Biological Effects Of Neural Progenitor Cell Based On Controllable Graphene Scaffold

Transplantation of Neural progenitor cell(NPC)combined with electrical stimulation is a promising therapeutic strategy for neurodegenerative diseases.The three-dimensional(3D)scaffolds have been developed to effectively address the common limitations in transplantation including indirected flow of cells and poor cell survival.Conductive and biocompatible 3D graphene scaffolds have been expected as NPC scaffolds in transplantation owing to their unique characteristics of enhancing the electrical activity of neural cells,as well as promoting neurite outgrowth and the differentiation of neural stem cell into neurons.However,neurons differentiated from NPC tend to grow along the skeletons in 3D scaffolds and it is difficult to form a dense neural network across gaps.In addition,the size effect of skeletens on NPC behaviors in 3D scaffolds is less reported.In this thesis,an integrated 3D hybrid graphene(3D-HG)that can be precisely controlled in submicron size was designed and manufactured using Micro/Nano technology.The graphene film in 3D-HG can provide the support for neurite bridges across spacings.We investigated the NPC-differentiated neural network in 3D-HG and explored the size effects of skeleten width on NPC using 3D-HG.The prolongation of NPC-differentiated neuronal processes taking advantage of wireless electrical stimulation was also explored.The main content of the thesis can be summarized as follows:(1)Preparation of 3D-HG and its biological effects:3D nickel templates with customized width,length,height,gap and shape were prepared by micro/nano techniques such as magnetron sputtering,ultraviolet lithography,electroplating,plasma etching,etc.The novel hybrid copper-nickel catalyst was formed after high-temperature bonding of copper foil and nickel templates.After graphene growth by chemical vapor deposition,copper and nickel were etched to form 3D-HG with graphene film at the bottom.3D-HG has excellent conductivity and biocompatibility;The NPC in 3D-HG simultaneously underwent rapid changes in calcium transients triggered by the electrical stimulation on 3D-HG,indicating that 3D-HG can effectively transmit external electricity signals to the NPC;The 7-day differentiated NPC could form neural network spanning multiple gaps in 3D-HG;The results of scanning electron microscopy indicated that the neurites could penetrate the graphene film and form a connection with the cells outside the 3 D-HG;The results of immunofluorescence,Western Blot and RT-qPCR experiments on 14-day differentiated NPC showed that the presence of graphene film in 3D-HG significantly promoted the expression of synaptic proteins and facilitated the formation of neural network.(2)Size effects of skeleten width on NPC in 3D scaffold:3D-HG with different skeleten widths were prepared to study the effect of skeleten width on NPC behaviors.After 7 days of proliferation,both the EdU cell proliferation assay and the WST-1 cell proliferation assay showed that the skeleton width did not significantly affect the NPC proliferation when the width exceeded the cell size.After 7 days of differentiation,the results of immunofluorescence experiments showed that the width change did not cause a significant difference in the astrocytic differentiation rate,but narrower skeletons increased the number of astrocytic processes per cell.Besides,the results of immunofluorescence and RT-qPCR experiments showed that NPC preferred to neuronal differentiation on narrower skeleton.(3)The effects of wireless electrical stimulation on NPC differentiation:The annular graphene was prepared by chemical vapor deposition.Then,the induced current through the graphene could be generated by the change of the outside magnetic field.The magnitude and frequency of the induced current could be effectively controlled by adjusting the alternating current through the conductive coil outside the culture dish.Stimulation of NPC on graphene substrate by induced current not only exerts the advantage of graphene material to promote neurite outgrowth,but also combines the application of electrical stimulation to promote neurite outgrowth,leading to the average length of NPC-differentiated neuronal processes up to 296.4±18.9?m.Novel graphene scaffolds have been constructed through the cross-application of cell biology,physics and materials science.We realized the formation of neural network across spacings in 3D scaffold,and studied the effects of skeleton width and wireless electrical stimulation on NPC.Therefore,this research not only provides powerful tools for exploring the physical effects on cell behaviors,but also provides new opportunities for expanding the application of graphene in tissue engineering.