| Neurological diseases(NDD)occurs in the central nervous system,the peripheral nervous system,the autonomic nervous system with sensory,motor,consciousness,autonomic nervous dysfunction as the main performance.The main reason is that the neuron damage or loss in the injured site,and the nerve impulse cannot complete transmission.Neurodegenerative diseases are common clinical diseases,and there are about 5 million new cases every year in China alone.As aging intensifies,the treatment of neurodegenerative diseases is also urgent.Many articles have long reported the significant role of mesenchymal stem cells in repairing neurological diseases.However,due to difficulties in operation and the immaturity of related technologies,we have not yet found significant therapeutic effects.The most effective treatment for nerve injury is nerve tissue repair and reconstruction.Tissue engineering is to combine the principles with technologies of life science to inoculate stem cells on biological materials,and to realize the reconstruction of the structure and function of tissue defects by regulating stem cells on scaffold materials.As three elements of tissue engineering,seed cells can provide cell sources,scaffold materials can provide spatial support for the growth of seed cells,and growth factors can regulate the growth and differentiation of seed cells.The coordination of these three factors will promote the application of tissue engineering in nerve repair.Mesenchymal stem cells from a wide range of sources have the most potential applications in neural tissue engineering.Most of the existing methods for inducing neural differentiation of mesenchymal stem cells have disadvantages such as difficult operation,complex mechanism and short survival time of induced nerve cells.Electrical stimulation in physical factors has been proved to be used in the treatment of neurological diseases,but the existing treatment methods require external power supply or equipment,complex and inefficient operation,and cannot be widely used.In order to make stem cells efficiently differentiate into functional nerve cells in the nerve defect area of the body,we need to use nerve scaffold materials to build Bridges between nerve breaks,support and fix seed cells,and guide and promote the growth of axons.Therefore,combining with neural scaffold materials,exploring methods that could accelerate the rate of neural differentiation and improving the quality of neural differentiation of stem cells has become another research hotspot in neural tissue engineering research.Due to the electrical activity of neurons,electrically conductive biomaterials combined with electrical stimulation as a new thought in neural differentiation of mesenchymal stem cells have been paid more and more attention in recent years.The inconvenience or secondary injury caused by external wire makes the need of in-situ wireless electrical signal input more urgent in vivo.And external stimuli can be converted into electricity,such as electromagnetic induction,in which a conductor produces induced electromotive force and induced current when it cuts magnetic induction line.How to efficiently convert mechanical energy into electrical energy?That requires conductors with good conductivity.At present,graphene,which is a two-dimensional material with single atomic layer composed of carbon atoms hybridized by SP2 and connected by covalent bonds,is known as the best electrical conductivity.It has attracted great attention in the field of biomedical science,including biosensors,drug delivery,et al.Moreover,it has proved that graphene-based materials have good biocompatibility and can be degraded in vivo through the degradation of peroxidase(MPO)in the human body,and the degradation products are non-toxic and have no any side effects,indicating the prospect of biological safety and clinical application.Recent studies have shown that using an external electricity to give graphene foam an appropriate amount of electrical stimulation(10?A current,30 minutes everyday and for 21days)can induce human neuron precursor cells to differentiate into neurons.Based on this report,we infer that cutting the magnetic line of induction with graphene-based materials with good biosafety and electrical conductivity may induce the electrical signals generated by stem cells to differentiate into nerve cells.In summary,this thesis utilize a rotating magnetic field as a driving source,uses the magnetic-electric coupling effect between graphene-based nanomaterials and magnetic wire cutting,realizing electromagnetic induction driving graphene mediated by conducted pulse electrical stimulation or induced electromotive force to induce mesenchymal stem cells to differentiate into nerves;we propose to use graphene as a medium to transform the external magnetic field into endogenous electrical signals,and realize the radio stimulation of stem cells through magnetoelectric coupling conversion,and to study the regulation mechanism of such signals on differentiation of stem cell,which may fundamentally solve the neural tissue damage.It not only has important innovative value in theory,but also may produce important social and economic benefits.In this paper,two-dimensional nanomaterial graphene was used as the environment for cell attachment,and its excellent electrical conductivity and other advantages were used as the medium for converting external magnetic field into endogenous electrical signals to achieve radio stimulate stem cells,so as to regulate cells differentiation.The main works of this paper mainly includes the following two parts:1.Using the principle of electromagnetic induction to explore the differentiation of m BMSCs induced by magnetic field mediated micro electric field on graphene substrate:The 2D-graphene materials have good electrical conductivity and biocompatibility,and have attracted much attention as neural interface materials in biomedical field.In this experiment,graphene film was synthesized by chemical vapor deposition.In order to make it easy to transfer and achieve the purpose of cell inoculation,we used polydimethylsiloxane(PDMS)with excellent physical and chemical properties,hardness and good biocompatibility as the substrate to drop on the surface of graphene film to modify the material.The prepared PDMS/graphene film substrate not only has excellent conductivity,but also has smooth graphene surface.After oxygen treatment with plasma,it also has high hydrophilicity.Using rat bone marrow mesenchymal stem cells(r BMSCs)as seed cells in tissue engineering,the adhesion,proliferation and neural differentiation of r BMSCs on PDMS/graphene film substrate were studied.The results showed that the biocompatibility of PDMS/graphene film substrate was good.Compared with the cells seeded on glass(TCP)and PDMS substrate in the control group,the adhesion and growth of r BMSCs were not affected.On this basis,we carried out the cytoskeleton staining and the live/dead staining under the condition of magnetic field treatment,and found that the morphology of cells changed and appear axon-like structure.Subsequently,real-time quantitative PCR was used to analyze the expression of neural related genes on the third,fifth and tenth day after the cells were grown on different substrates with magnetic field treatment.It was found that only the cells seeded on PDMS/graphene substrate,under the action of electromagnetic induction induced micro current,the neural stem cell specific markers nestin,MAP2 gene,which expressed predominantly in the cell body and dendrites of neurons and type III?-tubulin(Tu J1)and glial fibrillary acidic protein(GFAP)increased significantly.Especially the expression of MAP2 and Tu J1 was obvious.On this basis,in order to make the results more intuitive,we carried out the immunofluorescence staining of the four specific genes at the protein level.Then we found that only the cells seeded on PDMS/graphene substrate could be stained with specific dye under magnetic treatment.In the immunofluorescence staining image of nestin and MAP2,stem cells seeded on the PDMS/graphene substrate could be stained with red and green dyes representing nestin and MAP2 respectively on the fifth day under the treatment of rotating magnetic field,indicating that stem cells at this time contained nestin.With the extension of the experimental days,the ratio of cells stained with green dye increased,indicating that more r BMSCs contain MAP2 gene and more r BMSCs differentiate into neurons.In the same way,in the experiment of immunofluorescence staining of Tu J1 and GFAP,we can get that the stem cells inoculated on the PDMS/graphene substrate were stained with red(Tu J1)and green(GFAP)dyes after magnetic treatment for five days,indicating that the stem cells had begun to differentiate at this time.With the extension of experimental time,the ratio of cells dyed red became more,indicating that under the condition of magnetic treatment,the r BMSCs inoculated on the PDMS/graphene substrates were more differentiated into neurons.These results indicate that the micro electric field produced on graphene substrate by electromagnetic induction can induce the differentiation of r BMSCs.2.The fate of human adipose derived mesenchymal stem cells(h ADMSCs)is regulated by electrical signal mediated by graphene film induced by electromagnetic induction;In order to further explore the induction current generated by graphene under rotating magnetic treatment can drive neural differentiation of h ADMSCs,we got ADMSCs and carried out the following research:(1)Graphene with a thickness of about 400nm was prepared by chemical vapor deposition(CVD).PDMS was fused and dropped onto the surface of graphene.After solidification,it was transferred to a mixed solution of Fecl3-6H2O and diluted hydrochloric acid for etching to obtain PDMS/graphene film substrate.The materials were characterized by SEM,AFM,XRD and Raman,and the induced current of 10?was measured by nanovoltmeter;(2)The substrate of PDMS/graphene film has good cell compatibility.the cell counting kit-8(CCK-8)and the live/dead cellular staining were performed to confirm that h ADMSCs survived well in the condition of no magnetic field treatment.And on the third day of culturing cells,the death rates were 97.8%,98.0%and 96.2%respectively seeded on glass cover glass(TCP),PDMS substrate and PDMS/graphene film substrate.the CCK-8 assay also showed that the growth status of h ADMSCs was not affected.The image of cytoskeleton staining and SEM scanning also showed that there was no difference in cell morphology and the cell adhesion was similar.It shows that the substrates have good biocompatibility.(3)In addition,in order to further verify whether the graphene film substrate can induce the neural differentiation of h ADMSCs under magnetic treatment,an external magnetic field was constructed to simulate the effect of magnetic stimulation on the differentiation of ADMSCs in vitro.The results of cytoskeleton staining and CCK-8measurement showed that the cultured stem cells showed different growth status and proliferation trend compared with the control group.The number of stem cells decreased and the morphology of stem cells also appeared neuron like,which made us further carry out the experiment of neural differentiation.The results of PCR showed that nestin,MAP2,Tu J1 and GFAP were expressed in the cells seeded on PDMS/graphene substrates with magnetic field treatment.In particular,when the cells seeded on PDMS/graphene substrates were cultured for 10 days under rotary magnetic field,the expression of nestin,MAP2,Tuj1 and GFAP were 180,165,90 and 275 times of those in the control group,respectively.Immunofluorescence staining results showed that under magnetic treatment,graphene film mediated micro electric field could promote the expression of nestin,MAP2,TuJ1 and GFAP proteins in h ADMSCs.However,with the prolongation of treatment time,the expression of specific protein representing neurons became more prominent and gradually stabilized.In addition,under the stimulation of neurotransmitters gamma aminobutyric acid and dopamine,these hADMSCs derived neurons showed strong calcium sparks,which indicated that the micro current generated by graphene based electromagnetic induction could induce cells to differentiate into functional neurons.From the above results,we can conclude that 300rmp/min rotating magnetic field can make the adipose derived mesenchymal stem cells on graphene film differentiate into neurons.The purpose of this work is to solve the problems of neurodegenerative disease repair and neural tissue reconstruction at this stage.Using the theoretical knowledge of tissue engineering,we use micro nano materials as the external environment for cell growth,and use the physical principle to construct a simple external field to directly generate internal electrical signals to stimulate stem cells,regulate the fate of stem cells.That provides new ideas for clinical medicine and regenerative tissue medicine... |
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