Photo-Induced Co-Crosslinking Of Silk Fibroin/Gelatin And Its Application In 3D Printing

Silk fibroin(SF)materials have great potential in bioink applications due to their excellent biocompatibility,outstanding mechanical properties and controllable biodegradability.However,single-component silk fibroin solution cannot meet the requirements of printing rheology and structural stability,while the composite silk fibroin systems have different degrees of biocompatibility.In this paper,a printing method for the fine structure of silk fibroin/gelatin biomimetic scaffolds was established by adjusting the printing rheological properties of silk fibroin solution using the temperature sensitivity of gelatin components.Besides,based on the photochemical reaction characteristics of amino acid side groups of silk fibroin and gelatin,the structure-activity relationship between the co-crosslinking structure of silk fibroin and gelatin macromolecules and its application performance was studied,providing a useful exploration for the development of silk fibroin/gelatin bioink and its 3D bioprinting application.The main conclusions are as follows:(1)The rheological properties of SF/Gel system in extrusion printing process were studied under steady shear and dynamic oscillation conditions.The results showed that the addition of gelatin component would significantly increase the viscosity of SF/Gel composite system and endowed it with shear thinning properties.Based on the temperature-sensitive properties of gelatin component,the triple helix structure of gelatin can be deconstructed or restored by temperature-controlled incubation,which affected the formation of β-sheet structure,and then realized the regulation of printing rheological properties of SF/Gel composite system.In addition,the theoretical equilibrium modulus G’∞ of temperature-induced SF/Gel composite gel was quantified and predicted,providing a basis for the regulation of physical gel modulus during extrusion process.(2)Blue light induced crosslinking behavior and crosslinking mechanism of SF/Gel were studied by photorheological test system and three-dimensional fluorescence spectroscopy.The results showed that the photo-crosslinking efficiency of SF/Gel composite system increased integrally with the increase of photoinitiator,irradiation intensity and irradiation time.However,due to the filter effect’ of Ru(Ⅱ(bpy)32+,the photo-crosslinking efficiency might decreased at a high Ru(Ⅱ)(bpy)32+dosage.Fluorescence spectroscopy analysis showed that the blue light induced gelation process of SF/Gel composite system was caused by the cocrosslinking reaction between silk fibroin and gelatin macromolecules,which was mainly based on the free radical coupling reaction of tyrosine in silk fibroin and gelatin structure.Based on the dynamic oscillatory rheological test and analysis,an exponential function G=γ exp(-δt/tgel)+G’∞ was established to describe the photo-induced SF/Gel gel process and predict the theoretical equilibrium modulus G’∞.(3)The relationship between the structure and properties of SF/Gel cocrosslinked hydrogels was analyzed by compression test,drug release test,in vitro degradation test,CCK-8 test and Calcein/PI staining.The results showed that with the increase of gelatin content and photoinitiator dosage,the crosslinking degree of SF/Gel composite hydrogel increased,and its compression resistance increased,while its degradation rate and drug release rate decreased.The maximum compressive strength of SF/Gel co-crosslinked hydrogel was 1769.3±31.6 KPa.The highest degradation rate was 96.8±2.8%after 5 days of in vitro degradation.The highest cumulative release rate of the drug was 47.6±4.2%after 5 days of sustained release.In addition,the biocompatibility of SF/Gel co-crosslinked hydrogels was closely related to the photoinitiator content in the composite system.Under the optimized amount of photoinitiator,the SF/Gel co-crosslinked hydrogel had excellent biocompatibility,which could be further improved with the dissolution of the photoinitiator.After L929 cells were co-cultured with the hydrogel for 3 days,the cell viability reached 108.1±12.7%.(4)The effects of inlet simulated pressure on the flow rate,viscosity and shear rate of SF/Gel composite system were analyzed by COMSOL fluid simulation software.The results showed that the flow rate of SF/Gel at the outlet of the nozzle increased with the increase of inlet simulated pressure,and the pressure decreased gradually from the sample chamber to the nozzle,and the viscosity was in a low range(10"3 Pa s).The thixotropy test and the rheological regulation strategy were used to optimize the printable modulus range(G’ between 1000 Pa-4000 Pa)and incubation conditions(incubation at 20℃ for 10 min)of SF/Gel composite system.Under the optimized printing parameters and SF/Gel composite formula,the smooth extrusion of SF/Gel composite hydrogel line can be achieved with smooth surface and uniform diameter,realizing the 3D bioprinting of fine bionic scaffold structure.