Electrical Stimulation-Mediated Differentiation of Neural Cells on Conductive Carbon Nanofiller-Based Scaffold

An important strategy in neural tissue engineering involves imparting electrical properties to the regenerative template to encourage cell proliferation and differentiation. Several clinical studies have confirmed that direct or indirect electrical stimulation therapy greatly impacts the treatment of peripheral and central nerve injury. Nerve regeneration can be accelerated by the application of electrical stimulations of different methods and with varying parameters. For a long period, electrical stimulation along with conventional conductive polymers have played an important role in nerve tissue regeneration, due to their conductivity. However, the low biocompatibility of these materials has brought attention toward the need for the alternative of the conductive polymers. Carbon nanofillers (graphene, nanotubes, and their derivatives) have been showing promising results in bioimaging, biosensing, and composites, simultaneously, proving themselves as a prospective biomaterial in neural tissue repair and regeneration due to their excellent electrical and mechanical properties, alongside, biocompatibility. Therefore, carbon nanofiller-based scaffolds synchronized with electrical stimulation may lead to a breakthrough in the treatment of nerve injury. This review article focuses on the influence of the electrical properties of carbon-based material on nerve tissue engineering. In this article, we explicitly focus on the different methods to deliver electrical stimulation and the pathways involved in the differentiation of neuronal cells. Emphasis is given to the analysis of the suitable fabrication strategies of carbon-based neural scaffold and the way its interfaces interact with neurons for promoting neuronal differentiation. Furthermore, this review summarizes the ongoing advancements to augment the conductivity and biological activities (cell adhesion, proliferation, neural differentiation, neurite outgrowth), as well as to reduce the potential toxicity in conductive carbon nanofiller-based scaffolds.


Introduction
Spinal cord injury (SCI) and peripheral nerve injury (PNI) are the primary reasons for nerve dysfunction all over the world.Disruption of nerves of the spinal cord can cause permanent disability; whereas, in case of peripheral nerve injury, motor functions can be reduced or impaired at a particular site [1].Primary injuries are mostly the results of compression, laceration stretch, shearing, or hemorrhage from any traumatic events, such as accidents or sports.Secondary injury is the result of the by-product of the chemical and physical events during primary injury.After the injury, the formation of glial scar by the activated astrocytes restricts the cell expansion, migration, proliferation, and axonal extension, by producing an inhibitory environment [2,3].
Gunjan Kaushik and Chandra Khatua have contributed equally to this work.

3
The treatment of SCI should be provided in an acute phase, within a very short time after the SCI.But that is not practical often due to limited resources [4].So, the main focus has been given to treat efficiently, in the chronic phase.The existing treatment is based on neuroprotective and neuroregenerative strategies [5].Early surgical compression and treatment with anti-inflammatory drugs are necessary for neuroprotective measurements at the acute phase [6].But these strategies do not provide the gold standard treatment for the SCI.Many researches have been going on in this field to provide a stable treatment and some of these are currently in preclinical stage.
In case of PNI injury, the end-to-end direct suturing is used to join the gaps less than 1 cm.But, autologous nerve grafts are being considered as a golden treatment to join gaps more than 1 cm.However, there are constraints, like minimal supply of donor tissue, size mismatch between donor and recipient, and chances of possible neuroma formation [7,8].To counter these issues, the development of artificial nerve conduits has come into the light, as it is easily reproducible and cost effective [9,10].An artificial nerve guidance conduit can be fabricated from biodegradable biomaterials, such as collagen [11,12], chitosan [13], polyglycolic acids [14], and polycaprolactone [15,16].These biomaterials have shown clinical potential in nerve grafts.However, application of these alone have failed to regenerate long-gap injuries [17].The hollow tube structure or nerve wraps lack the axonal guidance and supportive cues and this might be the reason behind their failure [18].
It was noticed that scaffold alone is not very much effective for the treatment of both kinds of nerve injury.Exhibition of combinational therapies, such as cells (Schwann cell, IPSCs, NSCs) or bioactive molecules (growth factors) or therapeutic drugs carrying biomaterials or synthesizing polymer nano-composite by reinforcing conducting nano phase within the biocompatible polymer matrix [19][20][21][22], play an exceptional role in the recovery [23,24].In addition, several studies have demonstrated that one of the biophysical cues (ultrasound stimulation, electrical stimulation) has the ability to influence neuroplastic changes [25].Application of electrical stimulation directly onto the injured nerve with certain parameters is a promising strategy to accelerate central nerve regeneration, as well as peripheral nerve regeneration.In the peripheral nerve injury, it accelerates Schwann cells to stimulate neurotrophic factors at the site of nerve injury, thus encouraging the nerve regeneration [26,27].Tissue engineering uses both the ways synergistically with the help of electroconductive scaffold.Electrically conductive biomaterials can efficiently deliver electrical signals to cells and improve electrical communication among the cells [28,29].Some conducting polymers, such as polypyrolle, polyaniline, and poly (3,4-ethylenedioxythiophene) have been used in preparation of scaffold, since a very long time [30][31][32].However, besides the numerous advantages, we face low biocompatibility in use of these polymers.Cracks or delamination of conducting polymers, under long-term stimulation, restrict the electrode performance [33].Carbonbased scaffolds, such as carbon nanotube and graphene, are of high interest based on the chance of biofunctionalization and drug loading [34,35].The manipulation of carbon nanofiller-incorporated scaffold are likely to have a great influence on neuroregenerative techniques and other biomedical applications.Carbon nanofillers have been found to interact with the nervous system, promoting the neural development [36,37].Furthermore, the outstanding conductive properties make carbon nanofillers favorable for neural growth and stimulation [38].In this review, we are providing an overview of the electrical stimulation-mediated differentiation of neural cells on conductive carbon nanofiller-based scaffolds.This review article aims to discuss on the use of different carbon nanofillers (graphene, carbon nanotubes) in the scaffold for the neural tissue engineering and their role in efficient neural regeneration.

Electrical Properties of Nerve Cell
Neurons and neuroglia are the basic unit of CNS and PNS, which regulate the functions of the nervous system.Among all the units, neurons have the intrinsic property to communicate among themselves, muscles and organs via electric potentials, or action potentials or impulse [39].Neurons are composed of soma, axon, and dendrites.Soma receives signal from the dendrites of the other neural cells and pass to the axon, while axon transmits this electrical signal away from the soma.Neuroglial cells act as supporting cells and are classified into astrocytes, oligodendrocytes (CNS), and Schwann cells (PNS).Stimulus (external or chemical)-generated impulse is received by receptor cells on the neuron.This modified signal is transduced into an electrical impulse at the plasma membrane of the axon where it joins with the soma.It conducts the impulses away from the soma or dendrites.As the axon passes the impulses to the terminal end, it triggers the secretion of the different chemical substances called neurotransmitter.At the end, signal (neurotransmitter) are transmitted from presynaptic terminal to postsynaptic terminal of the soma or dendrites located on the adjacent neuron [40].
In the nervous system, electrical impulses are generated through the exchange of ions (Cl − , K + , Na + ) from the semipermeable neuronal membrane.As a result, potential difference is formed and maintained by active sodium-potassium pump, passive voltage-dependant sodium, potassium channel, calcium channels, and anion channels on the nerve membrane [41].In the extracellular matrix, high concentration of Na + and Cl − is present, whereas in the cytoplasm, 1 3 K + concentration is high in comparison to Cl − to maintain the positive charge inside.Usually, the charge at the inside of the membrane is negative due to less sodium ions.In the absence of nerve impulse, nerve maintains the resting potential of about − 60 to − 75 mV.This resting potential is maintained by sodium-potassium pump, which discharge more sodium ions than potassium through the cell.Upon stimulating, the electrical responses are generated by activating ion channels in the membrane.These permit the influx of Na + ion into the cell that depolarize the membrane.Upon reaching the threshold potential − 50 mV, it triggers nerve potential or action potential.At the threshold point, voltagegated sodium channel opens, and influx of the sodium into the cell continues at the high speed.This makes the inner cytoplasmic environment positive and reverse the polarity.At that moment nerve fibers remain in the depolarized state and potential starts to increase up to + 30 mV.As it reaches to the peak of the action potential (+ 30 mV), sodium channel gets inactivated and potassium channel opens which allow the more influx of potassium ions.At this repolarization state, no second action potential can be induced by giving any stimulus.Another second action potential can be generated only after a refractory period, but the stimulus current should be higher than the previous one.This nerve impulse is transmitted like a wave on the nerve fiber.When one wave propagates, it follows the depolarization and leaves the repolarization state (Fig. 1).

Methods to Deliver Electrical Stimulation
Indeed, providing electric stimulation (ES) is a considerable method based on recent research.ES stimulate the differentiation, migration, and proliferation on applying directly to the cells.Combining the tissue engineering techniques with ES reduces the cost of treatment and enhances the capability of the treatment.There are three methods which are being used to deliver ES in the in vitro and in vivo, namely, (1) direct coupling, (2) capacitive coupling, and (3) inductive coupling (Fig. 2).

Direct Coupling
In this method, the electrodes are directly immersed in the culture medium and deliver ES either via electrodes or scaffold [33].In this system, electric field is generated between two electrodes, e.g., cathode and anode, which are directly in the contact with the cell culture dish.It is the easiest method to apply electric field to the cells.It has been observed that upon applying the direct current into the cells, in the culture, neurite outgrowth happened at a very significant level [43].However, direct contact of electrodes with cell may lead to many disadvantages, such as insufficient biocompatibility, increase in temperature, pH change, and production of harmful by-products.

Capacitive Coupling
In this process, the electrodes are placed outside the cellseeded scaffold.They do not remain in the direct contact of the cell.Thus, this coupling is biocompatible.This type of coupling is generally used in the regeneration of nerve cell in CNS and PNS injury.Hu et al. investigated the effect of ES on the dorsal root ganglion cells (DRG) of the rats by applying the stimulation of 100 or 200 mV/mm for 0.5, 1, or 2 h.They noticed that ES induced dorsal root ganglion (DRG) cell viability, which ultimately induce Schwann cell activity [44].In a study conducted by Al-Majad and group have shown that the ES of 20 Hz frequency leads to the motor and sensory neuron regeneration.They transacted the femoral nerve and applied ES.They noticed that the number of axons increased in the motor neuron early, at the distal surgical site [45].

Inductive Coupling
This type of coupling use electromagnetic system, which is placed around the cell culture plate and deliver pulsed electric field.This technique is called the pulsed electromagnetic stimulation (PEMF).This electromagnetic field is generated by conductive coil.The advantage of this coupling is to provide the stimulus in the pulsed form to mimic the natural potential transfer.Another advantage is that it does not directly apply to the target cells rather it applies potential to the adjacent cells.Sisken and group exposed the crushed lesion at the rates with pulsed electromagnetic (PEMF) frequency of 2 Hz and magnetic flux density of 0.3 mT for 4 h/ day.They noticed the recovery rate was increased by 22%, which was equivalent to that obtained in any reported drug and hormone treatment [46].Similarly, the pulsed electromagnetic stimulation is being used in the treatment of diabetic neuropathy [47] and central nervous injury [48].This technique comes with some drawbacks also, for example, it is time consuming as it can take up to 10 h for the session and is costly also.

Conductive Carbon Nanofiller-Based Scaffold
Natural and synthetic biomaterials have shown great potential for repairing and regenerating the peripheral and central nervous systems.As one of the promising biomaterials for nerve regeneration and repair, carbon-based nanofillers are attracting significant interest.Carbon-based nanofillers, including carbon nanotubes (CNTs) and graphene, draw attention to neural tissue engineering applications for their attractive electrical and mechanical properties.These nanofillers possess many unrivaled benefits, including mechanical strength, chemical stability, large surface-to-volume ratio, and high electrical conductivity [49].In the neural tissue engineering field, it holds in high regard and a continuous effort have been made to combine them with available materials for the application of scaffold reinforcements, tissue regeneration, drug delivery, and biosensor.

Graphene
Graphene is a two-dimensional single-layer honeycomb structure composed of carbon atoms and leads to garnering a great deal of interest in biomedical engineering.Graphene and its derivatives such as graphene oxide (GO), reduced GO, and graphite oxide possess remarkable features, like large surface area, ease of functionalization, and excellent electrical conductivity, making it more promising in the field of bioelectronics devices, neural tissue regeneration, cancer treatment, and regenerative medicine.

Electrical Properties
Graphene and graphene oxide encompass a thin layer of twodimensional sheets.They possess excellent electrical and thermal properties due to their sp 2 -hybridized structure and the presence of free π electrons [50].Graphene oxide has inferior electrical and thermal properties, as compared to graphene.However, the conductive nature of graphene and graphene oxide has made them suitable for tissue engineering applications in particular for promoting neural regeneration [51].Graphene exhibits high electrical conductivity as electrons and holes act as charge carriers.Electrical conductivity of graphene ranges from 10 7 to 10 8 (S/m) [52].Among the four valence electrons of carbon in graphene structure, one electron is available for electronic conduction as each carbon atom is linked with the other three carbon atoms on single-layer honeycomb lattice structure.This unbonded π electron forms an orbital perpendicular to the plane.The valence and conduction bands (bonding and anti-bonding state) of these orbitals determine the electrical behavior of graphene [53] (Fig. 3a).The energy band structure of graphene possesses zero bandgap.In graphene, the conduction and valence band meet at the Dirac point where electrons and holes both contain zero effective mass.Because of the zero density of states, electronic conductivity is poor at the Dirac points.Furthermore, due to lack of mass, electrons in graphene are considered to behave similarly to photons in terms of mobility.Charge carriers can freely move to submicrometric distances without any scattering, which is known as ballistic transport.At room temperature, graphene has an electronic mobility of about 15,000 cm 2 V −1 s −1 and the electron conduction rate can reach 1/300 of the speed of light.The carriers in graphene undergo a particular quantum tunneling mechanism that prevents backscatter, when they hit impurities, producing higher conductivity [54].

Fabrication of Scaffold
The exceptional conductive nature of graphene opens up a new route in neural tissue engineering application.Natural or synthetic biomaterials have been extensively studied for their excellent biocompatibility.But, their relative inertness restricted their uses in the field of neural tissue regeneration.The use of electrically conductive materials is a potential approach for neural tissue regeneration.Therefore, graphene-based scaffolds have been produced by various fabrication techniques to improve the conductivity, as well as biocompatibility.Graphene can be fabricated mostly by top-down and bottom-up methods.The top-down process generally involves detaching or exfoliating graphite crystals.Exfoliation process is associated with Scotch Tape method [55], activation of the graphite or graphite oxide sheets under ultrasound [56], or electrical arc discharge between graphitic electrodes [57].Another top-down process is the Hummers method where graphene oxide is converted into graphene by a reduction process in presence of reducing agents, like N 2 H 4 and NaBH 4 [58,59].On the other hand, through bottom-up process, graphene layers are grown directly on the surface of a substrate.This method is accomplished with epitaxial growth on metal crystals by chemical vapor deposition (CVD), in which graphene is precipitated on the surface of a transition metal from a hydrocarbon source [60].CVD is the most promising method in terms of operational control, throughput, and complex structure.The properties of graphene-like surface area, surface topography, and conductivity are strongly related to its fabrication processes [61].
In a classical approach, the incorporation of graphene and its derivatives into other materials could remarkably enhance the conductivity of composites by several orders of magnitude.The graphene-based composite scaffold has been Reproduced with permission from Ref. [98] broadly investigated for neural tissue repair [62][63][64].These composites are fabricated in different forms to enhance the efficiency of a scaffold.Several researchers developed doped graphene composites for the application of neural cell stimulation [65].Graphene oxide-doped poly(3,4-ethylenedioxythiophene) composite has been fabricated through in situ interfacial polymerizations and it promoted neural cell orientation and differentiation [65].Apart from this, graphene combined with poly(ε-caprolactone) has been developed into a composite, using layer-by-layer casting technique [60].But it is very difficult to reach the uniform dispersion of graphene for the formation of the composite.To overcome these issues, surface functionalization of graphene with some bio-macromolecules, like DNA and proteins, has been suggested [66].However, in comparison to planar 2D scaffolds, three-dimensional graphene-based scaffolds deliver superior properties due to their high interface area and ability to provide an in vivo microenvironment for neuronal regeneration.Researchers have exploited several processes to fabricate 3D scaffolds, like freeze-drying, salt leaching, fiber spinning, or 3D printing [62,63].Guo et al. assembled a porous scaffold with a layer of reduced graphene oxide nanosheets on the surface of collagen-based porcine acellular dermal matrix channels for larger neurite outgrowth (Fig. 3b) [67].Another approach for the fabrication of 3D porous scaffolds is non-solvent induced phase separation.Through this process, a piezoelectric scaffold, composed of PVDF/GO, has been fabricated for peripheral nerve regeneration and it showed remarkable enhancement of the cell viability [68].Apart from these, graphene-based conductive electrospun nanofibers provide an additional advantage of having a 3D architecture, which is similar to neurites found in the human body.This type of conductive fibrous scaffold has been fabricated by an electrospinning process.Graphene, combined with collagen and poly(ε-caprolactone), formed aligned fibers that were comparable with nerve fibers [69].An initial investigation in this direction has been made by graphene and silk fibroin composite nanofiber [70].Graphene is found well dispersed in the silk matrix, which improves the electrical conductivity and mechanical strength of the composite.3D printing offers a potent way to fabricate the 3D scaffolds, but in case of pure graphene, it is difficult to print [71].A potential solution to this problem is to combine graphene with other printable materials, such as polymers.Farzan et al. have used PEGylated GO to make 3D-printed conductive nerve guidance conduits with precise geometrical features that support cell elongation in a suitable direction [72].However, the fabrication of graphene-based composite in a complicated shape and larger construction remains a challenge and needs more research.

Effect on Differentiation of Neural Cells
Graphene is a unique material that has unique topographic and chemical properties that could act as a bridge between regenerating nerve cells.The benefit of graphene is its ability to allow electrical conduction between the damaged nerve ends due to its conductive properties, which promote neural regeneration.
Apart from chemical approaches (such as growth factors) [73][74][75], non-chemical approaches, such as manipulating surface topography at nanolevel [76,77], stiffness of the substrate [78,79], or electric current flow [80][81][82] have been shown to drive neural stem cell proliferation and differentiation.Among the non-chemical method, electrical stimulation has been considered as one the best method to stimulate the neural cells [83].In human body, electrical signals are crucial for cell communication and behavior, such as migration, proliferation, and differentiation [80,84].Electrical stimulation that is provided to cells through conductive biomaterials has the potential to have an effect on a variety of biological processes, including gene expression, metabolic activity, cell survival, and others [85].Thus, in order to promote neurogenesis, it is critical to synthesize electroactive scaffolds that can effectively transmit ionic and electrical signals.Graphene's remarkable electrical characteristics make it a good alternative for constructing conductive scaffolds for electroactive cell populations, such as neural cells.
Numerous evidence suggests electrical stimulation can help regenerate both central and peripheral neural systems.Moreover, electrical activity is intimately connected to the operations of the nervous system.According to research, electrical stimulation can increase stem cell proliferation [80], neuronal differentiation [80], cell migration [86], and axon outgrowth [87].For example, Lu et al. studied the effect of electrical stimulation on peripheral neuron regeneration in vivo [88].They used different frequencies of electrical stimulation for this purpose.They have shown that protocol of applying electrical stimulation is very crucial.In their study, depending on the application protocol, electrical stimulation was shown to have a beneficial or negative effect on regeneration.Precisely, they observed that the greater the frequency of electrical stimulation applied, the more likely it is that regeneration will be inhibited.In a different study, Chang et al. examined the effect of continuous or programmed electric current on neural stem cells [80].According to this study, biphasic electrical currents of 1-30 µA cm −2 for 200 µs at 100 Hz increased cell proliferation and neural development in embryonic neural stem cells.
Stimulating neural cells and observing the effect of stimulation on cells are crucial for developing clinical tools for diagnosis and treatments, and graphene's electrical characteristics make it ideal [89][90][91].In one study, graphene electrodes have been used to stimulate neural cells for their differentiation.After stimulation, intracellular Ca 2+ changes were used to assess cellular responsiveness.The result of the study showed that after electrical stimulation, graphenebased neurons displayed elevated calcium levels, indicating effective electrical connection [91,92].In another study, steady and pulsed electrical stimulation was used to differentiate PC-12 cells on a non-functionalized graphene film [93].It was found that constant electrical stimulation resulted in neuronal differentiation, neurite extension, and migration, particularly at 100 mV mm −1 .On the other hand, pulsed electrical stimulation was found to have greater effects on neurite length and extension compared to those with a constant waveform.Electrical stimulation also promoted neurogenic differentiation of human mesenchymal stem cells (hMSCs), as seen by increased expression of MAP2 and Tuj1 in both unpatterned and patterned (grid or column) graphene substrates [94].For the first time, it has been shown that rat MSCs grown on 3D-printed graphene circuit can transdifferentiate into neuronal like cells, simply by applying electrical stimulation to the graphene-integrated electrodes without any traditional chemical treatment [95].In a study, Heo et al. used a graphene/polyethylene terephthalate stimulator to apply electrical stimulation to promote neuronal cell-to-cell interaction in vitro [64].Existing cellto-cell couplings were strengthened by mild electrical field stimulation (4.5 mV mm −1 ).These effects may be due to well-tuned cytoskeleton-related gene and protein expression.Another study used laminin-coated graphene sheet for induced proliferation and differentiation of neural cells [96].Due to the low electrical sheet resistance of the graphene oxide foam (GOF) layers (170 Ω sq −1 ), rolled lamininfunctionalized GOF were used as electroactive scaffolds to transfer external electrical current (20 mA) to human neural stem cells (hNSCs).This resulted in enhanced division and differentiation of neural stem cells.Several other studies used graphene-coated polymer structures to supply electrical stimulation for the differentiation of neural cells [97,98] (Fig. 3c).Feng et al. synthesized a chemically reduced graphene oxide-coated poly(vinyl chloride) electrospun nanofibers to provide electrical stimulation to neural cells [97].Incorporation of graphene oxide to the nanofibers increased its electrical conductivity.The growth of primary motor neurons was speed up during long-term culture, when subjected to a mild electrical stimulation of 10 mV cm −1 pulses.This was in comparison to the development of primary motor neurons grown on tissue culture plate and 2D graphene film.It was hypothesized that these amplifications were caused by the three-dimensional fibrous architecture, which made it possible for an anisotropic electron transfer along the random fibers.This transfer might reduce the cell's capacity to adjust to the stimulus.In a different study, Guo et al. prepared composite microfibers with reduced graphene oxide and poly(3,4-ethylenedioxythiophene) (PEDOT) for the purpose of synthesizing human motion-driven selfpowered triboelectric nanogenerator [99].The stimulation resulted in a significant increase in the expression of neural proteins and genes, such as Tuj1 and GFAP, which led to a marked improvement in the neural differentiation of rMSCs.Several researchers also developed graphene reinforced polymeric composites for the application of electrical stimulation to the neural cells PC-12 cells cultured on a doped graphene oxide composite exhibited enhanced cell orientation and differentiation, under the influence of electrical stimulation (100 mV cm −1 ) [65].

Carbon Nanotubes
Carbon nanotubes (CNTs) in various forms, such as single walled (SWCNT) and multi walled (MWCNT), have been widely used in nerve tissue regeneration.The physical and chemical properties of CNTs can be tuned in terms of regulating their length, diameter, chirality, surface functionalization, and surface area.

Electrical Properties
CNTs are cylindrical structures consisting of rolled-up graphene layers with a diameter in the nanometre range.CNTs are conjugated with sp 2 carbon composed of a π-electron system with a controllable band gap which makes it either a high conducting or semiconductive one [101].SWCNTs are long and hollow structures where sp 2 -hybridized carbons are connected in hexagonal form, while MWCNTs are concentric cylindrical structures made of multiple layers of graphite sheets.CNT possesses electrical properties similar to metals, like copper.The electrical conductivity of the CNTs relies on their chirality, the degree of twist, and their diameter.CNTs with armchair structures appear to have better conductivity compared to other types of CNTs.The electronic structure and electrical features of CNTs are strongly related to the electronic structure of a graphene sheet [102].In the band structure (Fig. 4a), the valence (π) and conduction (π * ) bands are touched at six points in a hexagon shape, called Fermi point [103].In the ℾ-K directions, i.e., direction toward the Fermi points, the electrons move freely and CNT behaves like a conductor.In the ℾ-M direction, a semiconductor-like band gap prevents the electrons to pass through and CNT behaves as a semiconductor.The electrical conductivity of SWCNTs ranges from 10 2 to 10 6 S cm −1 , while MWCNTs have conductivity in the 10 3 to 10 5 S cm −1 range [104].The electrical properties of MWCNTs have received less attention than SWCNTs, due to their complex structure in which each carbon shell has a unique electrical character and chirality.MWCNT has been found to exhibit interwall interaction that redistributes the current over individual tubes in a non-uniform manner.In contrast, the current across the different parts of SWCNT remains unchanged.The high electrical conductivity value has made SWCNTs a promising filler material in neural scaffold fabrication.

Scaffold Fabrication
CNTs have exceptional strength, flexibility, and conductivity, which are being used exclusively or as a filler material in neural tissue engineering applications.It needs to have an appropriate platform to accelerate neural regeneration to repair the damaged nerve without showing any adverse biological effects.Apart from the exceptional electrical conductivity of CNTs, the morphological similarity of CNTs with neurites makes them favorable for nerve tissue repair.The conductive nature, biological activity, and neural regeneration efficiency of CNTs are greatly influenced by their fabrication techniques.CNT (MWCNT and SWCNT) can be synthesized by different processes, such as CVD, carbon arc discharge, and laser ablation [105,106].CVD process is a widely used technique to produce CNTs and controls their chirality, particularly for SWCNTs [107].In the past, its poor solubility and the presence of toxic metal impurities (Fe, Co, Ni, Y) associated with its fabrication process have restricted its biological application [108].It is extremely difficult to make a homogeneous dispersion of CNTs in aqueous solution or organic solvents, due to its inherent structural and chemical stability.This limited its utilization, due to its agglomerated form, making it very toxic even at low doses.However, dissolving CNTs in aqueous media is currently a processing challenge for their utilization.The insolubility of CNTs causes considerably harmful effects to cells and tissues [109].The purity and biocompatibility of CNT have been improved in recent times as a result of advancements in synthesis, purification, and surface modifications [110].For improving the dispersibility and neurocompatibility of the CNTs, either various functional groups were incorporated into it or positive and negative charges were induced in it [49].When carbon nanotubes are combined with hydrophilic polymers, like polyethylene glycol (PEG), their water solubility improves, making CNT-based scaffolds easier to Reproduced with permission from Ref. [117], c synaptic bridge formed between two MWNT bundles and the soma of the neuron is located along the edge of the MWNT.Reproduced with permission from Ref. [118] and d Schematic representation of the interacting mechanism of absorbed ECM proteins with CNT multilayers that controls NSCs biological processes Reproduced with permission from Ref. [137] fabricate [111].Other efforts to improve neurocompatibility have been focused on the biofunctionalization of CNTs that also simultaneously improve their water dispersibility [111].Because of the electrostatic interaction between the charged CNTs and the negatively charged plasma membrane of neural cells, CNTs were functionalized with the amine group, making them positively charged [112].The aminated MWCNTs were incorporated into a poly(ethylene glycol) diacrylate (PEGDA) matrix to fabricate 3D-printed microelectroporous scaffolds that facilitated differentiation and growth of neural cells [113].Ahn et al. fabricated a fibrous scaffold with aminated CNT embedded in phosphate glass for the treatment of PNI [114].Multiple initiatives have been reported to tailor the electrical conductivity of composites, and most of them have been focused on the reinforcement of CNTs.CNTs have been explored as a filler material and offer an electrically conductive framework that improves the neural cell interaction.Wu et al. investigated the potential usages of composite hydrogels reinforced by MWCNTs [115].In this study, this hydrogel was made of chitin-blended modified CNTs using hybrid solvents.It has been observed the complete dispersion of CNTs in the chitin matrix which make it a neurocompatible scaffold and constituents are held together by intermolecular interactions, such as hydrogen bonding, electrostatic interactions, and amphiphilic interaction [116].SWCNT was used as a conductive filler in a collagen-based hydrogel, resulting in an increased bulk conductivity (up to 1.7-fold) by 10-100 g mL −1 loading and neurite outgrowth enhanced 3.3-fold at 20 μg mL −1 SWCNT loading [117] (Fig. 4b).Further, a geometrical pattern of the scaffold guides neurite elongation along its edges.In the same way, the alignment of the carbon nanotubes can also drive neurite extension [118] (Fig. 4c).Micro-patterning techniques for the fabrication of patterned CNT have been employed to regulate the neurite growth direction.Vertically arranged MWCNT-based scaffolds have been fabricated by the combination of microlithography and CVD techniques [118].CNTs have also been combined with other materials to produce a designed scaffold which is promising for directional neurite growth.For this, aligned CNT-coated poly(llactic acid-co-caprolactone) electrospun nanofibers have been made that have strong influence on the growth of PC12 and DRG neurites [119].Topographical cues can be used simultaneously as templates for guiding and orienting neural outgrowth.Gabriela et al. produced three-dimensional micropatterned CNT micropillar templates to regulate neurite outgrowth by CVD method [120].However, taking all the requisites into consideration, fabrication process, scaffold design, and CNTs modifications are needed to be explored further to offer superior biological, mechanical, electrical, and topographical cues for nerve regeneration [121].

Effect on Differentiation of Neural Cells
Neuropathologies and nerve tissue damage are being treated with CNTs, which act as an attractive approach.The morphological similarities of CNTs with neurites and dimensional similarities of the CNT bundles with the dendrites further increase their potential for repairing, stimulating or reconfiguring neural networks, as well as providing insight into basic neuronal mechanisms.
In the tissue engineering field, CNT affects the neural cells differentiation pathways due to its electrically conductive properties, diameter of less than 100 nm, and aspect ratio (height/diameter) close to nerve fiber [122].CNT-based scaffold mimics the extracellular environment (ECM) and conductivity.In the NCS differentiation, CNT accelerated the expression of gamma enolase (ENO 2) and Methyl CpGbinding protein 2 (MECP2).These two proteins are found in mature neuron, which indicates the differentiation of NSCs in neurons [123].The linear alignment of CNT structure gives a proper medium for the cells to contact with each other through synapse.It improves the adhesion and interaction of cells on CNT due to its affinity toward different proteins, such as laminin and fibronectin [124].It also increases the expression of microtubule-associated protein (MAP2) [125].Astrocyte of the brain cells also gets affected on the CNT scaffold.Astrocyte cell area and perimeter increase in contact with CNT, which ultimately affect neuron differentiation.Thus electro-stimulated CNT impacts the growth of astrocytes, glial cells, and microglial cells [126].
CNT-incorporated scaffold takes part in neurite growth, extension, and restoration of its electrical properties [127,128].Several studies have found that CNTs promote the early differentiation of neural stem cells into mature neurons or provide guidance to neurons for extension with or without electrical stimulation [129,130].These processes cause some major morphological changes [128].Unmodified CNTs showed mitigation of the cell adhesion activity in vitro in comparison to modified CNT, due to less solubility of the former in water and potential toxicity.Functionalized CNTs provide the capacity to promote neuronal extension, neuronal survival, and neurite extension [110].Hu et al. varied charges on MWCNT and observed the effect on neurite outgrowth, number of growth cones, length, and branching.They noticed that positively charged MWCNT reflect the potential capability in synaptic transmission [131].Scaffolds mimicking the extracellular matrix enhance the cell interaction and growth [132,133].Similarly, scientists have attached different ECM molecules to CNTs in order to promote the neuroregenerative environment.It was demonstrated that laminin-modified PLGA/CNT scaffold showed higher cell viability of PC12 cells and significant neurite extension in the absence of NGF [134].
CNTs have been used directly as substrates for promoting neuron adhesion, cell differentiation, and facilitating the longer neurites [135].But, the potential toxicity of carbon nanotubes in biological systems limited their use as a substrate.Due to the unique electrically conductive nature, CNTs have been combined with biocompatible polymers to construct an electrically conductive scaffold.MWCNTs were used as fillers in the development of a poly(l-lactic acid)-based composite with carbon loading range from 0.1 to 5% [136].This composite was found to be biocompatible and promoted the proliferation of the SH-SY5Y human neural precursor cell.The addition of negatively charged CNTs in positively charged poly(diallyldimethylammonium chloride) (PDDA) could lead to offering an effective regulatory signal over neural stem cells, including adhesion, proliferation, differentiation, and outgrowth of neural stem cells [137].Furthermore, a probable mechanism was proposed in this study that focal adhesion kinases (FAK) are activated by integrin-regulated interactions between composites and neural stem cells (Fig. 4d).It results in the initiation of a series of signaling pathways, like MAPK signaling pathway, guiding NSC differentiation, the Wnt signaling pathway, controlling NSC proliferation, and the PI3K-AKT signaling pathway, governing cell survival.A CNT-based nerve guidance system is widely recognized due to many aspects of conduit features, including biomimetic surface topography, and electrical conductivity.Carbon nanofibers, fabricated by electrospinning, showed great promise in neural regeneration [138].It can support the growth of nerve cells from human endometrial stem cells.Recently the role of patterned CNT is being explored in spatially directed neuronal growth [118,139].Gabriela et al. worked on the strategy to provide the topographical, biochemical, and mechanical cues that mimic the structure of ECM for better cell adhesion [120].They tailored CNT in 3D microscopic pillars in different geometries and coated it with laminin to accelerate adhesion of neural stem cells (NSCs).They observed that neurites are anchored onto the top of the pillars and attached with adjacent neurites forming a neural network.Depending upon the structure of micropillars arrangement neurite can be grown in any shape.This may lead to its application in nerve injury [120].Considering the conductive nature of CNTs and the advantages of electrical stimulation on neural tissue engineering, integrating the conductive scaffold with electrical stimulation will produce a robust neural regeneration strategy.The combination of electric stimulation and CNT-based scaffold leads to accelerate the regeneration of the neural tissue [34].This synergistic effect enhanced cell proliferation, neuronal differentiation, and maturation along with stimulated neuronal gene expression levels.After applying 100 μA direct current, it enhanced the 35% cell proliferation as compared to without any electrical stimulation [34].The external electrical stimulation applied on the composite scaffold at 100 mV mm −1 and 20 Hz for 2 h day −1 increased the PC-12 cell proliferation and migration, as well as improved intracellular connections and neurite outgrowth [140].Imaninezhad et al. investigated the influence of scaffold stiffness, scaffold conductivity, and electrical stimulation on the alignment, the morphology of neural cells, and neurite directional outgrowth [141].They have found that polyethylene glycol-CNT composites stimulated by the direct current for 1 h at 30 V m −1 lead to align the cells perpendicular to the applied current and increased neurite length.The presence of CNTs in these hydrogels also resulted in improving total neurite outgrowth and longer neurites.Koppes et al. showed the effect of carboxylated SWCNT loading in neurite outgrowth under direct current stimulation [117] (Fig. 4b).When SWCNT loading was increased, the electrical conductivity, as well as neurite outgrowth, were improved compared to the SWCNT-free scaffold.When electrical stimulation was applied, SWCNTloaded scaffold showed 7.0-fold enhancement of outgrowth compared to the unstimulated one.Similarly, Wang et al. observed that the use of electrical stimulation of 40 mV enhanced the neurite extension [142].Besides this, applying an external electric field on PCL/CNTs nanofiber for 2 h at 20 Hz and 100 mV, it has been found to improve the remyelination and axonal regeneration in vivo [143].However, the mechanism of cell-material interaction under electrical stimulation is still required for further investigation.

Mechanism of Electrical Stimulation on Neural Differentiation
Electrical stimulation, a type of physical stimulus, is important for the overall development of tissues and it promotes the differentiation of neural cells.Electrical stimulation induces migration, proliferation, and differentiation of neural stem cells [80,84,144] Neuronal differentiation, in presence of electrical stimulation, is a complex mechanism that involves several steps, such as the activation of the receptor present on cell surface, reorganization of the cytoskeletal proteins, Ca 2+ efflux and influx, and activation of several downstream cell signaling pathways.This section of the review summarizes the potential mechanism of neural differentiation in presence of electrical stimulation (Fig. 5).
The mechanism behind electrical field-assisted neurite extension or cell migration changes depending upon the cell types.Several studies have identified different mechanisms of cellular migration and neurite extension in presence of an electric field.The phosphatidylinositol 3-kinase (PI3K)/ Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways have been identified as major cell signaling pathways responsible for the migration of the neural stem cells in the presence of electrical stimulation [146][147][148].In a study by Dong et al., it has been shown that Achaete-Scute Family BHLH Transcription Factor 1 (Ascl1) is one of the major mediators of electrical field-induced differentiation of neural stem cells [149].They observed that the amount of expression of Ascl1 was directly proportional to the strength of the electrical field applied.The activation of Ascl1 in turn activates the PI3K-Akt cell signaling axis, which ultimately leads to neuronal differentiation [149].In a different study by Rejnicek et al., a novel pathway, independent of PI3K, MAPK/ERK pathways were identified [150].In this study, neuronal growth cones were observed to be migrated toward the cathode.They claimed that this migration was due to the activation of the cell division cycle 42 (Cdc42), Rac, and Rho proteins.It was also reported that migration and differentiation of induced pluripotent stem cells and neurons were influenced by Rho-kinase signaling pathway [151].Wang et al. reported that biphasic electrical stimulation protected olfactory bulb neural progenitor cells against growth factordeprived apoptosis through the brain-derived neurotrophic factor (BDNF)-PI3K/Akt pathway and promoted neuronal differentiation [152].In another study, Chang et al. reported the combinatorial effect of nerve growth factor and electrical stimulation on neural differentiation.It was observed that this combinatorial approach promoted neurite outgrowth by upregulating the protein kinase C and pERK1/2 signaling pathway [153].
Calcium ions (Ca 2+ ) is a very important element of the body, participating in various cellular processes, including electrical field-induced migration (also known as Galvanotaxis) and differentiation of neural cells.The dynamics of Ca 2+ (efflux and influx) play a very important role in determining the fate of stem cells.Study showed that the cellular level of Ca 2+ and cyclic adenosine monophosphate (cAMP) increased in presence of an electrical field which in turn initiates neurite extension from the neural cells [154].In one study, Ca 2+ was observed to be a mediator of electrical stimulation and help in the differentiation process of embryonic stem cells (ESCs) [155].According to another study, galvanotaxis of neural stem/progenitor cells is induced by neurotransmitter binding to ionotropic receptors, especially NMDA.This neurotransmitter release is mediated by Ca 2+ influx [156].There is some speculation that glutamatergic, GABAergic, and cholinergic receptors possibly play a role in electrical field-induced differentiation and migration of neural stem/progenitor cells [144,157].Along with Ca 2+ ions, Sodium and Potassium ions also play critical role in electrical field-induced neurogenesis.According to the literatures, ion pumps and exchangers, particularly Na + /K + -ATPase pumps and Na + /H + exchangers, assist in establishing the electric fields in vivo to trigger galvanotaxis [158,159].
Several research groups studied the effect of electrical stimulation on these signaling pathways after culturing and differentiating neural cells on carbon nanofiller-reinforced polymeric scaffold.For example, in their study Shao et al. showed differentiation of NSCs on CNT-reinforced PDDA platform in presence of electrical stimulation.The differentiation of the cells occurred through MAPK and PI3-Akt pathway [137].In another study, Ghosh et al. observed enhanced neurogenic differentiation of HT-22 hippocampal neurons cultured on CNT-reinforced PCL-collagen-casted scaffold in presence of electrical stimulation.This work also showed the differentiation of the cells follows PI3-Akt pathway [160].
Overall, the underlying method of electrical stimulation promoting differentiation of stem cells toward neuronal cells is complex and therefore a more in-depth investigation is necessary to completely comprehend it.For that existing investigation methods should be combined with recently emerging techniques such as single-cell sequencing and gene editing and this may aid in the identification of electrical stimulation-induced genes that regulate the differentiation of stem cells toward neuronal cells.

Conclusions and Future Perspectives
This review has elucidated that how electrical properties of the carbon-based nanofiller have been utilized for neural tissue regeneration.Recent advances in neural tissue engineering have led to the fabrication of carbon nanofiller-based biomimetic constructs as an alternative of present clinical therapies.New fabrication techniques and modification of the materials play a key role in these advancements.Although, the researchers have only focused on the effects of electrical stimulation of carbon nanofiller-based scaffold on neural cell differentiation, more study is needed to determine the cytotoxic behavior of the carbon-based nanofillers.Researchers have tried to mitigate the cell cytotoxicity by functionalization of the nanofillers.Yet, comprehensive in vivo studies are required for evaluating of long-biocompatibility and cytotoxicity effects.Many studies have discussed the low solubility of CNT and graphene in water and organic solvents, which leads to their limited use in tissue engineering applications.The surface area, surface topography, and conductivity of the carbon nanofiller strongly depend on its fabrication processes.The incorporation of carbon-based nanofiller into other materials could remarkably enhance the conductivity of composites which can have a potential influence on neural tissue regeneration.The topographic and chemical properties of the carbon-based nanofiller-based scaffold could act as a bridge between regenerating nerve cells.However, the mechanism by which carbon nanofillers interact with neural tissue has not been explained properly till now.There is a dire need of more study on the effect of the interaction of cells with different surface chemistries with diverse topography of carbon-based nanofillers.Carbon nanofiller-incorporated scaffold takes part in neurite growth, and extension and geometrical pattern of this scaffold have the great role to regulate the neurons extension.In light of the conductive properties of carbon-based fillers and the benefits of electrical stimulation for neural tissue engineering, the combination of electric stimulation and carbon-based nanofiller-based scaffold have been produced as an efficient neural regeneration strategy.But the mechanism of cell-material interaction under electrical stimulation is still not clear.
Despite having all these concerns, carbon-based nanofillers successfully deliver the desired mechanical strength and conductivity with different polymers for the fabrication of regenerative templates.These properties are being utilized in additive manufacturing to fabricate the 3D structures, making its application area broader.In a nutshell, after addressing all the challenges, carbon-based nanofillers have a great prospect and can play an astounding role in electric stimulation-based nerve tissue regeneration.

Fig. 1 Fig. 2
Fig. 1 General outline of nerve action potential based on electrochemical mechanism.Figure reprinted from Ref. [42] under a Creative Commons Attribution v3.0 International License (CC BY 3.0)

Fig. 3 a
Fig. 3 a Band structure of graphene, K and Kʹ are also known as the Dirac points.Figure reprinted from Ref. [53] under a Creative Commons Attribution v3.0 International License (CC BY 3.0), b neurite orientation and outgrowth on the PLCL-graphene composite scaffold (SEM and confocal micrographs by DAPI-FITC staining).Fig-

Fig. 4 a
Fig. 4 a Band structure of CNT. Figure reprinted from [103] under a Creative Commons Attribution v3.0 International License (CC BY 3.0) and b influence of SWCNT and electrical stimulation combination on neurite outgrowth.Reproduced with permission from Ref.[117], c synaptic bridge formed between two MWNT bundles and the soma of the neuron is located along the edge of the MWNT.Reproduced with permission from Ref.[118] and d Schematic representation of the interacting mechanism of absorbed ECM proteins with CNT multilayers that controls NSCs biological processes Reproduced with permission from Ref.[137]

Fig. 5
Fig. 5 Schematic diagrams of different pathways involved in electrical stimulation-induced neural differentiation.Figure reprinted from Ref. [145] under a Creative Commons Attribution v3.0 International License (CC BY 3.0)