Regulation Of Silk Fibroin Nanofibrils And Application In Tissue Repairing Biomaterials

The micro/nanoscale topography of scaffolds plays an important role in controllingthe growth, proliferation and differentiation of cells and the reconstruction of theextracellular matrix (ECM) in tissue engineering. Mimicking the micro/nanoscalestructure of the ECM to fabricate the microenvironment for the growth of cells andtissues is a new direction for fabricating the tissue repairing substrates. Nanofibril is thefundamental unit of living systems. Nanofibrous materials have the similarmicrostructures with the ECM, providing more suitable enviroment for tissueengineering. Silk fiber is one of the strongest and toughest biological materials withhierarchical structures where nanofibril with size of below20nm is crifical factor indeterming the excellent mechanical properties. Although silk nanofibrils have beenfound in natural and regenerated silk solutions, there is no effective way to activelycontrol nanofibril formation in aqueous solution, which limits the application of silkmaterials in tissue engineering.Here we show a simple but effective method to prepare silk nanofibrils byregulating silk self-assembly process. Through a repeated drying-dissolving process,silk fibroin solution composed of metastable nanoparticles was firstly prepared and thenused to reassemble nanofibrils with different sizes and contents of β-sheet under varioustemperatures and concentrations. These nanofibrils have similar morphology with thatin natural fibers, providing a suitable unit for further assemble hierarchical structure invitro. Several important issues such as the relationships between silk nanofibrils,secondary conformations and viscosity are also elucidated, giving a new insight into theself-assembly process.Then, through different post-processing methods, we further regulate the secondarystructures and mechanical properties of silk nanofibrils to obtain silk nanofibrous filmswith different mechanical stiffnesses and then discuss their effects on regulating theneural stem cell (NSC) behavior. The results showed that nanofibrils with differentstructures and mechanical properties significantly affected the behaviour of NSCs. The nanofibrils treated with water-annealing or80%methanol significantly improved theNSCs’ migration. The nanofibrils treated with50%methanol-annealing or80%methanol significantly improved the NSCs’ differentiation to neurons. More importantly,the nanofibrils treated with80%methanol provided better microenvironment for NSCs,not only improving the NSCs’ differentiation to neurons, but also restraining theformation of astrocytes, which would be suitable matrices for neural tissue repairing.We then mixed the nanofibrils with silk fibroin solutions to induce the further silkself-assembly and improve the formability and stability of three-dimensional scaffolds.With the increase of nanofibril content, the microstructures of the scaffolds changedfrom separate sheets to porous structures, implying the improved formability. At thesame time, the content of nanofibrils also dramatically affected the mechanicalproperties and the water-solubility of scaffolds. The scaffold with11.8%of nanofibrilsachieved best mechanical properties and above85%insolubility in water and retainedthe original structure and porous microstructure without any post-processing. Comparedwith the traditional silk fibrous scaffolds, this scaffold with lower crystallinity and fasterdegradation performances shows better hydrophilicity and biocompatibility, implyingpromising future applications of the scaffold in soft tissue engineering. Then thestructures and properties of nanofibrous scaffolds were controlled throughwater-annealing or80%methanol immersion treatments to further regulate themechanical properties, secondary structures as well as degradation behaviors of thescaffolds. In vitro results showed that these scaffolds had good biocompatibility.In conclusion, this work provided an effective way to prepare silk nanofibrilsbased on repeated drying-dissolving process. Through adjusting the content of thenanofibrils and then treating the silk materials with different post-processing methods,we prepared nanofibrous silk materials with different secondary structures andmechanical properties. These results indicated that various microenvironmentsaccording to different tissues’ requirements could be prepared based on silk fibroinscaffolds, further improving the feasibility of silk in different tissue regenerationapplications.