Silk Fibroin Scaffolds With Well-aligned Fibers Loaded With Dual Factors For Peripheral Nerve Repair

Peripheral nerve injuries frequently occur following trauma or surgical procedures and may lead to a serious loss of sensation or motor control even lifelong disability. Various grafts including autograft, allograft, biological tissues and artificial nerve grafts were used for peripheral nerve injuries repairs, but have many limitations. Therefore, developing superior tissue engineered nerve grafts for peripheral nerve repair is very necessory. Silk fibroin, an attractive biomaterial, has good biocompatibility, controlled degradability, nontoxicity and versatile processability in different forms. Thus, silk fibroin has wide applications in the biomedical fields such as bone, skin, nerve and vascular repair. However, silk fibroin-based conduits achieved finite success for nerve regeneration compared to autograft.In this thesis, two silk fibroin scaffolds were prepared by traditional and coaxial electrospinning using regenerated silk fibroin(RSF) aqueous solution for nerve regeneration. To get the aligned scaffolds, the electrospun fibers were collected on a roller at a rotating speed of 2000 rpm. The morphology, structure, performance of the scaffolds were characterized by scanning electron microscope(SEM), wide Angle X-ray diffraction(WAXD), material testing machine, respectively. SEM images indicated that the aligned RSF fibers were distributed throughout the scaffolds with uniform fiber diameters. The diameter distribution of the fibers in traditional electrospun scaffolds was quite narrow and the average diameter of the RSF fibers was 1.8 ± 0.5 mm. The RSF fibers were typically cylindrical with smooth surface. The average diameter of the fibers in coaxial electrospun scaffolds was 2.5 ± 0.7 μm. Moreover, some uneven and ribbon-shaped silk fibers were observed. WAXD results showed that after water vapor annealling, the crystallinity of the traditional and coaxial electrospun RSF scaffolds were up to 57% and 53%, respectively. The stress-strain curves of the two types RSF scaffolds showed significant differences between parallel and perpendicular directions to the aligned fibers. The traditional RSF scaffolds had a strong tensile strength of about 12.3 MPa and a low elongation at break of 1.5% at the parallel direction. At perpendicular direction, the scaffolds had poor mechanical properties with a tensile strength of 3.4 MPa and an elongation at break of 1.0%, respectively. In the case of the coaxial electrospun RSF scaffolds, a strong tensile strength of about 9.8 MPa and a relatively low elongation at break of 1.4% were exhibited at the parallel direction. At the perpendicular direction, however, the coaxial scaffolds showed a weak tensile strength of 2.1 MPa and an elongation at break of 1.2%.To boost nerve tissue regeneration, brain-derived neurotrophic factor(BDNF) and vascular endothelial growth factor(VEGF) were simultaneously loaded into scaffolds for sustained releasing. On the one hand, BDNF was used to maintain neuronal survival and promote nerve regeneration. On the other hand, VEGF as a potent angiogenic growth factor has ability to increase vascular permeability and induce angiogenesis. When the two factors were encapsulated in silk-based scaffolds, the bioactivity of BDNF and VEGF can be maintained and site-specific delivery can be provided. An ELISA assay measurement of two factors from the RSF scaffolds in vitro demonstrated that the release of the dual factors from the scaffolds was well maintained up to 2 weeks. The cumulative release of BDNF and VEGF approached about 15.9% and 11.9% of the drug loading capacity over 16 days, respectively.Schwann cells(SCs) morphologies, viability and proliferation on RSF scaffolds were obtained using SEM, laser scanning confocal microscopy(LSCM) and MTT, respectively. The scaffolds were implanted in a mouse model to support nerve regeneration and angiogenesis in vivo, which was evaluated by histological and immunohistochemical(IHC) analyses. All retrieved scaffolds were evident without chronic inflammatory responses. Compared to the bare scaffolds, the dual-factor loaded scaffolds significantly promoted nerve regeneration and possessed great potentials in peripheral nerve repair, regeneration and other tissues repair.To boost the synergistic effect, it is necessary to investigate how the release order, dose and time of the double factors affect the nerve regeneration efficiency. Inner-VEGF/Outer-BDNF Coaxial Scaffolds(IVOB) and Inner-BDNF/Outer-VEGF Coaxial Scaffolds(IBOV) were prepared by simply switching the position of BDNF and VEGF for either core or shell domain using coaxial electrospinning to achieve time-programmed dual release formulation. The release of BDNF and VEGF were tested using ELISA kit. As the dual-delivery order is different, the release profiles of the factors from the scaffolds were very unique. The factors in the shell of coaxial fibers showed a higher release rate than that in the core domain. For IVOB, the cumulative releases of BDNF and VEGF close to 9.3% and 7.0% of drug loading capacity, respectively. For IBOV, the cumulative releases of BDNF and VEGF were close to 11.2% and 4.6% of drug loading capacity, respectively. An in vitro assay showed that the IVOB scaffolds obviously accelerated SCs growth, proliferation, and spreading owing to the rapid release of BDNF. However, in vivo subcutaneous implantation in mouse back demonstrated that IBOV scaffolds facilitated significantly more angiogenesis in the early days, and promoted subsequent more nerve regeneration based on great benefit of angiogenesis. Results showed that dual-delivery order of VEGF and BDNF was quite important to promote peripheral nerve regeneration. And the delivery system of multiple drugs possessed great potential in improving the drug efficiency and reducing the associated side effect of drugs.