Wireless Electrical Stimulation Mediated By Nanomaterials Induces Neural Differentiation Of Neural Stem Cells

Neural stem cells(NSCs)are a type of cell in the nervous system with the potential for self-renewal and multidirectional differentiation,which can promote the differentiation of NSCs into neurons,glial cells and other nerve cells.Exogenous NSCs can be implanted into the adult nervous system,and their differentiation direction can be regulated by local niche to achieve the therapeutic method based on NSCs.This method holds great promise for the treatment of hard-to-cure neurodegenerative diseases and nerve injuries.The therapeutic benefits of transplanted NSCs may be attributed to the specific neuronal differentiation of NSCs,which replaces damaged neurons in neural circuit,or the secretion of neurotrophin that support survival of neurons.However,depending on the spontaneous differentiation ability of NSCs,the proportion of transplanted NSCs that differentiate into neurons is very low,and the differentiated neurons have almost ineffective,which limits the clinical application of NSC-based therapy.A lot of electrophysiological evidence demonstrates that electrical activity plays an essential role in the process of neural development,repair and regeneration,since Luigi Galvani first explored bioelectricity phenomena in the 1790s.So far,based on the exploration of bioelectricity phenomena,electrical stimulation has been applied to promote neuronal development,synaptogenesis,neuronal recruitment,and neuronal survival et al.Especially in regulating the fate of NSCs,electrical stimulation has become one of the most direct and effective ways to promote the neuronal differentiation of NSCs,since the neurons are electrically active cells.However,most traditional methods of applying electrical stimulation require a pair of wires to connect the external signal supplier and the stimulus site in vitro,which is highly susceptible to infection and limits clinical application.Although various materials that can mediate wireless electrical stimulation have emerged,it is regrettable that existing materials with photoelectric,piezoelectric,or magnetoelectric responses have shortcomings such as low utilization of external energy,difficulty in using for electrical stimulation of NSCs in deep tissues,high coupling requirements for external energy emitting devices,unclear mechanisms of action,and a lack of evidence of the functional nature of the differentiated neurons.Due to the above reasons,there has been no exploratory research on the application of materials to promote the differentiation of NSCs into functional neurons through wireless electrical stimulation in neurodegenerative disease or injuries.Based on the above problems,this thesis aims to design and prepare nanomaterials that can effectively mediate wireless electrical stimulation.The nanomaterial-mediated wireless electrical stimulation is used to promote neuronal differentiation of NSCs.The RNA sequence and Western Blot analysis confirmed that the mechanism of neuronal differentiation is that the wireless electrical stimulation activates voltage-gated ion channels and triggers downstream signaling pathways.The flexible nanomaterials that mediated wireless electrical stimulation was further prepared and used in the exploration of wireless electrical stimulation assisted transplantation of NSCs for repair of nervous system injuries in vivo.Finally,the cell-nanomaterial two-way interactive interface was further constructed to realize cell adhesion traction triggering wireless electrical stimulation to localized regulate its own fate.This thesis mainly includes the following three aspects:(1)Gold nanostrip array-mediated wireless electrical stimulation for accelerating functional neuronal differentiationFor the first time,the Au nanostrip array(AuNS array)with strip width of～550 nm was prepared by a nanoimprint lithography(NIL)technique using DVD as template.Only place a piece of rotating permanent magnet on the top of AuNS array,wireless electrical signals can generate on the AuNS array by leveraging the effect of electromagnetic induction,which can be used to promote neuronal differentiation of NSCs.CCK-8 assay and live/dead staining results demonstrated the good biocompatibility of the AuNS array with NSCs and the lack of a negative influence of the rotating magnetic field on cell viability.In vitro experiments,the AuNS array-mediated wireless electrical stimulation can upregulate the expression of neuron-specific markers Tuj1 and MAP2 in NSCs at gene and protein levels,which indicate AuNS array-mediated wireless electrical stimulation accelerate the neuronal differentiation of NSCs.The differentiated neurons exhibited multipolar characteristics of longer axons and increased neurite arborization.The Ca2+ fluorescence intensity changes in the differentiated neurons under the process of two neurotransmitters(acetylcholine and y-aminobutyric acid)indicated that the mature functional AChergic or GABAergic neurons can be obtained through wireless electrical stimulation within only 5 days without any biological growth factor or chemical small molecule,which at least 5 days earlier than the cells without treatment.RNA sequence was used to further explore the mechanism of neuronal differentiation by wireless electrical stimulation.The results showed that the wireless electrical stimulation activates voltage-gated ion channels,triggers downstream signaling pathways.Western Blot analysis further confirmed that the protein expression of c-Fos(Ca2+influx marker)and phosphorylated calmodulin-dependent protein kinase Ⅱ increased during the process of accelerating neuronal differentiation by AuNS array-mediated wireless electrical stimulation.The increased protein expression was blocked by treatment of NSCs with CoCl2(non-specific voltage-gated Ca2+channel blocker),which further demonstrated the importance of activation of voltage-gated ion channels and triggering of Ca2+inflow in the acceleration of neuronal differentiation by wireless electrical stimulation.The above work reveals a new strategy to accelerate the differentiation of NSCs into mature functional neurons,which will help to improve the efficacy of NSC-based therapy in the treatment of neurodegenerative diseases.(2)Flexible gold nanostrip array-mediated wireless electrical stimulation assisted neural stem cell-based therapy in the repair of dural defectsIn order to evaluate the performance of the AuNS array-mediated wireless electrical stimulation in promoting neuronal differentiation of NSCs in vivo,the Au nanostrip array was fabricated on biocompatible and flexible PI(Soft-AuNS array)using the same nanoimprint lithography technology to better fit the neural tissues.Soft-AuNS array can generate wireless electrical stimulation under the action of rotating magnetic field.By combining Soft-AuNS array as mechanical support layer with NSCs as biological integration interface,living artificial composite dura was constructed.In vitro experiments,the Soft-AuNS array-mediated wireless electrical stimulation can accelerate neuronal differentiation of NSCs at gene and protein levels.In addition,the Soft-AuNS array-mediated wireless electrical stimulation promoted the transformation of the neuroprotective A2 reactive astrocytes and reduced the formation of the neurotoxic A1 reactive astrocytes,which helps the transplanted NSCs exert better therapeutic benefit in the biointegration interface with the nervous system.Living artificial composite dura was used to repair dura defect.After one weeks in vivo,micro-CT images showed that the living artificial composite dura in right dural defect fit perfectly under the skull without displacement and prolapse,in the view of transverse,sagittal,and coronal plane,which main attribute to the role of Soft-AuNS array in the living artificial composite dura that act as the mechanical support layer to contact the skull.Moreover,the differentiated mature neurons facilitated the integration of living artificial composite dura with brain tissue and avoided the encapsulation of glial scar and alleviated inflammation in the implantation study in vivo.Soft-AuNS array-mediated wireless electrical stimulation could even promote the survival and differentiation of transplanted NSCs in an extreme environment of inflammatory cell infiltration.To further explore the therapeutic benefits of Soft-AuNS-mediated wireless electrical stimulation assisted NSC-based therapy outside of the dura defect repair,living artificial composite dura was used as implanted dura for spinal cord injury repair,which effectively improved the sensory and motor nerve functions of mice after spinal cord injury 4 weeks later.This provides inspirations for the use of wireless electrical stimulation-assisted NSC-based therapy in the treatment of neurodegenerative diseases and injuries in future.(3)Location-committed differentiation of neural stem cells by cell-traction-triggered wireless electrical stimulation on patterned nanopillar arraysIn order to realize the regulation of NSCs fate by nanomaterial-mediated wireless electrical stimulation without external energy input,the two-way interactive interface between NSCs and nanomaterials was constructed.PLLA nanopillar with long axial to diameter ratio(5:1)array(PLLA long nanopillar)was prepared by the method of high temperature nanoimprint using anodic aluminum oxide nanopore array as template,which could maximize the adhesion traction of NSCs to achieve optimal shear piezoelectric response.XRD results showed that the crystallinity of PLLA long nanopillar was increased after the high temperature compression process,which is conducive to higher net polarization under shear stress,thus showing better shear piezoelectric response.PFM results showed that the shear piezoelectric response of PLLA long nanopillar is significantly improved compared with PLLA planar film due to the synergistic effect of shape structure and crystal structure.After constructing the two-way interactive interface between PLLA long nanopillar and NSCs,it is found that PLLA long nanopillar has good biocompatibility with NSCs,and the adhesion traction of NSCs can effectively trigger the shear bending deformation of PLLA nanopillar.In vitro experiments,the PLLA long nanopillar-mediated wireless electrical stimulation can accelerate NSCs differentiation at gene and protein levels and increase the proportion of neuron differentiation.Furthermore,the PLLA long nanopillar with customized patterns was prepared by combining the high temperature nanoimprint method with micromachining technology.The results of immunofluorescence staining showed that more mature neurons were formed in the PLLA long nanopillar region and more astrocytes were obtained in the PLLA planar film region,which means that the PLLA long nanopillar with customized patterns achieves the location-committed differentiation of NSCs.In addition,Ca2+fluorescence probe staining was used to intuitively verify the increase of Ca2+content in NSCs during the process of PLLA long nanopillar-mediated wireless electrical stimulation accelerate NSCs differentiation,which further demonstrated the importance of activation of voltage-gated ion channels and triggering of Ca2+inflow in the acceleration of neuronal differentiation by wireless electrical stimulation.Finally,in order to eliminate the influence of nanopillar with long axial to diameter ratio introduced in the process of accelerating the neuronal differentiation of NSCs,PDLLA nanopillar with long axial to diameter ratio(5:1)array without piezoelectric response was prepared.In vitro experiments,the PDLLA long nanopillar had no significant promoting effect on the differentiation of NSCs into mature neurons,which confirmed that the effective acceleration of neuronal differentiation by PLLA long nanopillar was contributed to the wireless electrical stimulation generated by effective piezoelectric response,rather than the influence of the nanopillar structure.The above work lays a foundation for further exploration of the precise localized regulation of NSCs fate without external energy input.In summary,this thesis has prepared a nanomaterial capable of mediating wireless electrical stimulation,conducted an in-depth study on its biocompatibility and promoting effect on neuronal differentiation of NSCs,revealed the mechanism involved in wireless electrical stimulation promoting neuronal differentiation,and further explored the nanomaterial-mediated wireless electrical stimulation assisted NSC-based therapy in the repair of nervous system injuries.Finally,a cell-nanomaterial two-way interactive interface was constructed to realize localized regulating of NSCs fate by adhesion traction triggering electrical stimulation without external energy input.These studies and explorations provide guidance for realizing the clinical application of electrical stimulation-assisted NSC-based therapy in neurodegenerative diseases and injuries,and also provide inspirations for the innovation and development of other implantable medical electronic devices.