared. The result suggests that the morphology of high molecular weight three-dimensional porous scaffold is dense, compression propertyis excellent, the secondary structure is mainly Silk I and the crystallinity ishigh. With the increase of temperature, the space becomes bigger,compression property becomes worse, the secondary structure is alsomainly Silk I and the crystallinity decreases. Solubility test results showthat the higher molecular weight of three-dimensional porous, thesolubility is lower.In summary, the effect of degum and dissolution on the molecularweight and its distribution of silk fibroin is studied in this study and initialexploration of the effect of molecular weight and its distribution on theproperties of silk fibroin three-dimensional porous scaffold has beenstudied. Although we have done some work about the high molecularweight and controllable molecular of regenerated silk fibroin, it ispreliminary. In the future we hope further work can be done to realizeautomation and standardization of the process.