Preparation Of Regenerated Silk Fibroin Fibers And Its Application On Artificial Ligament

Anterior cruciate ligament(ACL) is the core structure of knee-joint, which plays an important role in the physiological and mechanical function of the knee. At present, the number of patients presenting with ACL injuries has been on the rise in resent years due to the growing popularity of sports, and the increased incidence of traffic accidents. Moreover, the serious damage of ACL can also affect the function of knee-joint. Therefore, the demand for ACL repair materials and reconstruction are increasing dramatically. Silk fibroin(SF) has good plasticity and biocompatibility, overcoming early graft immune rejection problems in migration process. Therefore, regenerated SF scaffolds are recognized as ideal materials for ACL repair in clinical applications. In this study, the composite degradable SF scaffolds with regenerated SF fibers and native silk was studied, which combined the degradable property of regenerated SF fibers and excellent mechanical property of native silk.In this paper, our work found a novel solvent system(Ca Cl2-formic acid, Ca Cl2-FA) for silk dissolution at room temperature, more importantly, which characterized by preserving the natural fibrillar structure. At the same time, regenerated SF fibers are prepared by wet-spinning and electrospinning, respectively. The effect of dissolved parameters on the effect of SF fibers solubility and analyzes the solution properties of SF to influence the regeneration process, the morphology and mechanical property of regenerated SF fibers are disscussed. Based on these researches, the degradation and biocompatibility of regenerated SF fibers are confirmed by vitro degradation and cell culture experiments. The higher performance materials are prepared by using the fibrillar structure of SF in solution, which is obtained through Ca Cl2-FA dissolution method. Therefore, artificial ACL is constructed by using the three kinds of materials containing SF filaments, regenerated SF nanofibers, and natural SF fibers. This method is provided a new way for SF-based ACL reconstruction.Unfortunately, the present solvents(Ca Cl2-C2H5OH-H2O) for silk dissolution share a common feature of dissolution at the molecular level, which leads to the complete destruction of the silk hierarchical structure and noticeable degradation of the fibroin molecules. Moreover, the traditional dissolution methods need higher salt solution, longer dissolve time and higher temperature. Fortunately, the Ca Cl2-FA dissolution method can dissolve silk at room temperature and characterized by preserving the natural fibrillar structure and removing the β-sheet crystal structure. In the dissolution process, with the solvents enter the inner of fibers, fiber expansions and volume increases by hydration of polar ion, the hydrogen bonds between molecules are weaken, the β-sheet structures are destroyed. Silk is disassembled into fibrils in Ca Cl2-FA solution instead of down to the isolated fibroin molecules as is the case in traditional solvents. At the same time, the fibrillar structure of SF is clearly observed in SF solution and its formed films. The dimensions of fibrils show a trend of decreasing with the increase of Ca Cl2 concentration and dissolved temperature. The viscosity of SF solution, prepared by Ca Cl2-FA dissolution method, is higher than solution prepared by traditional method. The solubility of SF fibers is increased with the increase of Ca Cl2 concentration and dissolved temperature. Additionally, regenerated SF fibers prepared by Ca Cl2-FA solution is insoluble, FTIR and XRD results depict the secondary structure of SF materials is transferred from random coil to β-sheet, when is treated by deionized water.After wet-spinning, these fibrils in solutions easily re-assembled into fibers after physical shear and coagulation in water. Furthermore, the molecular orientation and mechanical property of SF filaments are improved by post-drawing treatment. In our experiment, SF solution with 15.0 wt.% had better spinnability. After drawing 4 times, the diameter of spinning fiber is 10.8±1.4 μm, and its breaking strength and elongation at break reaching 358.4±43.2 MPa and 24.8±4.2 %, respectively, is improved obviously relative to spinning fiber. More important, the coagulation bath of water has the advantages of inexpensive, simple in operation and environmentally friendly.Electrospinning is employed to prepare electrospun nanofibers by dissolve SF films prepared by Ca Cl2-FA redissolved in FA. The spinning solution preserved fibrils improved that the fibrils are stable in FA solution. The custom-made cylinder collector is used for electrospun nanofibers collecting to obtain oriented fibers. The structure of electrospun nanofibers is similar to extracellular matrix(ECM). The influence of Ca Cl2 and SF concentrations on fiber forming is investigated. In addition, the influence of speed of cylinder and stretching on fiber orientation is analyzed. The results show that SF spinning is smoothly done at the condition(SF concentration 6.0 wt.%, Ca Cl2 concentration 5.0 wt.%, and roller speed 1000 rpm). The nanofiber diameter is 221±20 nm. After drafting 1 time, the oriention of fibers are improved significantly, and the breaking strength and elongation at break of nanofibers are 18.6±3.8 MPa and 15.1±2.5%, respectively, is higher than fibers prepared by Ca Cl2-C2H5OH-H2 O dissolution method.Regenerated SF fibers show a slight and slow weight loss with time in PBS solution, after 60 d degradation, the weight loss of nanofibers and filaments is 15 % and 8.9 %, respectively. Nanofibers adhensive each other a little, and there is fition on the surface of filaments, which only a little degradation happened on the fiber surface. However, the severe and rapid weight loss is observed in protease XIV solution. Little nanofibers are existed after 48 h degradation, and the weight loss of filament is nearly 61.5 %, most of the filaments are break down. Compared with disordered electrospun nanofibers, cells are quickly proliferated on oriented electrospun nanofibers. Moreover, SF filaments and natural SF fibers have excellent biocompatibility.Based on the high performance regenerated SF fibers, in this paper, composite SF ligament are constructed by using vertical spindle knitting machine, which is containing three layers: SF filaments, natural SF fiber braid fabrics, and electrospun SF nanofibers. The breaking strength of composite SF ligament is reached 2581.7±23.7 N, meeting the ACL demand of human body. SF nanofibers with different diameters have exhibited different degradation rates in protease XIV solution. Regenerated SF materials with rapid degradation rate provide favorable growth space for cell proliferation, however, natural SF fibers with lower degradation rate provide the mechanical properties of ACL materials. Finally, it realizes the design requirement for ACL repair materials with different degradation rate.Through above researches, this paper clarifies a novel dissolved method for producing regenerated SF materials from Ca Cl2-FA solution. A simple and efficient method for constructing silk scaffold with different fiber scales is presented. It exhibits superior mechanical properties, biocompatibility, and biodegradability. Therefore, this novel SF-based scaffold material can be used in the application of ligament tissue engineering. At the same time, this research may provide theoretical support and experimental data for the preparation of ACL repair materials.