Recent advances in the application of MXenes for neural tissue engineering and regeneration

Abstract

Transition metal carbides and nitrides (MXenes) are crystal nanomaterials with a number of surface functional groups such as fluorine, hydroxyl, and oxygen, which can be used as carriers for proteins and drugs. MXenes have excellent biocompatibility, electrical conductivity, surface hydrophilicity, mechanical properties and easy surface modification. However, at present, the stability of most MXenes needs to be improved, and more synthesis methods need to be explored. MXenes are good substrates for nerve cell regeneration and nerve reconstruction, which have broad application prospects in the repair of nervous system injury. Regarding the application of MXenes in neuroscience, mainly at the cellular level, the long-term in vivo biosafety and effects also need to be further explored. This review focuses on the progress of using MXenes in nerve regeneration over the last few years; discussing preparation of MXenes and their biocompatibility with different cells as well as the regulation by MXenes of nerve cell regeneration in two-dimensional and three-dimensional environments in vitro. MXenes have great potential in regulating the proliferation, differentiation, and maturation of nerve cells and in promoting regeneration and recovery after nerve injury. In addition, this review also presents the main challenges during optimization processes, such as the preparation of stable MXenes and long-term in vivo biosafety, and further discusses future directions in neural tissue engineering.

Introduction

Peripheral (PNS) and central (CNS) nervous system injuries can cause loss of motor and sensory function, greatly affecting patient quality of life. The PNS and CNS and have insufficient capacity to regenerate after injury; therefore, novel approaches are needed to reconstruct cellular structures and restore function. Neural tissue engineering (NET) has emerged as a promising approach for nervous system regeneration. I It is feasible to apply biomaterials to regulate the interactions with nerve cells and additionally use these signals to direct the development, maturation, proliferation, and differentiation of cells (Kim et al., 2012; Santos et al., 2016; Xiao et al., 2020). Various biomaterials have been used as substrates for neural reconstruction and neuron regeneration (Subramanian et al., 2009). In the development of the nervous system, electrical activity plays a marked role in regulating signal transmission and neuronal network activity. Therefore, electrical nanomaterials like graphene, MXenes, and their derivates have been extensively used to build microenvironments for nerve cells (Fabbro et al., 2016; Olabi et al., 2020; Xiao et al., 2022).

Two-dimensional (2D) transition metal carbides and nitrides (MXenes) are crystal nanomaterials with a number of surface functional groups like fluorine, hydroxyl, and oxygen, which can be used as carriers for proteins or drugs (Huang et al., 2018). MXenes have diverse structures and compositions, and their general formula is M_n+1X_nT_x, where M is the transition metal, X is the carbon or nitrogen, T_x is the surface terminations of the outer transition metal layer, and n ranges from 1 to 4 (Naguib et al., 2014; Anasori et al., 2017). MXenes have high conductivity, surface hydrophilicity, and excellent mechanical properties; the surfaces of MXenes can be easily modified and functionalized, as such MXenes can be used in various areas, such as environmental science (Rasool et al., 2016), physics (Kumar et al., 2017; Xia et al., 2018), and energy production (Anasori et al., 2017; Pang et al., 2019). Moreover, MXenes have considerable application prospects in biomedical engineering, such as in nanomedicine (Han et al., 2018), (electrochemical) biosensing (Wu et al., 2018), antibacterial treatments (Liu et al., 2011; Gao et al., 2022), diagnostic imaging (Dai et al., 2017), theranostics (Huang et al., 2018) and drug delivery system (Li et al., 2018).

The potential MXenes for inducing nerve regeneration include 2D MXene substrates and 3D MXene hydrogel systems also incorporating MXene nanosheets (Guo et al., 2022; Li et al., 2022; Liao et al., 2022; Xiao et al., 2022; Zhang et al., 2022). However, most studies have focused on the cellular level, and there are few in vivo studies. Herein, we comprehensively summarize the preparation and biocompatibility of MXenes and their regulation of nerve cells in MXene-based 2D and 3D culture systems. Finally, we discuss the challenges and future development directions to improve the preparation and biocompatibility of MXenes and applications of MXenes in NTE (Figure 1 and Table 1).

Figure 1.

Applications of MXenes for neural tissue engineering and regeneration.

Application of two-dimensional and three-dimensional MXenes in peripheral and central nervous system. Investigations on the effects of MXenes on neural stem cells, cortical neurons, hippocampal cells, dorsal root ganglion, spiral ganglion neurons (SGNs), organoids, and spinal cord. Created using Adobe Illustrator CS6.

Table 1.

The applications of MXenes in neural tissue engineering

Types	Models	In vitro/In vivo	Toxicity test	Key findings	Studies
Ti3C2Tx MXene	Neural stem cells (NSCs)	In vitro	Yes	Biocompatible, capable of supporting neuron growth and network formation; ensured normal growth of NSCs and enhanced proliferation, led to higher neuronal differentiation efficiency, promoted NSC maturation; promoted neuronal differentiation during strong depolarizations and increased synaptic release frequency, enhancing synaptic transmission.	Wang et al., 2021; Guo et al., 2022; Li et al., 2022
Ti3C2 MXene	Primary cortical neurons	In vitro	Yes	Primary cortical neurons could adhere, grow, and form neuronal networks on Ti3C2 and polystyrene. This material provided a neural interface that has a high-resolution for neuroelectronic devices.	Driscoll et al., 2018
Ti3C2Tx MXene	Dorsal root ganglion	In vitro	Yes	Both flakes and films could generate photothermal stimulation of dorsal root ganglion neurons even under the low incident energy densities.	Wang et al., 2021
Ti3C2Tx MXene	Hippocampal cells	In vitro	Yes	This MXene preserved basic physiological functions in neuronal circuit development.	Xiao et al., 2022
Ti3C2Tx MXene-Matrigel hydrogel	Spiral ganglion neurons	In vitro	Yes	3D Ti3C2Tx MXene system could promote the formation of mature synapses and intercellular signal transmission in spiral ganglion neurons.	Liao et al., 2022
Ti3C2Tx MXene-Matrigel hydrogel	Organoids	In vitro	Yes	Promoted their proliferation, maturation and formation of organoid hair cells, facilitated the establishment of neural connections between hair cells and spiral ganglion neurons, and increased the efficiency of synapse formation.	Zhang et al., 2022
GelMA-MXene hydrogel	Spinal cord	In vitro and in vivo	Yes	Effectively repaired complete transection spinal cord injury and connections between the repaired and regenerated nerves.	Cai et al., 2022

Retrieval Strategy

An online search of the PubMed database was performed to retrieve articles published from inception until December 1, 2022. The following words and their combinations were used to maximize the specificity and sensitivity of the search: “MXenes, NTE, nerve, neuron, nerve regeneration, neural cells, neural stem cells, spiral ganglion neuron, hydrogels, organoid, and spinal cord”. The authors screened the related studies to identify potentially useful studies, first screening titles and abstracts and full texts using the keywords. Only studies addressing MXenes in relation to nerves were included to assess the research on the role of MXenes in NTE. There were no restrictions on language or study type.

Preparation and Properties of MXenes

Preparation of MXenes

MAX phases are a large family containing more than 50 members consisting of transition metal carbides, nitrides, or carbon nitrides (Naguib et al., 2011; Anasori, et al., 2017). The “M” is transition metal element, the “A” is a main family element, and the “X” is nitrogen or carbon (Figure 2A; Naguib et al., 2014). The basic formula can be expressed as M_(n+1)AX_n. Moreover, in the perspective of biomedical engineering, the most popular materials in this family are Ti₃SiC₂, Ti₃C₂T_x, and the multilayer Ti₃C₂ (Huang et al., 2022). MXenes are typically prepared using a top-down stripping method with selective stripping of the A element layer from the precursors to convert to an MAX phase or non-MAX phase (Naguib et al., 2014).

Figure 2.

Preparation and characterization of MXenes.

(A) Schematic representation of MAX phases and MXenes. (B) Characterization of MXenes. i) Scanning electron microscope (SEM) micrograph of the Ti₃AlC₂ showing a typical multilayer block structure. ii) SEM micrograph of multilayered Ti₃AlC₂ showing a multilayer structure in the shape of an accordion. iii) SEM micrograph of the two-dimensional delaminated ultrathin Ti₃C₂T_x MXene nanosheets after ultrasonic exfoliation. iv) Representative transmission electron microscopy image of the Ti₃C₂T_x MXene. The upper right corner is the SAED pattern. v, vi) X-ray diffraction and X-ray photoelectron spectroscopy patterns of Ti₃AlC₂ and Ti₃C₂T_x MXene, exhibiting the elimination of the Al layer after etching. Reprinted with permission from: (A) Naguib et al., 2014, Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim; (B) Dong et al., 2021, Copyright © 2021 American Chemical Society.

This method can be classified according to three aspects: the classes of precursors, the composition of the etchants, and the composition of delamination intercalants (Huang et al., 2018). There are two types of precursor materials used for the fabrication of MXenes: MAX phases and non-MAX phases. The target MXene determines which M_n+1AlX_n with a metal composition is the effective MAX-phase precursor (like Ti₃AlC₂, Ti₂AlC) (Naguib et al., 2011). Single layers or several layered stacks of MXenes can be achieved through selectively etching gel-like ion-binding layers (Alhabeb et al., 2017). Instead of pure metal layers, some non-MAX phase precursors, such as Zr₃Al₃C₅, are etched with the metal-nonmetal layer from the precursor to obtain MXenes (Huang et al., 2018). Based on the composition of etchant, it can be classified into hydrofluoric acid-etching and non-hydrofluoric acid-etching. To avoid the harmfulness of hydrofluoric acid reagents and adapt these materials to biomedical applications, the reaction of the acid and fluoride or molten fluoride have been used to generate hydrofluoric acid to obtain selective etching (Ghidiu et al., 2016; Yu et al., 2017). In addition, the etchant mainly determines the surface termination of MXenes.

To produce monolayer MXenes, simple mechanical peeling is not efficient enough, and intercalators need to be used. Delamination intercalators mainly include metal ion intercalators (such as metal hydroxides or halide salts) and organic intercalators (such as TBAOH) (Alhabeb, et al., 2017; Yu et al., 2017). Dong et al. (2021) reported the procedure of converting multilayer Ti₃AlC₂ into the layered Ti₃C₂T_x MXene. MAX phases have a typical multilayer block structure, which is similar to an accordion structure, after selective etching and 2D nearly transparent ultrathin delaminates after ultrasonic exfoliation (Figure 2B; Dong et al., 2021). There are a few bottom-up synthesis methods of MXenes. Xu et al. (2015) synthesized ultra-thin α-Mo₂C 2D crystals with a transverse size of up to 100 mm on Cu/Mo foil using chemical vapor deposition. They made W and Ta into ultra-thin WC and TaC crystals. The MXenes prepared using their method have a large lateral size. Compared with top-down manufacturing methods, the bottom-up synthesis methods usually start from small organic or inorganic molecules/atoms (Huang et al., 2018). They also have the advantages of manipulating the size distribution and morphology of MXenes, but the surface termination is less controllable. This may be due to the complex composition of MXenes.

In summary, the preparation of MXenes is usually achieved using top-down selective etching to strip the A elemental layer from the precursor MAX phase or non-MAX phase. However, suitable methods are still required for the bottom-up synthesis of MXenes.

Surface modification tactics for MXenes

Proper surface modification can enhance the performance of MXenes mainly using methods such as additive-mediated intercalation and additive-aided chemical modification (Zou et al., 2022). To date, there have been three main classes of mediated intercalations used for MXenes. (1) Molecules (e.g., DMSO and H₂O) can increase the c-LP of Ti₃C₂T_x MXene (Wang et al., 2018). The water dispersion of MXenes can be effectively improved by polyethylene glycol (Xuan et al., 2016). A nanocomposite hydrogel (Ti₃C₂) composed of polyacrylamide and Ti facilitated drug delivery and release (Zhang et al., 2020b). The polymeric molecule polyvinyl alcohol can be used to regulate the thickness of MXenes through a vacuum filtration process (Ling et al., 2014). (2) Cations, including NH₄⁺, H⁺, Li⁺, Na⁺, K⁺, Mg²⁺ and Al³⁺, under appropriate pH conditions (Lukatskaya et al., 2013), can be intercalated spontaneously between Ti₃C₂T_x MXene layers in aqueous solutions (Ghidiu et al., 2014; VahidMohammadi et al., 2019). Larger polyatomic cations, like +N₂-phenyl-SO₃H and [(CH₃)₃NR]⁺, were used to replace the intercalated Li/Na ions and increase the layer spacing of MXenes (Wang et al., 2016; Ghidiu et al., 2017). (3) As an intercalator, the organic base isopropylamine can form ammonium ions (r-NH₃⁺) that can be inserted between the Nb₂CT_x layers (Mashtalir et al., 2015). Other organic bases, such as n-butylamine and choline hydroxide, can reduce the bond energy between the MXene layers (Naguib et al., 2015; Yu et al., 2017). Chemical modification is also effective. The introduced MXene surface terminal groups include halogen terminal (–F, –Cl, –Br), chalcogen terminal (–OH, –O, –S, –Se, –Te) and imino (–NH) groups (Agresti et al., 2019; Velusamy et al., 2019; Deysher et al., 2020; Kamysbayev et al., 2020; VahidMohammadi et al., 2021).

Properties of MXenes

The Ti₃C₂T_x MXene consists of the transition metals nitride and carbide. It has good electrothermal, mechanical, and chemical properties. The structure, composition, surface, and interlayer chemistry of MXenes can determine their physical and (electro)chemical properties. Unlike other 2D materials, the electric properties of MXenes rely on the M, X, and their surface ends (Huang et al., 2018). Because of defects in their surface ends, the synthesis process affects their electronic properties (Anasori et al., 2016). MXenes have optical properties such as light absorption, emission, and scattering (Maleski et al., 2021). MXenes display surface plasmon modes among the visible and near infrared ranges (El-Demellawi et al., 2018; Hantanasirisakul and Gogotsi, 2018) and exhibit strong absorption in the ultraviolet range (Han et al., 2020). The structure, M and X sites, and surface terminations are main factors influencing their optical properties. Studies on the mechanical properties of MXenes have used monolayers of Ti₃C₂T_x and Nb₄C₃T_x (Lipatov et al., 2018). Ti₃C₂T_x MXene films show a high tensile strength (Zhang et al., 2020a), indicating high fracture toughness (VahidMohammadi et al., 2021).

MXenes show good hydrophilicity, surface flexibility, high metal conductivity, biocompatibility, and other characteristics, with an extensive scope of possible uses, including in nanomedicine (Lin et al., 2018), biosensors (Hroncekova et al., 2020; Ramanavicius and Ramanavicius, 2020), biological imaging (Yu et al., 2017; Song et al., 2020), antimicrobial therapy (Rasool et al., 2017), and therapeutic diagnostics (Fu et al., 2019).

Biocompatibility of MXenes

MXenes have already shown great promise in biomedical applications, although their biocompatibility and potential toxicity must be taken into consideration. The biocompatibility of MXenes has been reported in a variety of cell types. Guo et al. (2022) dispersed the Ti₃C₂T_x MXene with abundant surface functional groups on tissue culture polystyrene (TCPS) and cultured neural stem cells (NSCs) on the material after coating it with laminin to investigate regulatory effects on the cell survival and behavior. The researchers found that these cells were cultured on both the Ti₃C₂T_x MXene and TCPS forms with stable adhesion, additionally exhibiting extensive spreading of their terminal extensions (Figure 3Ai). Histology of living and dead NSCs (Figure 3Aii) showed that Ti₃C₂T_x MXenes had good biocompatibility and were an excellent neural interface material. Although the NSCs on these two materials had similar proliferative abilities, the Ti₃C₂T_x MXene film showed more efficient neuronal differentiation (Figure 4A). Moreover, compared with TCPS substrates, cells on Ti₃C₂T_x MXenes had longer neurites, more branches and tips, and were more active, mature, and more highly differentiated, implying that Ti₃C₂T_x MXene was an efficient interface for NSC-derived neurons.

Figure 3.

Biocompatibility of MXene films for neuronal growth .

(A) Scanning electron microscope (SEM) and fluorescence images of neural stem cells (NSCs). i) SEM images of NSCs. ii) Fluorescence immunohistochemistry of live and dead NSCs. The cytoplasm of live cells was marked green, and the nuclei in dead cells were marked red. Scale bar, 50 μm. (B) Representative immunofluorescence images of nerve cells cultured on Ti₃C₂ for 7 days in vitro. Neurons, anti-Tuj1 (red); synapses, anti-synapsin-1 (green); nuclei, 4′,6-diamidino-2-phenylindole (DAPI; blue). A magnified view of the white-boxed region in panel i is shown in ii–iv, and a representation of panel iv is shown in v and vi. The use of arrows and boxes highlights the network formation. The scale bars for i, iv, and vi are 100, 50, and 25 μm, respectively. Reprinted with permission from: (A) Guo et al., 2022, Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. (B) Driscoll et al., 2018, Copyright © 2018 American Chemical Society. TCPS: Tissue culture polystyrene

Figure 4.

Effects of MXenes on neural stem cell differentiation.

(A) Representative fluorescence images of neurons from neural stem cells (NSCs) after 7 days of differentiation on tissue culture polystyrene (TCPS) and MXene film. Neurons, anti-Tuj1 (red); nuclei, 4′,6-diamidino-2-phenylindole (DAPI; blue). (B) Under the strong depolarization condition, Ti₃C₂T_x MXene selectively enhanced the peak value of NSC-derived neurons. i) Representative spontaneous spikes of neural cells (TCPS: black; Ti₃C₂T_x MXene: red). ii) Frequency of spontaneous spikes. iii) Representative voltage responses evoked. iv) Number of spikes (10–100 pA). (C) Representative fluorescence microscopy images of neurons. Neurons, anti-Tuj1 (red); mature neurons, anti-MAP2 (green); GABAergic neurons, anti-GABA (green); presynaptic VGLUT1, anti-VGLUT1 (green). Scale bar, 50 μm. Reprinted with permission from: (A) Guo et al., 2022, Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd.; (B) Li et al., 2022, Copyright © licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/lisceses/by/4.0/); (C) Xiao et al., 2022, reproduced with permission from Royal Society Chemistry, Copyright © 2022 Copyright Clearance Center.

Driscoll et al. (2018) performed nerve recordings in vivo using Ti₃C₂ MXenes and reported good biocompatibility with immortalized tumor cell lines and the rat brain. They investigated the cytotoxicity of Ti₃C₂ films on primary cortical neurons, and immunocytochemistry results exhibited a widespread network on both substrates (Figure 3B; Driscoll et al., 2018). Their results demonstrated that the primary cortical neurons could adhere to, grow on, and form neuronal networks on Ti₃C₂ and TCPS. This material provided a high-resolution neural interface for neuroelectronic devices. This research can greatly broaden the potential applications of the MXene families for neuroscientific research. Zhang et al. (2019a) implanted Ti₃C₂T_x MXenes into Sparague-Dawley rats and found that MXenes were actively taken up via cell-mediated phagocytosis. In another study, a Ti₃C₂T_x MXene film showed no cytotoxicity to NSCs (Zhang et al., 2022), and no cytotoxicity was detected in nerve tissue (Vural et al., 2020; Wu et al., 2020). The above studies show that MXenes are biocompatible and are capable of supporting neuron growth and network formation (Wang et al., 2021; Guo et al., 2022).

Applications of MXenes in Neural Cells

2D MXenes

Among biomedical materials, MXenes show a special potential in regulating the fate of stem cells (Driscoll et al., 2018; Guo et al., 2022; Li et al., 2022). Because of their unique surface structure—which can be functionalized and has great electrical conductivity—MXenes have been used to offer an efficient and appropriate physiochemical environment (VahidMohammadi et al., 2021).

A great challenge in exploring complex neuronal functions is the modulation of neuronal activity (Pisanello et al., 2016; Zimmerman and Tian, 2018). In the family of MXenes, Ti₃C₂T_x MXene can be used for optical modulation of neuronal electrical activity with a high spatiotemporal resolution (Wang et al., 2021). In addition to the aforementioned applications, Wang et al. (2021) measured the photothermal performance of Ti₃C₂T_x MXene at a single-flake level and observed that both the flakes and films could generate photothermal stimulation of dorsal root ganglion neurons, even under low incident energy densities. This method can be combined with biomedical and tissue engineering to build practical neural therapy models (Wang et al., 2019).

Electrical activity is involved with numerous aspects of early neuronal development (Spitzer, 2006), meaning that effective electrical stimulation (ES) can be a good way to regulate the behavior of excitatory cells, such as in nerve regeneration (Zhang et al., 2007; Ghasemi-Mobarakeh et al., 2009). In previous studies, the ES mode that effectively enhanced functional recovery usually employed high-frequency sine wave signals or low-frequency pulse wave signals (Guo et al., 2021, 2022). Several studies applying ES have been conducted in mammalian animal models to promote peripheral nerve regeneration (Capogrosso et al., 2016; Song et al., 2016; Bonizzato et al., 2021; Roh et al., 2022). ES has been reported to play a crucial role in inducing a suitable stem cell response, which greatly affects the proliferation, self-renewal, and differentiation of stem cells (Thrivikraman et al., 2018). Guo et al. (2022) reported that ES could enhance the function of NSCs. The combination of stem cell therapy and biological materials towards the treatment of various neurodegenerative diseases is promising. Therefore, it is necessary to determine whether MXene films can be used as an effective interface for electrical transmission.

Li et al. (2022) reported that ES coupled with Ti₃C₂T_x MXene could ensure normal growth of NSCs and markedly enhance proliferation in NSCs, which also exhibited higher neuronal differentiation efficiency, implying that Ti₃C₂T_x MXenes can promote the maturation of NSCs. Ion channels are known to be essential for the fate and function of nerve cells (Yin et al., 2019). Li et al. (2022) investigated effects of MXenes on ion channels, synaptic connections between cells, and neural network formation in newborn neurons using patch clamp electrophysiology (Figure 4B). There was no significant difference in voltage-gated Na⁺/K⁺ currents when cells were in close contact with Ti₃C₂T_x MXenes, but the amplitude of the voltage-gated Ca²⁺ current was selectively increased, which may account for longer neurites. Further, Ti₃C₂T_x MXene promoted neuronal differentiation during strong depolarizations and increased the frequency of synaptic release, thereby enhancing synaptic transmission. In addition, Xiao et al. (2022) applied an uncoated Ti₃C₂T_x MXene to explore the development of hippocampal cells. Using electrophysiological recordings, they tested the effects of uncoated Ti₃C₂T_x MXenes on cultured neuronal cells and the activity of neuronal microcircuits; this MXene was suggested to preserve basic physiological functions in neuronal circuit development (Figure 4C). Their work makes it possible to construct neural repair devices that can be applied in research, diagnosis, and therapy.

3D MXenes

Effects of 3D MXene on organoids

Organoids are mini-organs with various cell types that are derived from stem cells of various tissues and organs, such as embryonic stem cells or induced pluripotent stem cells (Huch and Koo, 2015; Nengzhuang et al., 2022). They have highly similar cellular compositions and physiological characteristics to real organs, providing marked advances in research on nervous system development and disease (Di Lullo and Kriegstein, 2017). Cochlear organoids (Cochlear-Orgs) have been successfully formed in 3D in vitro using induced pluripotent stem cells and embryonic stem cells (Lee et al., 2017; McLean et al., 2017). However, difficulties remain in the simulation of extracellular environments (Gjorevski et al., 2016; Roccio et al., 2018). To better simulate the cellular environment, Zhang et al. (2022) combined Ti₃C₂T_x MXene nanomaterials with Matrigel to establish a co-culture system between Cochlear-Orgs and spiral ganglion neurons (SGNs) in vitro. The 3D hydrogel containing a certain concentration of Ti₃C₂T_x MXene was appropriate for Cochlea-Orgs and had the ability to promote the formation, maturation, and proliferation of organoid hair cells (Figure 5A; Zhang et al., 2022). In addition, they demonstrated that this 3D Ti₃C₂T_x MXene could facilitate the establishment of neural connections between hair cells and SGNs, and it increased the efficiency of synapse formation.

Figure 5.

MXene-Matrigel regulates the formation of neural networks in hair cells and spiral ganglion neurons.

(A) Co-cultured cochlea organoids and modiolus formed synapse-like contacts. Neurons, anti-Tuj1 (red); hair cells (HCs), anti-Myosin 7a (Myo7a) (green); supporting cells, anti-Sox2 (blue). i–iii) bright field images and representative immunofluorescence images of co-cultured cochlea organoids and modiolus. The representative region is highlighted in iii. Scale bar, 100 μm. (B) 3D Ti₃C₂T_x MXene with electroacoustic stimulation (EAS) positively regulated the growth of spiral ganglion neurons (SGNs). i) 3D Ti₃C₂T_x MXene combined with EAS promoted the functional maturation of SGNs. ii–iv) Side view and SEM images of Matrigel and 3D Ti₃C₂T_x MXene hydrogel. Scale bar is 20 μm. v, vi) Representative immunofluorescence images of SGNs and its growth cones. Scale bar, 5 μm. Reprinted with permission from: (A) Zhang et al., 2022, Copyright © 2022 The Authors. Advanced Science published by Wiley-VCH GmbH; licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/lisceses/by/4.0/); (B) Liao et al., 2022, Copyright © 2022 The Authors. Published by American Chemical Society; licensed under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/lisceses/by/4.0/).

Effects of 3D MXenes on SGNs

The culture of neural cells remains restricted in our ability to regulate the survival, regeneration, and function of SGNs. Low-frequency bidirectional postoperative electrical stimulations has been found to stimulate axonal regeneration in neural cells and to reduce neuron degeneration (Kopelovich et al., 2013). Cochlear implants can convert sound information into electrical signals, which can be precisely delivered to targeting functional SGNs (Ramsden et al., 2012). This may serve as an effective means of stimulation. Liao et al. (2022) constructed a 3D electroacoustic stimulation system that consists of a cochlear implant device and conductive Ti₃C₂T_x MXene-Matrigel hydrogel. The 3D Ti₃C₂T_x MXene hydrogel positively regulated the growth and maturation of SGNs (Figure 5B). In addition, the SGNs exhibited a greater number of synapses and higher calcium oscillation frequencies, suggesting that the 3D Ti₃C₂T_x MXene system could promote the formation of mature synapses and intercellular signal transmission. This research showed the potential value of MXenes in promoting the regeneration and recovery of the auditory nerve after injury.

Effects of 3D MXenes on spinal cord

The spinal cord orchestrates walking and movements, and spinal cord injury can interrupt pathways from the CNS to the lumbar spinal cord (Kathe et al., 2022), leading to some severe neurological disorders such as chronic pain, impaired mobility, autonomic dysfunction, and paralysis (Tate et al., 2020; Zipser et al., 2022). In strategies to repair the spinal cord after injury, the reconnection of injured axons and regenerated neurons and the improvement of the microenvironment are the preferred methods (Zipser et al., 2022). Cai et al. (2022) fabricated a GelMA-MXene hydrogel nerve conduit based on a microgroove hydrogel film. In vitro experiments showed that this 3D MXene system could positively regulate NSC differentiation, direct NSC proliferation, and effectively repair the spinal cord after complete transection by increasing connections between the repaired and regenerated nerves. Therefore, this NSC-loaded 3D Ti₃C₂T_x MXene hydrogel system with a microgroove structure is a potential therapeutic strategy to treat spinal cord injury. However, to the best of our knowledge, there have been no other reports on the efficacy of MXenes in spinal cord injury. Therefore, more comprehensive research and exploration are needed on this aspect in the future.

Limitations

This review had several limitations that should be noted. First, the rapid publication rate of studies on MXenes in NTE means that new results and perspectives are frequently shown. Second, the studies reviewed here were based solely on cells or animal experiments, and there were no clinical trials, posing a high risk of bias or imprecision in the effects shown. Third, this review focused only on articles published in English, and it may lack comprehensive relevant international data.

Conclusions and Future Perspectives

Despite their unique properties, MXenes have received extensive attention in biomedical applications in recent years, including biosensors (Zhang et al., 2019b; Chia et al., 2020), antibacterial treatments (Mao et al., 2020; Yang et al., 2021), bioimaging (Xue et al., 2017; Soleymaniha et al., 2019), diagnostics (Dai et al., 2017; Lin et al., 2018) and therapeutics (Szuplewska et al., 2019). This review summarized the progress of research on using MXenes in nerve regeneration. The toxicity of MXenes in different nerve cells have been analyzed to evaluate the effects and potential applications of MXenes in neurobiology, expecting to provide a certain reference value to design safer MXenes. Although the biocompatibility of Ti₃C₂T_x MXene was confirmed in some neural cells, such as NSCs, SGNs, hippocampal cells, immortalized tumor cell lines, rat brain and spinal cord, most studies were based on cell experiments (Driscoll et al., 2018). The long-term biosafety of MXenes remains unclear and has not been systematically evaluated. This is essential for further neurobiological applications of MXenes. There are still many problems to be solved, such as chronic toxicity, biodistribution, immunocompetence, and other effects of MXenes in small- and large-animal models. Therefore, it is crucial to establish an effective animal model.

In terms of preparation methods, most MXenes are synthetized using top-down etching, which is difficult to control in experimental conditions and the stability of the obtained products needs to be improved. The preparation method, surface modifications, and material size are the key factors affecting the effects of MXenes on nerve regeneration. In the future, more synthesis methods, such as bottom-up synthesis, need to be explored for preparing more stable MXenes with controllable morphology, structure, and properties. Moreover, the properties of MXenes should be further detailed to optimize their applications in neurobiology.

Regarding the applications of MXenes in neuroscience, previous studies are still limited and mainly studied the utility of MXenes for nerve repair and regeneration at a limited cellular level. Further studies are needed to clarify the regulatory mechanisms of MXenes for directly interfacing with targeted neural cells (Fabbro, et al., 2016; Pampaloni et al., 2018). To sufficiently reveal the interplay of the nervous system and MXenes, it is necessary to strictly regulate the dimensions of the material, its components, and its surface functional groups. The material stress characteristics and further application status of 3D MXene systems also need further exploration. In addition, it is vital to establish animal models of nerve injuries to explore implantation routes and functional verification of MXenes in vivo, so as to provide further research basis for clinical applications.

In conclusion, due to their non-toxicity to some types of nerve cells, MXenes have the ability to promote neuronal proliferation, differentiation and regeneration and are materials with excellent biomedical carrier properties. MXenes show considerable application potential in biomedicine, especially in nerve repair and regeneration. The combination of MXenes and other functional materials, such as graphene, Matrigel, and methacrylated gelatin, make multifunctional biomedical applications possible. Overall, MXene-based materials will play an active role in nerve regeneration and repair applications.