The Effect Of Collegan-carbon Nanotube Composite Film On Embryonic Cortical Neuronal Neurite Development And Neural Stem Cell Differentiation

PurposeStudy and discuss the effect of collagen-carbon nanotube composite film on thedevelopment of cortical neuronal neurite and differentiation of neural stem cells inembryonic rat, which could be used to lay the foundation for building cortex in vitroand repairing central nervous system injury in vivo.Materials and MethodsFirst: The preparement of collagen-carbon nanotube composite film: we chosesingle-walled carbon nanotubes and collagen (type I, from rat tail) as raw materials,and made the preparation of collagen-carbon nanotube composite film by usingultrasonic methods and drying technology.Second: The physics and biological evaluation of collagen-carbon nanotubecomposite film: we performed scanning electron microscope to observe theultrastructure and electrochemical workstation examination to measure its conductivity.Also, we evaluate its biocompatibility on embryonic cortical neurons and neural stemcells.Third: The effect of collagen-carbon nanotube composite film on the developmentof embryonic cortical neuronal neurite in rat. We conducted microtubule associatedprotein2immunofluorescence staining on neurons after culturing for3days in vitro.And we performed morphology observation of cortical neurons by scanning electronmicroscope after culturing for7days as well as neurites’ number and lengthmeasurement by using IMAGE J software after culturing for3days.Finaly: The effect of collagen-carbon nanotube composite film on thedifferentiation of embryonic cortical neural stem cells in rats. We sed embryoniccortical neural stem cells on the surface of the material and cultured in the medium without epidermal growth factor and basic fibroblast growth factor. And we observedthe surface morphology of the stem cells on the material as well as analyzed thedifferentiation ratios of neural stem cells into different lineages of cells after culturing7days in vitro.ResultsFirst: The preparement of collagen-carbon nanotube composite film:a. The collagen (type I, from rat tail) is crystal clear, moderate viscosity and itsconcentration is about3mg/ml.b. There is no aggregation observed visually presence of the mixed solution ofcollagen-carbon nanotube, while large aggregates observed by TEM in the mixedcollagen-carbon nanotube solution of1.5mg/ml and2mg/ml in relatively.c. The collagen-carbon nanotube composite film is visually observed as a highdegree of uniformity and there are no impurities present.Second: The physics and biological evaluation of collagen-carbon nanotubecomposite film:a. Consistent with TEM results, the collagen-carbon nanotube composite filmgroups of1.5mg/ml and2mg/ml concentration emerge more aggregates thanothers.b. Electrochemical workstation conductivity measurement results showed that: theresistance (*m) of different groups of collagen-carbon nanotube composite film（from0mg/ml to2mg/ml）are:9.19x104,14.1,5.77x10-1,1.89x10-3and1.54x10-4.c. Alma Blue biocompatibility test results found that:After culturing7days in vitro, the redox ratio of embryonic cortical neurons (%)on different groups of collagen-carbon nanotube composite film(from0mg/ml to2mg/ml) are:25.69±0.92,28.54±1.05,28.15±0.96,24.27±1.66and24.02±1.21(P<0.01).After culturing7days in vitro, the redox ratio of embryonic cortical neural stemcells (%) on different groups of collagen-carbon nanotube composite film(from0mg/ml to2mg/ml) are:20.89±0.45,25.03±2.78,19.78±1.21,19.35±1.20and 19.42±0.60(P <0.01).Third: The effect of collagen-carbon nanotube composite film on the developmentof embryonic cortical neuronal neurite in rat after culturing3days in vitro.a. The average numbers of neurites per neuron in different groups ofcollagen-carbon nanotube composite film (from0mg/ml to2mg/ml) are:1.17±0.64,1.06±0.25,1.49±0.78,1.04±0.19and1.06±0.35(P <0.01).b. The average lengths (μm) of neurites per neuron in different groups ofcollagen-carbon nanotube composite film (from0mg/ml to2mg/ml) are:56.13±26.88,68.23±48.73,84.48±32.53,73.08±23.74and77.15±27.67(P <0.05).Finaly: The effect of collagen-carbon nanotube composite film on thedifferentiation of embryonic cortical neural stem cells in rats after culturing7days invitro.a. The proportions (%) of MAP-2positive neurons (from0mg/ml to2mg/ml) are:25.69±0.92,28.54±1.05,28.15±0.96,24.27±1.66and24.02±1.21(P <0.01).b. The proportions (%) of GFAP positive astrocytes (from0mg/ml to2mg/ml)are:44.02±0.61,27.59±0.11,32.19±1.23,30.25±2.23and23.89±1.33(P <0.01).c. The proportions (%) of OLIGODENDROCYTE positive oligodendrocytes(from0mg/ml to2mg/ml) are:25.98±11.41,39.78±6.74,35.91±4.64,32.90±7.14and27.07±3.21(P>0.05).ConclusionFirst: The biocompatibility of different groups of collagen–carbon nanotubecomposite film is different. And the0.5~1.5mg/ml groups are relatively better than the2mg/ml group.Second: The effect of different groups of collagen–carbon nanotube compositefilm on embryonic cortical neuronal development is different. The1mg/ml group canpromote the development of embryonic cortical neuronal neurite.Third: The effect of different groups of collagen–carbon nanotube composite filmon neural stem cell differentiation shows that0.5mg/ml and1mg/ml group canpromote embryonic neural stem cells to differentiate into neurons.At the same time, the0.5mg/ml,1mg/ml,1.5mg/ml and2mg/ml groups can inhibit embryonic neuralstem cells to differentiate into astrocytes, and the2mg/ml group is more obvious.Atlast, different groups of materials show no difference on the effect of embryonic neuralstem cells differentiating into oligodendrocytes.