Guidance Behavior Of Micro/Nano Topography Of Silk Fibroin Materials For Cell Mobility And Spinal Cord Repair

Spinal cord regeneration is a key challenge in regeneration medicine field. The micropatterning techniques are able to manipulate cellular response and guide axon regeneration; therefore, the nerve conduits with micro/nano guidance cues have been suggested for spinal cord repair. However, the development of directional guidance scaffolds depends on the understanding of cell-material interactions, the mechanism on how micro/nano features trigger cell contact guidance and influence their behavior should be clarified. This thesis was focused on the effect of micro/nano topography on cell responses based on 2D silk fibroin(SF) film materials, and further developed 3D multi-channel scaffolds with directional guidance cues for spinal cord injury(SCI) repair.Firstly, microsphere and micropillar pattern were prepared on SF films using polydimethylsiloxane(PDMS) molds, to investigate mesenchymal stem cells(BMSCs) response. The results showed that microsphere pattern promoted cell adhesion and proliferation, whereas the micropillar structure mimicking lotus leaf surface decreased cell adhesion, spreading and proliferation. The transition ability of membrane protrusion from filopodia to lamellipodia played important roles on cell adhesion and spreading. Cell could form lamellipodia from anchored filopodia sites to steer the direction of lamellipodial extension, then further control the direction of cell spreading and migration. Moreover, we unexpectedly observed the effect of micropillar structure on tunneling nanotubes(TNTs) extension. The TNTs could interact with micropillar surface, which provided supporting points for nanotubular bridging. Furthermore, the extension direction of TNTs was affected by the micropillar topography. TNTs were able to connect neighboring cells in a circuitous route rather than at the nearest distance. These results imply that the surface topography of biomaterials have potential influence on cell communication mediated by TNTs.Secondly, microgroove-patterned and aligned micro/nanofiber coated SF films were prepared using PDMS molds and electrospinning, to investigate BMSCs contact guidance behavior on the oriented micropatterns. The results indicated that micro/nanofiber coating significantly promoted cell adhesion and proliferation. Filopodia sensed guidance cues and then steered small lamellipodia advance along pattern, which ultimately resulted in directional cell elongation. These results revealed the critical roles of filopodia-steered lamellipodial polarization on contact guidance.Further, the LN-functionalized aligned SF micro/nanofiber mats were prepared to promote and guide axon regeneration. Alignment and deposition of fibers could be enhanced by adjusting rotation rate of drum. Under the condition of this study, the optimal rotation rate and corresponding surface linear velocity to achieve aligned SF fibers were 2000 r/min and 10.47 m/s, respectively. Furthermore, LN was successfully coupled onto the surface of SF fibers through EDC activation, which promoted the bioactivity of fibers. In vitro results demonstrated that alignment of SF fibers and LN fictionalization could guide and promote axon growth of PC12 cells.Finally, the multi-channel nerve conduits based on aligned SF micro/nanofibers were fabricated and further functionalized with LN for SCI repair. The multi-channel conduits had multi-scale channel diameters ranging one hundred micron to hundreds of micrometers. More importantly, all channels in the conduit possess directional guidance cues. In vitro results demonstrated that LN modification and oriented topography of SF conduits provided biochemical stimulus and physical guidance cues for axon growth. SF conduits were further implanted into hemisection site of spinal cords of SD rats to assess in vivo therapeutic effectiveness and the diffenence between single-channle conduits and multi-channle conduits. Functional measurements were assessed by the BBB tests, histological observation and immunohistochemistry during 12 weeks post-surgery. The results showed that the implantation of the both conduits decreased the formation of scar and cavity in injury sites. Compared to the single-channel conduits, the multi-channel architecture increased specific surface area of lumen, which enhanced the supporting of cell growth, facilitating the entry and growth of tissue repair cells and regenerated axons. The observed functional recovery highlighted the potential advantage of multi-channel guidance conduits. Histological observation and immunohistochemistry results showed that multi-channel architecture accelerated neogenesis and vascularization, and promoted axon entry. Consequently, the better tssue morphogenesis and more neovascularization and myelinated axons were observed in the multi-channel conduits. The in vivo results highlighted the potential benefits of multi-channel SF guidance conduits, which provided physical guidance cues and biochemical stimulus for promoting functional recovery of SCI.