Effect Of Orientation Of Silk Fibroin Nanofibers On The Development Of Neurons

Traumatic injury to the central nervous system (CNS) induces permanent functional deficits through axonal damage, disruption of the well-organized cytoarchitecture of the lesioned white-matter tracts and the presence of an environment that is hostile to axonal regeneration and tissue repair.Attempts to bridge the inhibitory environment of the lesion have included the implantation of oriented growth-promoting tissues (e.g., peripheral nerve) and, more recently, oriented biomaterials that are capable of supporting directional axon regeneration and tissue repair. Recently, attempts to design the ideal scaffold for such bridging strategies have prompted investigations of cell-substrate interactions.In the present study, we seeded astrocytes, neurons and neural stem cells (NSCs) on aligned/random silk fibroin nanofibers scaffold substrates to analyze the possible effect the alignment of silk fibroin nanofibers on the growth of neural cells and the development of neurons, and to detect the possibility of the application of the aligned silk fibroin fibers on the regeneration of CNS. Results showed that silk fibroin nanofibers support the adhesion, development and migration of neurons, astrocytes and neural stem cells. Thse cells display a high affinity to silk fibroin nanofibers, cell bodies and processes following the orientation of the nanofibers.In contrast with cells grown on random nanofibers, astrocytes showed a more elongated morphology, and neurons took an end-to-end configuration with longer length but fewer branches when they were cultured on parallel nanofibers, demonstrating that the alignments of silk fibroin nanofibers affect the growth and development pattern of neural cells. Moreover, migration of cells away from neurospheres was strongly influenced by the nanofiber alignments. Cells following the orientation of the fibers migrated for significantly longer distances than those that attempted to migrate perpendicular to the main axis of nanofiber orientation, resulting in an ovoid distribution of cells around the neurospheres.Due to the nature of the interactions between astrocytes and neurons and neural stem cells in the CNS, astrocytes can potentially be used as biological cues to provide a permissive environment for the regeneration of CNS axons. In addition, extracellular matrix and oriented tissue structures influence cell migration and axon outgrowth in vivo. Guidance of the growth of astrocytes by aligned substrates may, therefore, potentially promote survival and control neurite outgrowth of neurons. To confirm this hypothesis, we co-cultured neurons with astrocytes on the aligned or random silk fibroin nanofibers and analyzed the development of neurons. The results showed that astrocytes combined with silk fibroin nanofibers supported neuronal growth. After 4 days of culture, neurons on astrocytes adopted a typical morphology of spindle-like body with numerous neuritis and had a more complex morphology than those cultured alone on the silk fibroin nanofibers, and again the direction of the neurites were still stickly associated with the orientation of the nanofibers.Taken together, these results indicate that, not only the aligned silk fibroin nanofibers control the growth of neural cells but guide and direct the extension and migration of these cells, and therefore the aligned silk fibroin nanofibers can be promising materials for therapeutic approaches to promoting the regeneration of CNS after injury.