Preparation,Structures And Properties Of Silk Fibroin Based Biomimetic Tissue Engineering Scaffolds

In clinical practice,the depressed deformities caused by tumor resection,trauma,congenital malformation and other soft tissue injuries have a serious impact on the appearance and even the function of movement of patients,thus affecting their mental health and quality of life.Therefore,the repair and reconstruction of surgical soft tissues including skin,fat,muscle and others have become one of the important problems in plastic surgery.The depressed deformities caused by surgical defect need to be filled by tissue or biomaterial.In order to overcome the limitation of tissue donor and avoid the immune rejection as well as infection caused by tissue transplantation,scaffolds for soft tissue repair and reconstruction need to resemble the structures and functions corresponding to the target site,which can achieve the goal of personalized repair.Therefore,it is of great significance to study how to improve the biomimetic effect of scaffolds related to complex tissue using biomaterials of appropriate mechanical properties,biocompatibility and biodegradability.Regenerated silk fibroin protein(RSF)is a structural protein extracted from the natural biological material silk,which displays good biocompatibility and excellent mechanical properties.Therefore,RSF shows huge application prospect to prepare biomimetic tissue engineering scaffolds as raw material.Scaffolds prepared with the traditional strategies such as lyophilization and electrospinning lead to simple structures.It is a big challenge to produce highly biomimetic tissue engineering scaffolds.Therefore,the traditional preparation technologies need to be modified or new technologies have to be applied.In this study,RSF was chosen as the main raw material.Firstly,electrospun RSF scaffold with gradient pore size structures was constructed by low-temperature electrospinning(LTE).The pore sizes of the RSF scaffold were tailored based on the mechanism of ice crystal template in the LTE process with a cold collecting plate.Secondly,biomimetic scaffolds with hierarchical pore structures were reinforced with an addition of nanofiber network in RSF based hydrogels.Thirdly,the compositions of RSF-based ink were further simplified by analyzing the rheological behaviors of the ink with oxidized cellulose nanofibers.A novel method was suggested to print biomimetic scaffolds with anisotropic structure.Finally,a high-performance double network hydrogel was designed by combing covalent cross-linking synthetic polymer elastic network in RSF hydrogel,which was printed to construct strain sensors.The effects of the microfibers of degummed silk on the sensors' performance were investigated.The 3D printed scaffolds were further studied in the application of flexible sensing of tissue engineering.(1)Traditional electrospun scaffold has a dense structure,which prevents cell penetration,transport of nutrient and metabolic waste in scaffolds.LTE was used in this study to adjust the pore size of RSF electrospun scaffolds by controlling collector temperature and the consequent ice crystal size.The relative humidity(RH)of LTE was also adjusted to control the ice crystal size.RH of 50% resulted in an LTE RSF scaffold with pore size of 15.9?23.1 ?m,which increased significantly compared to the traditional counterpart.The macro pores in the range of millimeter-scale were formed in LTE RSF scaffold at the relatively higher collector temperature(-50 ?).LTE RSF scaffolds with pore size ranging from 6 ?m to 50 ?m were prepared through adjusting collector temperature.L929 cells just grew on the surface of traditional electrospun scaffolds while showed significantly improved cell infiltration performance in 3D structure of LTE RSF scaffold.Therefore,the scaffolds combined small pores,medium pores and large pores could be fabricated by multi-step electrospinning including traditional and low temperature electrospinning.The RSF scaffolds with gradient pore structures show great potentials in the repair of full-thickness skin.(2)Although the electrospun scaffolds with gradient structures could mimic the simple tissue as skin but could hardly mimic complex tissues.To prepare highly biomimetic hydrogel scaffolds,3D printing was applied for silk-based inks,in which mixture of RSF/gelatin was used as the initial ink and a small amount of bacterial cellulose nanofibers(BCNFs)were additive.BCNFs were used to improve the printability of RSF/gelatin inks thus to acquire the high shape fidelity of the scaffolds.Furthermore,the mechanical properties of RSF/gelatin/BCNFs were enhanced by BCNFs.The Young's modulus in tensile tests of print lines with RSF/gelatin/BCNFs ranged from(315.3±104.4)k Pa to(1627.8±433.4)k Pa as the contents of BCNFs in inks increased from 0 to 1.4 wt%.Based on the effects of different print inks,the shape fidelity of scaffolds promoted as higher ratio of BCNFs.However,too much BCNFs led to the chocking of micronozzle frequently during printing and consequent poor shape fidelity.Therefore,0.7 wt% BCNFs in RSF/gelatin inks contributed to the best printability and shape fidelity.The compressive strength at 30% strain of RSF/gelatin/BCNFs(BCNFs-0.7 wt%)was twice as higher than RSF/gelatin.The elastic modulus was(186.5±20.2)k Pa which matched the mechanical requirement of some soft tissues.The printed scaffolds presented better compression resilience due to the network structure of RSF/gelatin/BCNFs composite hydrogel.Furthermore,large amounts of hydrogen bonds formed between BCNFs and RSF/gelatin,which were reversible after being broken during compression thus dissipate lots of energy.The dissipated energy of BCNFs-0.7 wt% was 3.7 k Jm-3.In addition,the hierarchical structure with micro pore of 10?20 ?m in print lines and macro pore of 300?600 ?m between print lines were formed in BCNFs-0.7 wt% after lyophilization.The hierarchical structures of the scaffold were benefit to cell infiltration and tissue regeneration.Therefore,a few percent of BCNFs was introduced in the ink of RSF/gelatin could produce the higher performance scaffolds with hierarchical structures by 3D printing.It may have huge potential application in the reconstruct of complex soft tissue.(3)Posttreatments were necessary for the printed scaffolds of RSF/gelatin/BCNFs to make RSF/gelatin insoluble in water.To simply the components of inks,novel inks consisting of RSF and 2,2,6,6-tetramethylpiperidine-1-oxyl(TEMPO)-oxidized bacterial cellulose(OBC)nanofibrils were developed for 3D printing.RSF backbones were cross-linked using horseradish peroxide(HRP)/H2O2 to form printed hydrogel scaffolds.OBC increased the viscosity of the inks during the printing process and further improved the shape of the scaffolds.Three-dimensional construct with ten layers could be printed with ink of 1SF-2OBC.The composite hydrogel of 1SF-1OBC exhibited a significantly improved compressive strength of(267.0 ? 13.0)k Pa and compressive strain over 50%.Furthermore,OBC nanofibrils could be induced to align along the print lines,providing physical cues for guiding the orientation of lung epithelial stem cells(LESCs),which maintained the ability to proliferate and kept epithelial phenotype after 7 days' culture.The 3D printed SF/OBC scaffolds are promising for applications in lung tissue engineering.(4)A biocompatible covalent cross-linked polyacrylamide(PAM)network was introduced into RSF hydrogel.The PAM/RSF double network hydrogel was prepared by one-step cross-linking via white light.Silk micro fibers(SMF)were prepared using Na OH/urea solvent.The negatively charged SMF were combined with RSF thus formed the physical crosslinking in the double network hydrogels of PAM/RSF/SMF.The tough hydrogels had a breaking strength of(256.5?11.5)k Pa and a compressive modulus of(617.4?67.1)k Pa at strain of 5%?10%.The elongation at break was over 500%.On one hand,SMF improved the mechanical properties of PAM/RSF/SMF hydrogels.On the other hand,physical crosslinking of RSF-SMF enhanced the printability of PAM/RSF/SMF hydrogels.In addition,PAM/RSF/SMF hydrogels showed good ionic conductivity after the immersion in phosphate buffered saline(PBS)and displayed the capability of strain and pressure sensing.The hydrogels made it possible for in-situ monitoring of tissue regeneration under biomechanical stimulation.