Growth And Migration Of Neural Cells Along Non-Woven Nanofibers Of SF And TSF

The diseases and injuries of central nervous system (CNS) currently become highest fatal in the world. Compared to the peripheral nervous system (PNS), the regeneration capacity of CNS is poor because of the formation of glial scar and the role of the growth inhibitor. Clinical treatment methods mainly confined to the autologous and allogeneic transplant to spur the regeneration of the nerve axons, but as we all known the future is not optimistic. With the development of biotechnology, genetic engineering and biological tissue engineering have begun to use in the treatment of nerve injury, including use of biocompatible materials to support the regeneration of CNS. The biocompatility of silk protein with abundant amino acids have been proven in previous works. Here, we seeded astrocytes, neurons and neural stem cells (NSCs) on regenerated silk nanofibers scaffold substrates to detect the possibility of the application of the silk fibers on the regeneration of CNS.In our study, we successfully cultured the astrocytes, neurons and NSCs in vitro. Meanwhile, the shapes, growth characteristics and specific antigen expression (GFAP, NCAM,β-III-Tubulin and Nestin) have been identified on each of the cells. The results showed that: (1) Astrocytes were purified through the repeated passage; (2) Neurons with the typical characteristics of cells have obvious neurite and better growth state in vitro; (3) Primary NSCs with two cultured methods (suspension and adherent culture) can maintain undifferentiated state with serum-free medium. Meanwhile, cells can differentiate into neurons and glial cells under serum medium.In the next experiment, we applied silk non-woven nanofibers from Bombyx mori (SF) and Tussah silk (TSF) as the cells substrates and analyzed the behavior of differentiated neural cells and neural precursor cells grown on these nanofibers. Results showed that neural cells displayed a high affinity to silk nanofibers: they grew and differentiated in paralelle along the fibers and exhibited a well-arranged specific antigens expression. When part of the cell bodies was in a direction perpendicular to the nanofibers, sharp turns were observed, forming a clear border between cells and surroundings where the fibers were possibly enwrapped. Moreover, time-lapse video analysis showed that astrocytes randomly migrated the first several hours after plating. Once they came in contact with a fiber, they kept migrating by using the fiber as a rail. Meanwhile, the cells migrated from the neurospheres also go along the nanfibers with differentiating. Taken together, these data indicate that, not only do the regenerated silk fibroin nanofibers control the growth of neural cells but guide and direct the migration of these cells as well, and thus making these materials the promising candidates for therapeutic approaches to promote the regeneration of CNS after injury.In order to support the result that the biocompatibility of silk nanofibers and neural cells, we also cultured the brain slices on silk nanofibers and detected the attachment and activity of slices using live, and dead assay. Compared to the control groups, we can see that the brain slices on silk fibroin nanofibers shown better biocompatibility, and neural cells migrated from the slices moved along the fibers.We concentrated on the migration and spreading, in addition to the attachment, viability and proliferation of these cells. Our long-term goal is to identify the optimum conditions in the context of the properties of the nanofiber substrates that may allow for the manipulation of astroglial cells and ameliorate the nonpermissiveness of the CNS injury sites upon implantation. To our knowledge, this study is the first to evaluate the interactions of astroglial cells with TSF and SF nonwoven nanofibers. And we are convinced that the non-woven nanofibers of silk fibroin have the potentiality to support nerve regeneration and will be an alternative in nerve tissue engineering.