Preparation Of Neotype Three-dimensional Peptide-based Scaffolds For Nerve Tissue Engineering And NSCs Differentiation Induced With This Scaffolds In Vitro

Part one Isolation, Culture and Identification of the Neural Stem Cells from the Neonatal MiceObjectives: To isolate, cultivate and identify the neural stem cells (NSCs) from the cerebral cortex of S-D neonatal mice (1-3d).Methods: The cerebral cortexes of S-D neonatal mice were dissociated mechanically. Cells were cultivated in serum-free culture medium by suspension culture technique in order to acquire large numbers of self-proliferating cell clones. And cell clones are adherently cultivated to detect differentiated capability. Immunocytochemical method of anti-Nestin, NSE, GFAP was used to identify the cells clones.Results: Cells which were dissociated from the cerebral cortex of neonatal mice and were cultivated by suspension culture had potential of self-proliferation and formed large numbers of clones named as neurospheres, The Anti-Nestin positive cells found. Neurospheres were able to differentiate into nerve cells and glial cells after adherent culture, there were Anti-NSE and GFAP positive cells.Conclusions: The cells isolated and cultivated have potential of self– renewal, self-proliferation, and differentiation into nerve cells and glial cells. And they are the stem cells of central nervous system (CNS). Part two Self-assembly Hydrogel from the Neotype of the Synthetically Amphiphilic PeptideObjectives: The IKVAV-containing amphiphilic oligopeptide was fabricated and synthesized. The framework and mechanical properties of three-dimensional and porous hydrogel self-assembly from the amphiphilic oligopeptide on different conditions was explored.Methods: The peptide, whose molecule weight and purity were detected by MS and HPLC respectively, was synthesized in solid phase methods. Peptide was dissolved in 0.1M NaOH solution. 200 ul of 10,2,1,0.5wt% peptide solution, which were prepared, were added into the same volume of DMEM/F12, or placed into the vapor of 10M HCL(Hydrochloric acid), or coated in the surface of coverslip and set into the baking oven at 37℃. The self-assembly hydrogel was examined with TEM and SEM.Results: MS showed that peptide MW was 1438.31. HPLC testified that the peptide purity was 96%. The peptide solution was self-supported into hydrogel triggered with DMEM/F12 in few seconds, or the thin hydrogel after two hours in the vapor of 10M HCL, or not hydrogel in the baking oven at 37℃. SEM showed that the hydrogel self-assembly from 10 wt% peptide solution was composed of nanofibers that ranked in layers where there were thick voids. TEM showed that the hydrogel self-assembly from 2,1,0.5wt% peptide solution comprised of woven network nanofibers, and the nanofibers of hydrogel self-supported from1wt% peptide varied from 3 to 6 nm in diameter and 100 nm to 1.5 um in lengths, and the nanofibers of hydrogel self-supported from 2wt% peptide ranked closely, and there were big voids within the thin nanofibers of hydrogel self-supported from 0.5wt% peptide, The time frequency demonstrated that the loss modulus is nearly linear and much smaller than the storage modulus, indicating that the resulting hydrogels is rigid and heavily and hydrogels are formed of sufficient rigidity for cell seeding. Conclusions: The amphiphilic oligopeptide was synthesized and self-organized successfully into porous hydrogel characterized as"intelligent"tissue engineering scaffolds containing the bioactive ligand, which was triggered by DMEM/F12.Part three Effects of Two-dimensional Gel Self-supported from Peptides on the Growth and Differentiation of NSCsObjective: NSCs cultured in vitro were implanted in the surface of two-dimensional (2-D) and porous gels that were self-assembly from IKVAV-containing peptide. The effects of gel on the growth and differentiation of NSCs was studied.Methods: Neural cells harvested from the cerebral cortex of neonatal mice were dissociated mechanically and cultivated in serum-free media with suspension methods, which was identified by the immunohistochemistry SABC methods. The peptide was dissolved in NaOH solution where PH was equal to 9.0 and the content of peptide was 1wt%. 1wt% peptide covered with coverslip was triggered to be self-organized into gel by the addition of DMEM/F12, which was detected by TEM. In experimental groups (EG): Neural cells were transplanted into the surface of 2-D gels; in control groups (CG): Neural cells were grown in the surface of coverslip covered with polylysine. Growth and Differentiation of Neural cells implanted in EG and CG was observed by IPCM. Neural cells in EG and CG, which were stained with Immunocytochemistry methods, were viewed by LCM.Results: Neural cells were identified as Nestin-positive NSCs with immunohistochemistry SABC methods. TEM showed that the self-assembly gel was composed of network nanofibers. The neural cells extended process after seven days, observed by IPCM in EG, however, only undifferentiated neurospheres were found in CG. NSCs which were differentiated into Neurofilament (NF)-positive neurons with strong red fluorescence and GFAP-positive astrocytes with slimly green fluorescence in EG and Nestin-positive neurospheres with green fluorescence in CG were detected by LCM.Conclusions: It was the first time that NSCs were cultivated without the addition of trypsin or EDTA. TEM testified that gel comprised of nanofibers. The 2-D and porous gels were cytocompatible to NSCs which were differentiated into neurons and astrocytes in the surface of the gels. It was demonstrated that 2-D and self-assembly gels were acted as the top nerve tissue engineering scaffolds.Part four NSCs Differentiation Induced Three-Dimensionally by the Self-assembly Hydrogel from Peptide in vitroObjectives: The synthetic amphiphile peptide could be self-supported into 3-D porous gel, when it was mixed with DMEM/F12 containing NSCs. Effects of gel on the growth and differentiation of NSCs were investigated.Methods: Amphiphile peptide was synthesized in solid-phase way. Neural cells harvested from the cerebral cortex of neonatal mice were triturated and cultivated in serum-free media, identified with the immunohistochemistry method. In experiment groups (EG) including in A1 and A2 subgroups: 1wt% peptide solution was added into same volume of DMEM/F12 media containing NSCs in A1 and A2 subgroups. In control groups (CG) containing B1 and B2 subgroups: Neural cells were seeded in the surface of the coverslip coated with polylysine. Cells in EG and CG were stained with immunocytochemistry methods and observed with LCM and IPCM, the ultrastructural morphology of cells incapsulated within the hydrogel were observed with TEM and SEM.Results: the molecule weight of peptide was 1438.31 and the purity was 96%, showed with MS and HPLC respectively. Gel was composed of internnective nanofibers, observed with TEM. 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 B2 and only formed neurospheres in B1. LCM showed that NSCs were differentiated into NF-positive neurons and GFAP-positive astrocytes in EG and in B2 of CG, and they only formed Nestin-positive neurospheres in B1, TEM showed that the cell encapsulated in an IKVAVPA 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. It was testified that peptide-based gel was characterized as the top and neotype scaffolds of nerve tissue engineering.