| Nerve regeneration in the central nervous system (CNS) is more challenging, with the inhibitory environment formed after injury in the CNS often restricting nerve regeneration. In particular, the formation of a disorganized reactive astrocytic scar and the depositon of axon growth repulsive extracellular matrix (ECM) molecules in and around the lesion present a potent physical and molecular barrier to axon regeneration. Attemps to bridge the inhibitory environment of the lesion have included the implantation of oriented growth-promoting tissues and oriented biomaterials that are capable of supporting directional axon regeneration and tissue repair.Silk fibroin has been increasingly studied for new biomedical applications due to the biocompatibility and slow degradability. The most widely studied silks are cocoon silk from the silkworm Bombyx mori and dragline silk from the spider Nephila clavipes. Structurally, silk fibroins from these species are composed of eighteen short side-chain amino acids. However silk fibroins from the modification of native silk fibroins are beginning to provide new options to further expand the utility of silk fibroinbased materials for medical applications. Altering the compositon and processing parameters of silk fibers maybe make cell adhesion, survial and proliferation differently. Therefore the objective of this study was to determine how diameters of silk fibroins affect morphology, orientation, proliferation and migration of astrocytes and neurons.In this experiment, we cultured astrocytes, neurons, and neural stem cell in vitro. The results showed that: (1) A population of purified cortical astrocytes were obtained from 1-2-day-old Sprague-Dawley rat. Over 90% of cells cultured under these conditions were selectively labeled by GFAP antibody confirming their astrocyte identity. (2) Cells were isolated from the SVZ, purified using Percoll gradient centifugation and exhibited mature multipolar morphology during the first three days in vitro. (3) Suspension culture of neural stem cells formed high refraction character and well-defined spherical shape with uneven external rims. Also to indentify the multiple differention ability of NSCs, we determined their phenotype immunochemically using antibodies to different marker proteins.Furthermore, we planted the seeding cells on diameters of silk fibroins to analyze the behavior of differentiated and undifferentiated neural precursor cells on silk nanofibers. (1) Silk nanofibers can support the adhesion, development and migration of astrocytes. Cell proliferation and extension on 400 nm substrate were significantly different from those on 1200 nm substrate by day 6 (p<0.05). Three diameters of materials showed similar process length. (2) Neurons display a high affinity to silk nanofibers. Neurons on 400 nm substance exhibited a higher dendritic complexity than cells on 1200 nm. (3) However neurons can not prolife in vitro, we cocultured astrocytes and neurons to support neuronal growth. Numerous in studies have demonstrated that astrocytes can stimulate the survival, differention and regeneration of neurons. The coculture results showed small diameter 400 nm of silk fibroin make higher complexity during the experiment. (4) Meanwhile, neurosphere which cultured on the silk nanofiber also maintain the characters of NSCs. Neurospheres on 400 nm fibers can make larger spreading area and the maximum migration length compare with other fibroins during 24 h and 48 h.This research provides the insight that the diameter of SF is a promising materials for therapeutic approaches to promoting the regeneration of CNS after injury.
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