The Study On The Silk Fibroin Materials Crosslinked By Tyrosinase

Bombyx mori silk fibroin have been uesd as tissue engineering scaffolds due to itsgood biocompatibility. But the fibroin materials without crosslinking would dissolve inthe water easily, which limits the range of biomedical applications. So it is veryimportant to seek a new crosslinked way, which makes the biocompatibility andphysical properties of silk fibroin materials better and can regulate their biodegradationrate. Tyrosinase is a copper metalloenzyme, which widely distributes in themicroorganisms, plants, animals and the human body and is directly related to synthesisthe pigments of organisms. In this paper, tyrosinase catalyzes crosslinking reactionamong the silk fibroin macromolecules, and then the crosslinked silk fibroin materialsare prepared, at the same time, a systemic research on the structure, physical propertiesand in vitro enzymatic degradation behavior of the crosslinked materials is carried onand the cytocompatibility of crosslinked silk fibroin films is aslo studied.Firstly, the tyrosinase catalyzed crosslinked fibrion films were prepared by castingtyrosinase/silk fibroin mixture solution on a polyethylene plate and then air-dried. Watersolubility of the crosslinked films showed that the tyrosinase was able to improve waterresistance effectively. The fixation index of crosslinked films increased as the content oftyrosinase increased. The FTIR showed the tyrosinase catalyzed the oxidation oftyrosyl residues in SF to o-quinones and the oxidation products, subsequently, leading tothe Michael-addition reaction with N-terminal amino groups of SF molecules at thep-position of o-quinone residues. At the same time, the amino acid analysis showed thatthe tyrosine and lysine content of the silk fibroin films reduced after crosslinked, andfurther explained the mechanism of crosslinked. The condensed structure of the filmswas studied by XRD. The results indicated that the crosslinked silk fibroin films werestill mainly amorphous structure.The breaking strength of the tyrosinase catalyzedcrosslinked films increased, and their elongation did not change obviously. Secondly, the crosslinked porous materials were prepared by freeze-drying withdifferent fibroin concentrations and different pre-freezing temperatures. Water solubilityof the porous materials reduced significantly. The FTIR, XRD and Thermal analysisresults showed that the crosslinked silk fibroin porous materials were still mainlyamorphous structure. When the concentration of fibroin increased and the freezingtemperature dropped, the pore size and the porosity would become smaller.Thirdly, we chose collagenase IA as proteolytic enzyme to degrade silk fibroinmaterials, and inveatigated how the degree of crosslinking affected the biodegradabilityof silk fibroin materials. The degradation in vitro indicated, as the degree ofcross-lingking increased, the degradation rate slowed down and the content of freeamino acid decreased. Through regulating the degree of crosslinking, the biodegradationrate of silk fibroin films should be controlled.At last, this paper studied the cytocompatibility between L929cell and crosslinkedsilk fibroin films. The crosslinked silk fibroin films were able to support cell growth,adhesion and proliferation. Cell vitality on the crosslinked silk fibroin films was sameas silk fibroin films treatmented by alcohol by means of microscopy, MTT and Bradfordassay.The research in this paper provided a new crosslinked way, which made thebiocompatibility and physical properties of silk fibroin materials better and can regulatetheir biodegradation rate.