| Spinal cord injury (SCI) is a kind of severe lesion of central nerve system. In China,there are at least tens of thousands of people suffered severe SCI due to fall wounds, trafficaccidents, etc every year. Unfortunately, reconstruction of the SCI is still far from satisfied.The reconstruction of the SCI is one of the difficulties and hot spots in regenerativemedicine. Nerve autografts have always been considered as the golden standard for SCIrepairing recently. However, application of autografts is limited by donor shortage,structure difference and secondary surgery. Thus, to substitute the autografts and recoverthe structure and function of SCI, design of bioactive artificial grafts with SCIreconstructive activity and controlled biodegradability is the urgency need in nerveregeneration research. Many studies have initially confirmed that silk fibroin (SF) has ahigh potential for nerve tissue regeneration. It will meet the needs for nerve tissuereconstruction better if its ability to regulate cell behavior and guide newly tissueformation is enhanced. Accordingly, by a directional temperature field, SF scaffolds withoriented multichannels were prepared freeze drying. The influence of preparation conditionon the structure of SF scaffolds and mechanism of oriented multichannels formation havebeen studied. Influence of oriented multichannels of SF scaffolds on the behavior ofprimary hippocampal neurons has been investigated. Further, the activity and mechanismof oriented multichannels SF scaffolds for SCI repairing have been evaluated in vivo.Firstly, oriented ice crystals were formed by directional temperature field freezing.When the ice crystals sublimated, parallel multichannels were remianed. Interestingly, amyriad of ridges on the inner surface of the multichannels were formed during the process.The scaffolds with high swelling ratio and good pore interconnection whose average porediameters decreased from238.49μm to40.29μm and the porosity decreased from93.13%to54.29%with the increase of SF concentration in scaffold cross-sections. In the processof uniaxially freezing of the SF solution, the directional growth of ice crystals had a stressforce on the SF molecule segments and lead to the SF solution concentration, thus resultedin the aggregation of SF segments followed by binding and crystallization in new sites.Therefore, the β-sheet content in the SF scaffold was increased. Changing the distribution of chemical potential of SF solution, the ice crystal sizes were adjusted and the diameterand shape of the multichannels were controlledBesides, the surface of SF scaffolds with oriented multichannels was graft withlaminin. The primary hippocampal neurons were seeded in these SF scaffolds followed byimmunohistochemistry evaluation. The results showed that the morphology of the neuronswas normal and markers of neurons were expressed extensively. The newly formed axonsof the neurons grasped to the ridges and extended along the direction of the multichannels.The ridges and channels could guide the adhesion, migration and extension of the neurons.Further, the three kinds of SF scaffolds were implanted into the T8-T10SCI of SD ratin vivo. Hematoxylin/eosin (H&E) staining indicated that inflammatory cell infiltrationwas not observed, the immune response and inflammation reactions were mild in all thescaffolds70days post-surgery. The repairing cells could migrate into the scaffolds andspinal cord tissue was constructed. Modified SF scaffolds with oriented channels weremore competitive in supporting the regenerated axons through the defect area andvascularization in the scaffolds. The results of BBB score of different scaffolds inreconstruction of SD rat SCI demonstrated that the score of scaffolds with orientedchannels were higher than scaffolds with non-oriented pores (P<0.05).Finally, to study the SCI repairing mechanism of the scaffolds, kinds of markers indefect area were tested and analyzed by immunohistochemistry. The expressions of GAP43,NF, NSE and β-III-tubulin in the scaffolds with oriented multichannels were higher thanthat in scaffolds with non-oriented pores (P<0.05), while GFAP was lower than controlgroup (lesions without scaffolds)(P<0.05). These evidences suggested that theimplantation of scaffolds could inhibit the scar formation in SCI lesions, and the scaffoldswith oriented multichannels had a bioactive promotion of axons regeneration and spinalcord reconstruction. The molecular mechanism was that the scaffolds absorbed activecomponents and created a good micro-environment for the repairing cells growth andsecretion of growth factors, then the scaffolds with oriented channels enriched growthfactors and guided the extension of axons, neo-tissue formation and SCI regeneration.In this study, the formation mechanism of oriented multichannels of SFscaffolds wasilluminated. The ridges on the inner surface of SF scaffolds oriented channels could guidethe cell adhesion, differentiation and migration. The scaffolds with oriented multichannelscould promote the SCI regeneration by adsorbing active components, enriching growth factors, inducing the repairing cells to migrate into the lesions, and then it could act as atemplate to regulate the cells behavior and neo-tissue formation. A novel scaffold wasprovided for SCI reconstruction in this work.
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