Electrical Signals Generated On 3D Graphene Network Driven By Magnetic Fields To Regulate Neural Differentiation Of Stem Cells

The nervous system,consisting of a complex network of millions of neurons,is responsible for the physiological and cognitive functions of the body.Damage to the nervous system can be fatal and irreversible when injury or degenerative disease occurs.Neural tissue engineering is a promsing straterg to repair neurological deficits and promote neural regeneration,which depends on the interaction of stem cells,nanomaterials and stimuli.Several types of stem cells with different differentiation potential are available for neural tissue engineering,such as embryonic stem cells（ESCs）,induced pluripotent stem cells（i PSCs）,mesenchymal stem cells（MSCs）,and neural stem cells（NSCs）.Autologous nerve transplantation is the gold standard for peripheral nerve regeneration using autologous cells in neural tissue engineering.However,the central nervous system has a limited regenerative capacity and can only self-repair at small scales using limited quiescent NSCs.It is necessary to develop more efficient strategy for large-scale neural dysfunction by explanted stem cells and scaffolds,which brings a new problem about the neural functions of differentiated cells.It should be noted that in addition to physical supporting,bioscaffold materials have been shown to activate signaling pathways and transcription factors involved in nerve proliferation and differentiation.The discovery of bioelectricity in the nervous system has refocused researchers’attention on conductive biomaterials.Graphene,with good electrical conductivity and heat transfer properties,has received a lot of attention in recent years.It is a carbon-based material with a honeycomb-like lattice structure in two dimensions.The aromatic structure’s sigma bond,along with three other carbon bonds,determines graphene’s planarity,unique physical and mechanical properties,large surface area,thermal conductivity,and chemical stability.Graphene is an ideal material for supporting stem cell proliferation and differentiation due to its excellent biocompatibility,flexibility,electrical conductivity,degradability,and unique physicochemical properties.The above-mentioned studies used wire connections to introduce electrical stimulation,which can cause secondary injury or inconvenience to the patient.Is there such a thing as wire-free wire-mediated electrical stimulation?Inspired by the principle of electromagnetic induction and generators,three-dimensional graphene foam（GFs）nanomaterials are placed under a rotating magnetic field to cut magnetic induction line and to produce magneto-electric coupling effects.The study aims to achieve radio stimulation of stem cells by using GFs,a three-dimensional nanomaterial,as a cell adhesion environment and its excellent electrical conductivity as a medium to convert external magnetic fields into endogenous electrical signals,thereby regulating stem cell fate.This dissertation’s specific work is divided into two major sections,which are as follows:1.A magnetic field-driven 3D graphene scaffold mediates radio signals to promote neural stem cell differentiation into functional neurons.The most ideal seed cells for tissue engineering to treat neurodegenerative diseases are NSCs.However,NSCs are slow to differentiate autonomously.Clinical application of NSCs for neural tissue repair can miss the optimal time for treatment.It has been demonstrated that some growth factors are able to accelerate the differentiation of NSCs,but they are costly,have short half-lives,have unpredictable behavior in vivo,and are complex to manipulate.Therefore,this study proposes to use GFs as a scaffold material combined with a rotating magnetic field to generate endogenous electrical signals to promote the differentiation of NSCs to functional neurons.When the rotation rate of the external rotating magnetic field is300 rpm,the magnitude of the electrical signal generated on the GFs surface is about 8μA.Under these experimental conditions,CCK-8 testing and live-dead staining were performed to verify the cytocompatibility of the GFs scaffold material,and cell morphology was observed by cytoskeleton staining.The results demonstrated that the GFs had good biocompatibility and the cellular morphology of NSCs changed significantly under the experimental conditions.Immunofluorescence staining results showed that electrical stimulation enhanced the expression of MAP2 in mature neurons.The feasibility of electrical stimulation to enhance the differentiation of NSCs was further demonstrated.Western Blot for quantitative analysis of protein expression showed the same results as immunofluorescence staining.Magnetic fields drove the GFs surface to generate electrical signals and stimulate cell growth after 7 days.The MAP2 protein expression of cells in the experimental group was significantly higher than that of NSCs grown on the GFs surface without magnetic field treatment;in contrast,the expression of the glial cell-associated protein GFAP decreased by electrical stimulation treatment protein expression,a result that demonstrates that electrical stimulation enhances the directed differentiation of NSCs toward neurons.As a safe and effective way to regulate stem cell differentiation,electrical stimulation has wide application prospects in the treatment of neurodegenerative diseases.2.Wireless electrical signals induce functional neuronal differentiation of BMSCs on 3D graphene framework driven by magnetic field.MSCs are well qualified for neural tissue engineering seed cells.At the same time,MSCs perfectly solve the problem of the scarcity of NSCs.However,there are difficulties in inducing MSCs to differentiate into functional neurons.Previous work has demonstrated that electrical stimulation can enhance the differentiation of NSCs toward functional neurons.Therefore,GFs was used as a scaffold material to mediate local radio signals to induce MSCs to functional neural differentiation.It was shown that currents above 10μA can effectively induce differentiation of MSCs.The electrical signal size generated on the surface of GFs is about 10μA when the rotational rate of the rotating magnetic field is 400 rpm.MSCs cultured on GFs effectively promoted neural differentiation of MSCs by 15 min/day of rotating magnetic field stimulation without the aid of other induction factors.The experimental results demonstrated elevated expression of neuron-related genes/proteins.In addition to this,when dopamine neurotransmitter was added to the neurons induced by MSCs,a significant Ca²⁺inward flow was observed in the neuronal cells.This result fully demonstrated that the differentiated neurons were functional neurons.Next,it was demonstrated by animal experiments that BMSCs were inoculated on GFs scaffold material and implanted into rats with in vitro rotating magnetic field treatment.After 13 days of treatment,Many BMSCs survived and some of them successfully differentiated into nerve cells and formed nerve-like tissues in vivo.This study solves the problem of lack of autologous NSCs in adult neurodegenerative patients and provides a simple and safe strategy for inducing neural differentiation of BMSCs,which is important for the clinical application of neural tissue engineering.