Blue Light Induced Photocrosslinking Of Silk Fibroin And Its Applications In 3D Printing

Silk fibroin(SF)is an ideal material for 3D bioprinting tissue engineering scaffolds due to its excellent biocompatibility,degradability and adjustable mechanical properties.However,the conventional crosslinking methods,physical,chemical and enzymatic crosslinking can't meet the requirement of high crosslinking efficiency for SF-based 3D printing.Although the photosensitive modification technology of SF displays relatively high crosslinking efficiency for SF-based 3D printing,there are still some controllability problems,such as uncontrollable grafting rate and grafting distribution,remaining to be resolved.In this study,blue light induced photocrosslinking of SF based on coupling reaction between amino acid residues was proposed.The main research contents include the construction of efficient blue light photoinitiation system,the exploration of amino acid photochemical reaction characteristics and coupling crosslinking mechanism,the establishment of structure-activity relationship of photocrosslinked SF,and the application of three-dimensional rapid printing of photocrosslinked SF.This work is expected to provide theoretical and applied basis for the regeneration and utilization of silk fibroin in the field of biotechnology.The main conclusions of this study are as follows.1.Blue light induced photocrosslinking of SF was studied using five bicomponent photoinitiators,camphorquinon(CQ),riboflavin-5'-phosphate sodium(FMN),curcumin(CC),eosin Y(EY),and Ru(bpy)₃Cl₂(Ru(?))as photosensitizer,and diphenyliodonium hexafluorophosphate(DPI)or potassium persulfate(KPS)as electron acceptor.The spectral absorption and photochemical properties show that all of the five photosensitizers possess the efficient electron transition in the blue light region,resulting in effective spectral absorption and emission behavior.Photoinduced electron transfer controlled by free diffusion occurs between the five photosensitizers and electron acceptors.Among them,Ru(?)/KPS system possess the highest electron transfer efficiency,followed by CQ/DPI and FMN/DPI systems,EY/DPI and CC/DPI systems.The results of photorheological test showed that Ru(?)/KPS system had the highest crosslinking efficiency of SF,showing the shortest gelation time(?1 s)and the highest storage modulus(G'is about 570 Pa under 10-min blue light irradiation).2.The photochemical characteristics of 18 amino acids were studied with using Ru(?)/KPS as an efficient blue light photoinitiator.The results show that tyrosine,tryptophan and phenylalanine had the highest photoreaction activity.Their N-acetylated derivatives,N-acetyl-L-tyrosine and N-acetyl-L-phenylalanine,could be oxidized to the dimer of N-acetyl-L-tyrosine by Ru³⁺and OH·;N-acetyl-L-tryptophan could be self-polymerized to melanin through the coupling reaction between hydroxyindole(or indole quinone)under the oxidation of OH·.Three photocrosslinking pathways of SF based on coupling reaction between amino acids were proposed:(1)coupling crosslinking between tyrosine residues,which was initiated mainly by Ru³⁺,supplemented by KPS and SO₄^-·;(2)phenylalanine residues could be oxidized to tyrosine residues via the oxidation of OH·,and then followed by the coupling crosslinking reaction;(3)tryptophan residues could be oxidized to hydroxyindole or indole quinone via OH·,and then followed by the coupling crosslinking between hydroxyindole(or indole quinone)residues.In addition,the transformation of SF macromolecules from the random coil conformation to?-sheet was promoted by the crosslinking reaction between the amino acid residues,thus achieving the physical and chemical double-crosslinking of SF hydrogels.3.The viscoelastic behavior and gelation behavior of blue light induced photocrosslinking of SF were systematically studied by using the photorheological testing system.The photocrosslinking kinetic models of SF were established with storage modulus G?,shear stress and complex viscosity as indexes.The results show that the photocrosslinking conversion increases exponentially with the irradiation time while the photocrosslinking rate increases first and then decreases.The"filter effect"that accompanied by high concentration of photosensitizer and the uneven crosslinking phenomenon that accompanied by high light intensity have great influence on the photocrosslinking conversion.Carboxymethyl cellulose sodium(CMC-Na)was grafted with tyramine hydrochloride(TA)to prepare photoreactive tyramine cellulose(CMC-TA).With the increase of CMC-TA content,the photocrosslinking efficiency,the mechanical properties,water absorption and shear stability of SF/CMC-TA composite gel were also significantly improved.4.Combining the blue light induced self-crosslinking characteristics of SF and 3D printing technology,a real-time crosslinked 3D extrusion-based printing technology suitable for pure SF gel scaffolds and a 3D extrusion-based printing technology with post-photocrosslinking suitable for silk fibroin/xanthan gum(SF/XG)composite gel scaffolds were developed.By modifying the printing equipment,adjusting the curing speed,air pressure and printing head moving speed,the semi-transparent and elastic SF hydrogel scaffold materials with different filament diameters were prepared respectively,which providing an innovative idea for 3D bioprinting of pure SF gel.Silk fibroin/xanthan gum(/tyramine cellulose)composite precursors were developed with xanthan gum as the natural thickening component,which showed good shear thinning properties and shear thixotropy.The inter-layer distribution of 3D printed composite gel is closely packed with relatively uniform filament size.The good wetting stability and pressure bearing performance of composite gel were expected to meet the requirements of high structural stability and fine structure of 3D bioprinted scaffold.