Neotype Peptide-based Scaffolds For Nerve Tissue Engineering And NSCs Growth And Differentiation Induced With This Scaffolds Surface In Vitro

Part one Isolation, Culture and Identification of the Neural Stem Cells from the Neonatal MiceObjectives:①To establish the methods of in vitro culture and identification of neural stem cells isolated from the cerebral cortex of neonatal mice (1-3d).②To approach the characteristic of the proliferation and differentiation of neural stem cells cultured in vitro,as a fundamental research for neural stem cell transplantation. Methods: The cerebral cortexes of neonatal mice were dissociated mechanically. Cells were cultivated in serum-free culture medium by suspension culture technique containing DMEM/F12, growth factors,bFGF and/or EGF,addB27 in order to acquire large numbers of self-proliferating cell clones,cells were collected.and incubated at 1×107/L,1×108/L,1×109/L,1×1010/L. obseve the cells morphous underinverted phase contrast microscope. 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:①Cells which were dissociated from the cerebral cortex of neonatal mice into floating neurospheres in the serum-free medium DMEM/F12 with the presenceof bFGF and/or EGF had potential of self-proliferation and formed large numbers of clones named as neurospheres, neurospheres could proliferate and passage in vitro.Immunocytochemistrystudy indicated that the neurospheres wereNestin-positive and could differential with mufti-directions.②MTT assay discover:When the neuraI stem cells planted at the concentration of 1×109/L ,fast grow after 3 days ,the growth rate of the cells was the highest of all concentrations after 1 week.③The 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 Neural stem cells(NSCs)Part two Self-assembly Hydrogel from the Neotype of the Synthetically Tissue engineering PeptideObjectives: The IKVAV-containing oligopeptide was synthesized and fabricated. The mechanical properties and framework of hydrogel self-assembly from the amphiphilic oligopeptide on different conditions was serached.Methods: The peptide, whose molecule weight and purity were detected by MS and HPLC respectively, was synthesized in solid phase methods(shanghai BOOTECH bio-technoloy company limited). 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, or coated in the surface of coverslip and set into the baking oven at 37℃. The self-assembly hydrogel was detected with TEM and SEM.Results: MS showed that peptide MW was 1351.72. HPLC testified that the peptide purity was 95%. The peptide solution was self-supported into hydrogel triggered by DMEM/F12 in several 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 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 network nanofibers, and the nanofibers of hydrogel self-supported from1wt% peptide varied from 3.5 nm to 5 nm at diameter and 95 nm to 1.5 um at lengths, and the nanofibers of hydrogel self-supported from 2wt% peptide closely ranked, and there were big spacing within the thin nanofibers of self-supported from 0.5wt% peptide, The time scanning demonstrated that the loss modulus is nearly linear and much smaller than the storage modulus, indicating that the resulting hydrogels is heavily and that are formed of sufficient rigidity for neural stem cell seeding.Conclusions: The peptide was synthesized and design successfully into porous hydrogel bioactive spinal cord tissue engineering scaffolds which was triggered by DMEM/F12.Part three the Growth and Differentiation Effects of NSCs on the surface of Self-assembled PeptidesObjective: NSCs cultured in vitro were implanted in the surface of porous gels that were self-assembly from IKVAV peptide. The effects of gel on the growth and differentiation of NSCs was serached.Methods: NSCs gained from the cerebral cortex of neonatal mice were mechanically dissociated and cultivated with suspension in serum-free media methods, which was identified by the new immunohistochemistry SABC methods. The peptide in NaOH solution where PH was amount to 9.0 and the concentration of peptide was 1wt%. 1wt% peptide spread on coverslip was triggered to be self-assembled by the addition of DMEM/F12, which was detected by TEM. In experimental groups: NSCs were transplanted into the surface of gels; in contrast group: NSCs were grown on the surface of coverslip covered with polylysine. Growth and Differentiation of Neural cells implanted in experimental groups and contrast group was observed by IPCM. NSCs which were drumed with Immunocytochemistry methods, were detected by LCM.neurospheres were transplanted into the surface of coverslip covered with polylysine.when neurospheres induction differentiation according to these groups:(1) 5μmol/ml Forskolin,20ng/ml bFGF,20ng/ml EGF 200 ng/ml HRG, abbreviate combination factor group;(2) 10%serum, abbreviate serum group;(3)20 ng/ml bFGF,NSCS culture medium, abbreviate bFGFgroup.Above all groups culture medium are DMEM/F12+B27 additives counting the neuron with MAP2,and counting the total cells with Hoechst33342+ccell mark.the differentiation rate of neuron= MAP2+ cells/Hoechst33342 mark cells analyze the MAP2+ cells cell body area, perimeter,and cells the length of the longest synapseResults: NSCs were appreciatied identified as Nestin-positive NSCs with new SABC immuno-histochemistry methods. TEM detected that the self-assembly gel was constructed of several network nanofibers. The NSCs extrusited process after 7 days, detected by IPCM in experimental groups, only undifferentiated neurospheres were found in contrast group NSCs which were differentiated into NF-positive neurons with powerful red fluorescence and GFAP-positive astrocytes with weak green fluorescence in experimental groups and Nestin-positive neurospheres with green fluorescence in contrast group were examined by LCM.combination factor group :immunofluorescence display: MAP2+ cells transference follow the S100+synapse,growth and differentiation periphery,after induction 10days, MAP2+ cells erupt more synapse, in those cells display TH+. serum group: immunofluorescence display: lenient and applan cells present S1O0 hadro-masculine. Hoechst33342 stained display: big cell nucleus, less MAP2+ cells,round and well-stacked cell body.bFGF group: immunofluorescence display: after induction 3～5days, present S100+,big cell body, thickness and short synapse.Conclusions: NSCs were cultivated by mechnical isolated. TEM showed that gel comprised of network nanofibers. The porous gels were cytocompatible to NSCs which were differentiated into neurons and astrocytes on the surface of the gels. It was confirm that self-assembly2-Dgels were acted as a nerve tissue engineering scaffolds.the growth and differentiation of NSCS in combination factor group, serum group, bFGF group present significant contrast.