The Regulatory Rules And Mechanism Of Cochlear Implant-based Electric-acoustic Stimulation System On Behaviors Of Neural Stem Cells

Cochlear implant is one of the most successful and effective clinically implanted functional artificial organs,which can transform sound signal into electric-acoustic stimulation directly on spiral ganglion neurons and help deafness patients gain or regain sound sensation.The cochlear implantation is considered to be the most effective therapeutic method for profound sensorineural hearing loss in current days.However,mammals don't have the ability to spontaneously regenerate spiral ganglion neurons when they're damaged or degenerated.So insufficient numbers of functional spiral ganglion neurons hinder the clinical effects of cochlear implantation,which has become one of major medical problems.Stem cell transplantation is believed to provide a novel strategy for neural degenerative diseases,including sensorineural hearing loss.As a result,the technological innovation system that combine cochlear implantation with stem cells transplantation will expand the range of its application,and will undoubtedly shed more light on this sensorineural hearing loss with less functional spiral ganglion neurons.Accumulating fundamental researches has demonstrated the effectiveness of spiral ganglion neurons after stem cells transplantation,these stem cells included inner ear progenitor cells,induced pluripotent stem cells,embryonic stem cells,mesenchymal stem cells and neural stem cells?NSCs?.However,some obstacles impeded the further application of stem cells transplantation in sensorineural hearing loss,for example,selection gist for stem cells and transplantation ways,a low survival rate,hard to migrate into Rosenthal's canal,uncontrolled differentiation and ineffective function of regenerated neurons.Considering these preclinical problems,it is the vital to manipulate the behavior of stem cells.Advanced and tunable tissue-engineered materials provide an effective regulatory way for stem cells.Their nature features are used to mimic cellular microenvironment,including stem cell niche,and gift cells necessary physical support,three-dimensional space,physical and chemical factors,and so on.Furthermore,the excitability of neural cells makes conductive tissue-engineered materials to be the best choice for spiral ganglion neurons regeneration.In this study,to build cochlear implant-based electric-acoustic stimulation system in vitro,she used graphene,an experienced conductive tissue-engineered material,and Ti₃C₂T_x MXene,a new conductive tissue-engineered material,as neural interface materials at the same time.This system will be helpful to study the regulating characteristics of electric-acoustic stimulation on NSCs and provide theory basis for mentioned new technological innovation system.To achieve these goals,the methods of immunofluorescence,western blotting,electrophysiological recording,calcium imaging were firstly performed to explore the regulatory rules and mechanism of graphene and Ti₃C₂T_x MXene on NSCs.Secondly,she built a cochlear implant-based electric-acoustic stimulation system with good biocompatibility on neural cells for the first time,in which graphene or Ti₃C₂T_x was used as conductive substrates.Finally,the regulatory rules that cochlear implant-graphene electric-acoustic stimulation on NSCs were revealed by various methods of cellular and molecular biology.In the first part,she synthesized Ti₃C₂T_x MXene substrates by mounting MXene films to traditional TCPS substrates,which gets excellent electrical conductivity,abundant surface functional groups and irregular topology on the surface.Compared with TCPS,Ti₃C₂T_x MXene possess comparable biocompatibility for NSCs,importantly,Ti₃C₂T_x MXene could maintain them stemness and proliferative ability.However,the data of calcium imaging told us that cultured NSCs on Ti₃C₂T_x MXene were more active and synchronous than those on TCPS substrate.To explain this phenomenon,she analyzed the effects of Ti₃C₂T_x MXene on NSCs differentiation and maturation of new-generated neurons.The results demonstrated that Ti₃C₂T_x MXene could significantly accelerate neuronal differentiation and these neurons'complexity,not including synapse development.Collectively,these results shown that conductive Ti₃C₂T_x MXene is an efficient neural interface material for modulating stem cells behaviors.The regulatory patterns of Ti₃C₂T_x substrate on NSCs are similar to that of graphene,in which they can't modulate the proliferation of NSCs,but have the potential to improve the level of neuronal differentiation.In order to have a better understanding of the mechanism of conductive materials on behaviors of NSCs,conductive graphene film was fabricated by chemical vapor deposition with simple structure and identical atoms component.The most direct effects of biomaterials on cells were considered to be on the cellular membrane,ion channels and pumps in the membrane plays an important role in controlling cellular function.Based on this,she focused on both the passive and active electrical activities of cellular membrane of neural stem cell,and tried to demonstrate the special relationship between the development of bioelectric properties of NSCs and subsequent changes of behaviors by elucidating the phenotypic changes in NSCs when they were grown on the graphene film.About the passive electrical activities,she found that graphene didn't affect the capacitance and input resistance of NSCs and their progeny,but resting membrane potentials were more strongly negative under both proliferation and differentiation condition.Furthermore,she illuminated that it may be because that graphene accelerated significantly the expression level of TREK-1,a kind of mechano-gated potassium channels.About the active electrical activities,she shown that graphene could accelerate the maturation of active electrical activities of NSCs and their progeny in the course of development,but only slightly influence the electric features of mature new-generated neurons.To further confirm the consequent neuronal development and maturation,it was found that graphene indeed promoted the neuronal maturation and function of 21-day differentiated neurons by spine density,synapse expressions and spontaneous postsynaptic current.Finally,she put forward a novel hypothesis about the regulatory mechanism of conductive materials on NSCs based on our results and COMSOL multiphysics model:conductive graphene could enhance the distribution and intensity of micro-electric field produced by NSCs,followed by the maturation of electrical activities of cellular membrane,and modulate the structure and function of NSCs and their progeny.In the second part,she designed a cochlear implant-based electric-acoustic stimulation system in vitro for the first time with graphene as neural interface and reference electrode.This system will greatly reduce the cost of experiments and time because they could be used for different experiments at the same time.And then she defined three fundamental stimulation parameters,pulse with,amplitude and frequency,based on clinical case and the latest debugging date from 50 samples of four or five-year old prelingual deaf children with cochlear implantation.Next,she used spiral ganglion neurons,the direct targets of cochlear implant,as subjects to optimize its biocompatibility for exploring the regulatory patterns and mechanism of electric-acoustic stimulation on spiral ganglion neurons and NSCs.Furthermore,she found that neurite extensions of SGNs were accelerated with long-time stimulation,which maybe contributed by the development of growth cones.Remarkably,this system is suitable to any other conductive tissue-engineered materials,including Ti₃C₂T_x MXene.In the third part,she summarized the regulatory patterns of cochlear implant-graphene electric-acoustic stimulation on NSCs.Full-band stimulation not only caused cell death,but also reduced proliferative ability of NSCs.In the process of exploring reasons,it was found that neurotoxicity depends on time duration and amplitude of electric-acoustic stimulation,reduced amplitude could recover parts of neurotoxicity and cellular proliferative ability.An intensive study demonstrated that neurotoxicity resulted from high-frequency stimulation,while low-frequency could promote proliferative ability of NSCs.This effect is also verified on Ti₃C₂T_x MXenes-based electric-acoustic stimulation system.For the behavior of differentiation of NSCs,she illustrated that full-band stimulation also suppressed differentiated ability of NSCs,while low-frequency stimulation accelerated both neuronal differentiation and synapse development of new generated neurons.In summary,the survival,proliferation and differentiation of NSCs could be regulated by specific parameters of cochlear implant-based electric-acoustic stimulation,including frequency,amplitude and time duration.It is promising to control transplanted stem cells,new generated neurons in animal models.