Self-assembly Hydrogel From The Amphipathic Peptide Scaffolds For NSCs Proliferation And Differentiation In Vitro

Objectives:Amphiphilic peptide contains active ligands self-assembly into porous gel ,when it was mixted with DMEM/F12 containing neural stem cells (NSCs). Investigate the effects of this gel on the growth and differentiation of NSCs.Methods:Neural stem cells harvested from the cerebral cortex of neonatal mice were triturated and cultivated in serum-free media, identified by immunohistochemistry. Experimental group (EG): including a1, a2, A1, A2 sub-groups and b1, b2, B1, B2 sub-groups, 1%wt peptide solution was added into same volume of DMEM/F12 media containing NSCs in a1, a2, A1, A2 and b1, b2, B1, B2 sub-groups, a1, a2, A1, A2 sub-groups were build into three-dimensional(3-D) gel culture system; In b1, b2, B1, B2 sub-groups, neural stem cells were inoculated on the two-dimensional(2-D) gel surface; the control group (CG):containling c1, c2 and C1, C2 sub-groups, NSCs were inoculated on poly-L-lysine surface. Cells in EG and CG were stained with immunocytochemistry methods and observed with Laser Confocal Microscopy (LCM) and Inverted phase contrast microscope (IPCM),the growth of cells were detect with Cell counting kit-8(CCK-8) ELISA and protracted growth curve, the ultrastructural morphology of the gel and cells incapsulated within the hydrogel were observed with Transmission Electron Microscope (TEM).Results:Neural cells were Nestin-positive NSCs. IPCM showed that neural cells formed neurospheres in two days and showed the typical phenotype of neuron seven days later in the surface or within the 3-D gel in EG, and in CG they revealed the neuron-like appearance in c2 and only formed neurospheres in c1. LCM showed that NSCs were differentiated into NF-positive neurons and GFAP-positive astrocytes in EG and in c2 of CG, and they only formed Nestin-positive neurospheres in c1. CCK-8 ELISA method showed that: In the third to seventh days of cultivation , cell proliferation in three-dimensional gel culture systems were more active than others, and the difference was significant (p<0.05);There was no significant difference between A1 and A2 (p>0.05); In the third to seventh days of cultivation , cell proliferation in B1, B2 ,C2 sub-group were more active than C1 sub-group, but there was no significant difference (p> 0.05);There was no significant difference among B1, B2 and C2 (p>0.05).TEM showed that gel was composed of internnective nanofibers, cells encapsulated in gel at 7 days had normal abundant processes.Conclusions:The self-assembly gel scaffolds from the synthetic amphiphile peptide was able to induce NSCs differentiation, and 3-D gel environment more conducive to the proliferation and differentiation of NSCs. It was testified that peptide-based gel was characterized as the top and neotype scaffolds of nerve tissue engineering.