Carbon nanomaterials for nerve tissue stimulation and regeneration

Materials Science and Engineering C 34 (2014) 35–49

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Review

Carbon nanomaterials for nerve tissue stimulation and regeneration Aneta Fraczek-Szczypta ⁎ AGH University of Science and Technology, Faculty of Materials Science and Ceramics, Department of Biomaterials, Al. Mickiewicza 30, 30-059 Krakow, Poland

a r t i c l e

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Article history: Received 10 July 2013 Received in revised form 11 September 2013 Accepted 28 September 2013 Available online 8 October 2013 Keywords: Carbon nanomaterials Nerve regeneration and stimulation Central and peripheral nerve system regeneration

a b s t r a c t Nanotechnology offers new perspectives in the field of innovative medicine, especially for reparation and regeneration of irreversibly damaged or diseased nerve tissues due to lack of effective self-repair mechanisms in the peripheral and central nervous systems (PNS and CNS, respectively) of the human body. Carbon nanomaterials, due to their unique physical, chemical and biological properties, are currently considered as promising candidates for applications in regenerative medicine. This chapter discusses the potential applications of various carbon nanomaterials including carbon nanotubes, nanofibers and graphene for regeneration and stimulation of nerve tissue, as well as in drug delivery systems for nerve disease therapy. © 2013 Elsevier B.V. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Central and peripheral nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nanomaterials as next generation biomaterials for nerve tissue regeneration and stimulation . . . . . . . . . . . . . Carbon nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Carbon nanotubes (CNTs) and nanofibers (CNFs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Graphene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Application of CNTs, CNFs and graphene in regeneration and stimulation of nerves . . . . . . . . . . . . . . . . 5.1. CNT in nervous system regeneration and stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1.1. Influence of carbon nanotube properties on nerve tissue regeneration and stimulation—selected examples 5.1.2. Functionalization of CNT surfaces for nerve growth stimulation . . . . . . . . . . . . . . . . . . . 5.2. Carbon nanofibers (CNFs) in nerve system regeneration and stimulation . . . . . . . . . . . . . . . . . . . . 5.3. Graphene in nervous system regeneration and stimulation . . . . . . . . . . . . . . . . . . . . . . . . . 6. Questions relating to the use of carbon nanomaterials in the nervous system . . . . . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Due to the complexity of the nervous system anatomy and function, repairing damaged nerves, as well as recovering the full function of injured nerves, have been challenging compared with the treatment of other tissues. Worldwide, approximately two million people live with a spinal cord injury [1]. In the United States alone, there are about ⁎ Corresponding author. Tel.: +48 126173759. E-mail address: [email protected]. 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.09.038

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250,000–400,000 people living with a spinal cord injury and nearly 13,000 additional people are subjected to spinal cord injuries each year [2]. Patients are typically in two age groups: young people up to the age of 15 and middle-aged between 30 and 50years of age [3]. Injury in the peripheral nervous system (PNS), though generally less potentially debilitating than injury to the central nervous system (CNS), is much more common. Tens of thousands of peripheral nerve repair procedures are performed each year [4]. With the increasing age and population of the world, a greater number of patients will need various neural implants allowing for the full restoration of damaged nerve function.

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The human nerve system consists of the CNS, including the brain and spinal cord, and the PNS consisting of the nerves and ganglia outside of the brain and spinal cord. Nerve injuries are complicated and are serious clinical and social problems in the world. Injuries of the PNS are often capable of spontaneously healing after traumatic injury; damaged CNS tissue does not regenerate in the same manner. The current treatment of CNS injury, particularly that of the spinal cord, relies on minimizing secondary injury and implementing physical therapy designed to help a patient function with limited mobility. Treatment to restore healthy tissue and regain sensory and motor function is still not a reality. Developments in CNS tissue regeneration aim to ultimately provide a method for repairing functional tissue and restoring sensory and motor function [4]. The CNS system lacks Schwann cells to promote axonal growth and, more importantly, the thick glial scar tissue may be formed mainly by astrocyte and meningeal cell activity resulting in an unfavorable environment, which inhibits neural regeneration [5]. Nerve tissue defects within the PNS may heal without surgical intervention if the loss is small. However, where the defect is greater, it is required to connect the nerve stumps with tubular prostheses. Usually allografts and autografts, such as cutaneous nerves or blood vessels, serve as connectors. However, very often the allografts and autografts are not sufficient, therefore, tubular implants are used. Despite intensive research on tubular implants with natural and synthetic polymers, an implant that meets all the requirements for nerve regeneration has still not been designed. There are high hopes for the progress of research on regeneration and stimulation of the nervous system associated with nanotechnology, in particular nanomaterials. It is expected that nanomaterials will be able, on the one hand, to prevent the activity of astrocytes (in CNS) and, on the other, to stimulate axon growth and the restoration of synaptic connections. Carbon nanomaterials are proposed as promising candidates for nerve stimulation and regeneration. Currently, the best known are the three types of carbon nanostructures: carbon nanotubes (CNTs), carbon nanofibers (CNFs) and graphene. Carbon nanostructures have unique mechanical, electrical and physicochemical properties, and their shape (CNTs and CNFs) is similar to neurites. Biostable CNTs are attempted to be used as implants where long-term extracellular molecular cues for neurite outgrowth are necessary, e.g. in regeneration after spinal cord or brain injury [6]. Moreover, these materials can be fictionalized and modified chemically using biomolecules stimulating neurite growth. The chemical and biological modification of carbon nanomaterials produces various surface charges affecting the nerve response. Moreover, the surface charge can influence the length of neurite outgrowth, their number, branching and the number of synaptic connections. 2. Central and peripheral nerve regeneration Problems with regeneration in the CNS are the result of several processes that take place after injury. These processes are designed to restore the blood–brain barrier and prevent further (secondary) tissue damage as well as to create an environment that is not favorable for nerve regeneration. Nerve damage induces astrocytes (glial cells) to increase their activity and, thus, to create a glial scar. Astrocytes migrate, proliferate, increase in size, and produce a glial scar rich in extracellular matrix (ECM) proteins, myelin and oligodendrocytes. Active astrocytes release proteoglycans including chondroitin sulfate proteoglycans (CSPGs), which are known as aggressive inhibitors of axon outgrowth. Glial scars can prevent axon regrowth through the creation of chemical and physical barriers. To be able to rebuild nerves, it is necessary to have free space through which axons can grow. The goals of all regeneration strategies in the CNS are generally to moderate reactive gliosis while promoting axon growth and tissue regeneration [4]. Peripheral nerve damage is typically, but not exclusively, caused by traumatic injury. It may also be a result of complications of orthopedic

surgery. The peripheral nerve regeneration process runs in a few phases. In the first phase, multiple pathophysiologic events occur that are described as Wallerian degeneration. This process is connected with myelin sheaths and nerve fibers break up that greatly depends on cells called macrophages. These cells, excluding their phagocytic properties, play an important role in supporting the nerve fiber reconstruction process, called regeneration. They produce various cytokines, which stimulate Schwann cell proliferation and also production of neurotrophic substances, such as NGF (Nerve Growth Factor). These substances reach the cell body by retrograde axonal transport and stimulate the expression of genes whose products are responsible for axonal regeneration. Schwann cells build characteristic bands, or tubes of Büngner, within which axons grow [1,7,8]. The axon growth rate is estimated at about 0.5–1 mm/day [9]. It follows that in a few days several millimeters of distance can be overcome; however, reinnervation of target tissues may take place months. It is important to note that mature neurons do not undergo mitosis. Thus, supporting the regrowth of axons from existing cells to distal targets is the goal of a nerve guidance channel (NGC) [10]. Very often defects of peripheral nerves are greater than 2cm, therefore, the implant should be designed to provide directional axon growth from the proximal to the distal nerve stumps to allow for synaptic connections. Usually allografts and autografts, such as cutaneous nerves or blood vessels, are used as nerve conduits. Among the materials of no biological origin, natural and synthetic polymers are the most common for producing peripheral nerve prostheses. These materials include polymers such as silicone, collagen, alginate, laminin, chitosan, hyaluronic acid, polylactide (PLA), polyglycolide (PGA), copolymers PLA with PGA and polycaprolactone (PCL) and others [11–13]. Surgery and implantation in the nervous system have a variety of problems not currently satisfying the high-performance demands necessary for today's patient. Specifically, for autografts taken from other sites of the body, it is frequently difficult to obtain enough donor nerves, and this deficiency of available tissue may result in functional impairment [14,15]. In addition, allografts and xenografts are associated with a risk of transmission of diseases and stimulation of the foreign body response, which is often the main cause of implantation failure [16,17]. Some implants e.g., silicone prostheses, strongly stimulate fibrous tissue formation, which consumes tube space limits or even stops regeneration and the migration of axons from the proximal to distal stump. Silicon probes or other metal alloys of neural electrode-based prostheses are frequently encapsulated by a dense glial scar tissue in the brain [18,19]; this significantly decreases the electrical conductivity between the probe and tissue, significantly impairs the efficiency of electrostimulation and makes the probes useless during therapy. Other biomaterials, including various polymers used for manufacture of nerve conduits, may be limited by their mismatch of mechanical and electrical properties as well as lowered biocompatibility [2]. In contrast, in recent years, nanomaterials have become promising candidates for a variety of tissue engineering applications. Nanomaterials are materials that perform structural configuration and morphology at the nanometric range i.e., at least one dimension less than 100 nm. Due to a large range of properties, nanomaterials can be engineered to interact with cells and proteins with a greater degree of specificity [4]. 3. Nanomaterials as next generation biomaterials for nerve tissue regeneration and stimulation Nanoscale materials have a number of unusual properties that are unreachable for the materials at the microscale. Thanks to these properties, nanomaterials have the opportunity for use in areas of far reach of traditional materials. First, nanomaterials have a larger surface area in comparison with conventional materials. The specific surface area of a given mass of nanoparticles is greater than the specific surface area of the same dose of microscale particles. The increased

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surface-area-to-volume ratio and surface area allow for a greater degree of surface interactions. Large surface development of nanomaterials improves adsorption capability of proteins and other biomolecules on their surface, which is important in medical applications. Nanomaterials enable to reflect nature compared to micrometer-scale materials, therefore, they are deemed as biomimetic materials. A material with nanoroughness more accurately resembles native tissue, protein adsorption to nanomaterials compared to traditional materials. Extracellular matrix (EMC) proteins including laminin, fibronectin, and collagen fibrils have nanoscale dimensions. The presence of proteins on the material's surface is a prerequisite for cell adhesion and further processing such as flattening, growth, proliferation and differentiation of cells. All cells exist within an ECM, the three-dimensional network of proteins that provide structural support for a biological tissue. ECM proteins interact with cell-surface receptors to regulate gene expression and cell function. Cellular interactions with a nanoscale biomaterial may provide a more physiologically activated cell surface receptor for improved interactions and greater tissue regeneration. Therefore, these materials can provide a substrate for tissue regeneration [4]. Appropriate electrical conductivity is a very important factor in nerve cell responses, as recognized in many publications [20,21]. Enhanced neural tissue regeneration with applied electrical stimulation has been observed both in the CNS and PNS. Direct current electrical stimulation is known to enhance and direct neurite outgrowth [22]. In addition, in vivo models using dogs with injured spinal cords showed that an oscillating electrical field of 500–600mV/mm significantly accelerated the return of sensory and motor function at time points of 6 weeks and 6 months [23]. Some nanoparticles, especially carbon nanoparticles, are characterized by excellent electrical properties. These materials have a higher electron mobility potential compared to conductive polymers, the latter of which are sometimes used in nerve regeneration [24–26]. Recreating normal neural cell function depends not only on achieving cell attachment and growth, but also on maintaining appropriate resting membrane potentials and cell signaling [26]. The extracellular matrix is composed of proteins such as laminin, fibronectin and collagen, which support the growth of nerve cells but do not conduct electrical signals and are required for the many types of devices for nerve tissue stimulation and regeneration. Moreover, the surface properties for deposition of nerve cells have a huge impact on their future behavior. As shown in Fig. 1, CNT-based substrates offer the possibility of exposing cells to different kinds of stimuli that can be controlled by taking advantage of the unique properties of nanotubes increasing the number of results indicating that certain properties of the nanotubes are particularly important for neural cell growth and function. Of particular importance of such material properties is nanoscale topography, surface charge, bulk electrical conductivity, etc. [26]. CNT, CNF and graphene have the potential to provide a substrate that supports cell growth, while also electrically integrating the cells to their substrate [26].

4. Carbon nanomaterials 4.1. Carbon nanotubes (CNTs) and nanofibers (CNFs) Carbon nanotubes (CNTs) due to their enhanced thermal, electronic, mechanical, and biological properties are being produced in increasingly large quantities for many technical and medical applications. Until now, CNTs have not only attracted enormous research interest but also stimulated CNT-related applications and industrial development. This can be seen through the fact that more than 77,000 articles devoted to CNTs have been published (ISI database, February 2012), and many CNT-relevant products are available on the market [27]. Geometrically, a CNT can be constructed by rolling a piece of graphene to create a seamless nanometer-scale cylinder. Depending on the number of shells, CNTs can be grouped into single-wall CNTs (SWCNTs) and multi-wall

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Fig. 1. CNT substrates can be tailored to exhibit physical, chemical and electrical properties that, in turn, affect neural cell attachment, growth and function [26].

CNTs (MWCNTs). Industrial application and device integration of CNTs require large quantities of bulk materials and scalable production. The methods of synthesis of carbon nanotubes are primarily electric arc discharge, laser ablation and chemical vapor deposition (CVD). Electric arc discharge and laser ablation require very high energy input, replacement of the spent carbon target, and re-evaculation of reactors, which pose difficulties for economic large-scale production of CNTs. In contrast, CVD methods have gained enormous popularity due to their simple operations and flexible processing conditions performing the greatest potential to achieve continuous mass production and integration of CNTs [28]. The unusual mechanical properties of the CNTs result from strong interactions between the carbon atoms in the C–C sp2 hybridization. Young's modulus of free from defects, multi-walled carbon nanotubes, according to some sources is 1.8 TPa [29], and for single-walled carbon nanotubes is 1.25 TPa [30]. On the contrary, when the CNT structure contains the defects this value is reduced to 10–50 GPa [31]. The estimated theoretical value of thermal conductivity of carbon nanotubes at room temperature is 6600 W/mK for SWCNTs [32] and 3000 W/mK for MWCNTs [33]. Carbon nanotubes can transmit the electrical current at the densities higher than 107 A/cm2, with a diameter of 5–25 nm without evidence of destruction in their structure [34]. In the literature, there are reports on ballistic conductivity of carbon nanotubes without structural defects. Ballistic conduction occurs when the length of the conductor is smaller than the mean free path of the electron. It has been observed that the transport of electric charges in the nanotubes can proceed over a length of up to 10 μm without collisions with other atoms and structural defects. Thus, in the case of carbon nanotubes characterized by ballistic conductance there is no energy dissipation in an electric conductor. Joule heat emitted during the current flow is scattered in the electrical wiring connecting the ballistic conductor with elements of the macroscopic system [35]. The small size, big development area, high sensitivity, high speed signal generation and reversibility at room temperature of carbon nanotubes make them an ideal candidate for biosensors. One of the advantages of nanotube biosensors is their size and sensitivity making them able to respond to a very small amount of a substance that, in the case of conventional biosensors, would be impossible to detect. The

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mechanical resistance of the nanotubes also affects the time of “life”, and the destruction of the biosensor is limited during repeated contact with the substance to be assayed. Other advantages of biosensor elements made of CNT are their high chemical stability and a better conductivity than the traditional materials. At present, CNTs are investigated as drug and gene carriers [36–40], as membrane elements, materials for tissue engineering [41,42], as well as scaffolds for cell growth [43,44] using the effects of electrostimulation. In vitro experiments showed that carbon nanotubes can be used to mimic neural fibers for neuronal growth [45,46]. The surface of CNTs can additionally be subjected to chemical processing to create electrically charged sites, capable of fixing various biomolecules [47–49]. On the other hand, CNTs are very mobile within the living body and easily migrate through biological membranes, skin, hair follicles and the respiratory and alimentary tracts, and diffuse through biological tissue and cellular membranes [50,51]. These features make CNTs useful materials as drug and gene carriers. Their mobility within living systems is a highly advantageous feature in diagnostics, gene transport and drug delivery devices. Attempts have also been made to design CNTs with specific shapes and properties in order to produce a new class of biocompatible composite implant [41,52,53]. Polymeric matrices modified with CNTs proved to have a greater biocompatibility in contact with cell cultures than pure polymers [54]. CNTs, due to their relative large length-to-diameter aspect ratio and very large specific surface, are suitable for highly sensitive molecular detection and recognition. Consequently, a large fraction of the CNT surface can be modified with functional groups of various complexities, which would modulate its in vivo and in vitro behavior [55,56]. Carbon nanofibers (CNF) are manufactured using different methods, although the most popular is vapor grown carbon nanofibers (VGCNFs) [57–59] and carbonization of polymer-based nanofibers obtained during the electrospinning process [60–63]. The most popular copolymer precursor for the production of carbon nanofibers is polyacrylonitrile (PAN). In the electrospinning process, a high voltage is applied to create an electrically charged jet of polymer solution or melt, which dries or solidifies to form a polymer nanofiber [63,64]. The carbon fibers from the polymer precursor are obtained in two step process: the stabilization and carbonization, like in the manufacture of common PAN-based carbon fibers. During stabilization, the precursor fiber is heated to a temperature in the range of 180–300 °C from 0.5 to 2 h [60,62]. Too low temperatures lead to slowing-down of reactions and incomplete stabilization, whereas too high temperatures may lead to fusing or burning the fibers [60,65]. Carbon nanofibers have gained considerable importance in the past two decades due to their very high specific surface [60]. Nanofibers have found applications in many branches including textiles, environment and medicine. Vapor grown carbon nanofibers (VGCNFs) constitute a pyrolytic product made by pyrolyzing hydrocarbon compounds in the presence of a transition metal catalyst (Fe, Co, Ni) in a hydrogen atmosphere [59]. In general, the typical lengths and diameters of carbon nanofibers are in the ranges of 5–100 mm and 5–500 nm, respectively. Although CNFs have been known for a long time, their chemical similarity to CNTs, a unique structure and potential for particular applications have also drawn a lot of research interest in their synthesis [66]. VGCNFs are much cheaper and easier to synthesize than nanotubes even if their individual thermal, electrical and mechanical properties are not ostensibly as impressive. However, their properties, such as high strength and electric conductivity and special functional properties are of such interest that scientists have shown a great deal of attention to the mass production of these materials [59]. The diameter of the nanofibers is governed by the size of the catalyst particles, whereas for the mass production of VGCNF's the key process is the seeding of the catalyst particles [67]. The initial particle size of the catalyst must be very small (b15 nm for Fe) because a considerable decrease in activity is observed when the catalyst particle diameter increases. Thus, it

is very important to retain the small catalyst particle size in the flowing gas and to avoid particle coagulation to larger ineffective diameters [68,69]. Iron particles begin extruding long slender hollow filaments of graphitic carbon at temperatures below 900 °C. Fibers begin to grow rapidly for several minutes until the catalyst is deactivated or when they leave the reaction zone [69]. The scheme of VGCNFs is presented in Fig. 2. 4.2. Graphene Graphene, like carbon nanotubes, belongs to one of the allotropic forms of carbon. It constitutes a planar monolayer of carbon atoms arranged into a two-dimensional (2D) honeycomb lattice with a carbon-to-carbon interatomic length, aC–C, of 1.42 Å [70]. Each atom has one “s” orbital and two in-plane “p” orbitals contributing to the mechanical stability of the carbon sheet [70]. Research devoted to graphene structure began to rise in the late 20th century, and after 2004, when Geim and Novoselov in Manchester [71] for the first time isolated a single layer and studied it, interest in this nanoform of carbon became enormous (more than 3000 articles published in the last 5 years) [70]. Considerable interest in graphene is mainly due to its unusual properties including high electron mobility at room temperature (250,000 cm2/Vs), exceptional thermal conductivity (5000 Wm− 1 K− 1), and superior mechanical properties with Young's modulus of 1 TPa and strength of 130 GPa as well as the highest theoretical specific surface area (2600 m2/g) [71–73]. Various attempts were made to synthesize graphene including the same approach as for the manufacture of carbon nanotubes, CVD method on metal surfaces, or the thermal decomposition of SiC [72]. Currently, the most commonly used methods to obtain graphene are exfoliation of graphite structure and cleavage, CVD, and chemically derived graphene. The first of these techniques includes mechanical separation of weakly bonded graphene layers in graphite. With an inter-layer van der Waals interaction energy of about 2 eV/ nm2, the order of magnitude of the force required to exfoliate graphite is about 300 nN/μm2 [74]. This extremely low-impact force can be easily overcome by using an adhesive tape—a method used for the first time

Fig. 2. Scheme of the vapor grown carbon nanofibers. A—During the saturation phase, an iron particle is loaded with carbon from the gas phase. A graphitic carbon filament then precipitates and lengthens as more carbon is supplied by the gas phase. The graphitic planes are conical and intersect the central hollow along the fiber axis at a highly oblique angle. Finally, the filament is thickened by vapor deposited carbon. B—Concentric cones of graphitic planes [69].

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by Geim and Novoselov [71]. In addition to the mechanical exfoliation method to produce graphene sheets, several promising approaches including epitaxial growth from SiC and CVD on metal surfaces have been reported [72]. CVD technique is used for the large-scale production of both mono- and multi-layer graphene films. However, to manufacture a single graphene layer a metal catalyst is required. Nickel is the most commonly used metallic catalyst for the synthesis of graphene. Currently, the method of converting graphite to oxidized graphene is the most prospective method to provide substantial amounts of graphene [75]. This is a multistage process consisting of graphite oxidation, exfoliation and reduction. Chemical reduction of graphene oxide sheets has been performed with several reducing agents including hydrazine and sodium borohydrate [72,76,77]. The high electronic conductivity of graphene is due to its very high structural ordering and the low-defect density of its crystal lattice. Defects scatter and inhibit the charge transfer and as a result, the electron mean free path is small. High electron mobility in graphene at room temperature, sensitivity to field effects and large lateral extension turned graphene into an alternative structure to carbon nanotubes for field-effect transistor devices [70]. The mobility of charge carriers remains high even at the highest electric fieldinduced carrier concentrations, suggesting the possibility of achieving a ballistic transport regime on a submicrometer scale at 300 K [70,78]. Sensing is a complex function requiring the integration of a number of properties from interface accessibility to transduction efficiency, molecular sensitivity and mechanical or electrical robustness. Graphene has all the necessary features for designing sensors with high selectivity and single-atom sensitivity. Indeed, suspended graphene is a pure interface with all atoms exposed to the environment. In addition, graphene can be modified chemically by what obtained on its basis sensors are characterized by greater selectivity. Electronic and mechanical properties can be exploited to perform the transduction of the sensing signal. Compared to carbon nanotubes, which are currently considered for sensing platforms, graphene and its oxidized forms are produced at significantly lower costs [70,79].

5. Application of CNTs, CNFs and graphene in regeneration and stimulation of nerves Since natural neural tissues have many nanostructured features (such as nanostructured extracellular matrices that neural cells interact with), CNFs and CNFs, which also have such nanofeatures and exceptional electrical, mechanical and biological properties, are excellent candidates for neural tissue repair. CNTs can be produced using different techniques such as chemical vapor deposition (CVD) and arc discharge. In addition, by controlling multiple process parameters many features of CNTs such as their dimension, structure and properties can be operated in a controlled manner. Many properties such as electrical, mechanical, biostability and an appropriate length-to-diameter ratio make nanotubes important in the field of nerve tissue stimulation and regeneration [2,41,80,81]. Similarly, CNFs possess attractive parameters from the nerve tissue regeneration point of view, but their cost of fabrication is lower and their scale-up process is easier in comparison to CNTs [2,82]. Compared with the conventional technologies, CNFs and CNTs proved to have great potential as electrical and chemical neural interfaces, for their superior electrical, chemical, and physical properties as follows: (1) chemically stable and inert in physiological environment, (2) biocompatible for long-term implantation due to their covalent carbon structure, (3) electrically robust and conductive for signal detection, (4) 3D structures that allow intratissue and intracellular penetration, (5) high surface-to-volume ratio, which reduces contacting electrical impedance greatly, and (6) high spatial resolution due to their ultramicro-scale sizes [83].

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Besides already confirmed promising properties of CNTs and CNFs used in neural electrodes, graphene-based carbon constituent also offers another opportunity to develop friendly and special-purpose interfacial materials for neural systems, such as neural chips, implanted electrodes and drug/gene vectors [84].

5.1. CNT in nervous system regeneration and stimulation Deep brain stimulation (DBS) Electrodes for deep brain stimulation (DBS) and scaffolds for nervous tissue regeneration within the CNS and PNS are the most perspective application of carbon nanomaterials, especially carbon nanotubes and nanofibers. Current neuromodulation techniques for DBS have major drawbacks, such as large size, lack of feedback monitoring of brain electrical activity, high electrical current needs, and hemorrhage within the brain caused by the number of microelectrode passes through the brain. Moreover, the generator/battery, which is placed in the subcutaneous tissue of the chest or abdomen, must be replaced every 3–5 years by opening the incision on the chest. DBS uses electrodes approximately 1mm in diameter to continuously stimulate specific regions of the brain. Application of the DBS electrodes gives the possibility of treating diseases such as Parkinson's disease, essential tremor, dystonia, and similar movement disorders that have exhausted pharmacologic treatments. Moreover, other applications of DBS are intractable epilepsy, obsessive–compulsive disorder, severe depression, and obesity [85]. Carbon nanotube-based nanoelectrode arrays address these drawbacks and offer the possibility of monitoring neurotransmitter levels at the synapse/neuronal level in real time. Scientists from the NASA Ames Research Center in California have been working for several years on the electrochemical properties of CNTs, with the possibility of their use for neuromonitoring and neuromodulation in DBS [86]. The electrical capacitance of pristine CNT arrays is 10 times higher in comparison with noble-metal microelectrodes; whereas for CNT modified with polypyrrole (PPy) this parameter is over 100 times higher. Similarly, the CNT nanoelectrode arrays resistance is reduced as compared to standard metal microelectrodes. A preliminary study of CNT nanoelectrode arrays for neurotransmitter detection also shows the superiority of the device in relation to carbon microfiber (CMF) electrodes. CNT nanoarrays show a 10fold improvement in dopamine detection levels over the ~ 0.5 μM concentration of dopamine required for CMF detection [85,87]. Scaffold for nerve regeneration Carbon nanomaterials, especially CNTs and CNFs, can be used as scaffolds for nerve regeneration within the CNS and PNS. One of the major problems in surgical treatment of peripheral nerve injuries is nerve anastomose in cases when large nerve tissue loss between nerve ends exists [4,88]. Three-dimensional materials that are being used as scaffolds for tissue regeneration may take place of the actually used classical method, which is nerve anastomose with use of cutaneous nerve allo- and autografts [89]. There are many disadvantages of the classical method, such as limited availability and length of donor nerve tissue, skin denervation in the area related to harvested nerve, second operative location, and risk of infection in the region of nerve harvesting [90]. Several types of materials previously described have been proposed as peripheral nerve substitutes. However, these solutions are not fully satisfactory. CNS lesions represent a major challenge to modern medicine for several reasons that include their long-term impact on the quality of a patient's life, their widespread occurrence and their high medical and social costs [91]. Modern technology offers many solutions for rapid intervention to prevent the worsening of damage. In any case, the reconstruction of neurons in a functional network must meet the following conditions: regrowth of axons and dendrites, appropriate direction of their growth, recognition of the target cell by the axon and restoration

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of synaptic connections. The approach to deal with nerve damage in the CNS and PNS is to create a bridge between the lost nerve and to ensure proper axonal growth. The difficulty in axon regeneration in the spinal cord is largely due to glial scar formation and the release of inhibitory factors following spinal cord injury (SCI) [10,92,93]. Following SCI, the lesion is invaded by fibroblasts, macrophages, and glial cells, and a dense glial scar forms. The myelin sheath also degenerates. Both the glial scar and myelin breakdown products become barriers to axon regeneration [93–95]. Nerve tissue bridges need to provide a bioactive scaffold for axon regrowth and guidance to proper targets [96]. This material should mimic the properties of the extracellular matrix which allows for the growth of axons and at the same time reduce the growth of other cell types while preventing the formation of glial scars [97–99]. Ideal implants for nerve grafting should be biocompatible with extremely low inflammatory, immunogenic and cytotoxic responses, biodegradable, permeable, and stimulating nerve regeneration. Moreover, the implants should have ability for easy chemical incorporation of nerve growth factors, appropriate biomechanical and physical characteristics to mimic the overall properties of nerves and simultaneously stimulate neuronal interactions [12]. Already in 2000, Matson et al. pointed out that a large gap in our understanding of the regulation of neurite outgrowth exists because of the lack of methods for manipulating the growth environment at the nanometer scale [100]. Although there have since been a number of approaches in neural prosthetics at the nanoscale by using, e.g. carbon nanotubes and semiconductor nanoparticles, the material engineering of functional and stable neural/electronic interfaces remains a crucial research area [86,100–103]. The major challenge for the engineering and application of neuroprosthetic implants constitutes the establishment of a bi-directional flow of information between a conductive nanomaterial and the neural systems [104–106]. Carbon nanotubes are strong, electrically conductive and hollow structures of carbon that might conduct electrical signals to neurons stimulating axons to grow and regenerate. The structure of CNTs can provide a scaffold for nerve cell growth and elongation between the severed nerves [107]. Polymer modified with carbon nanoparticles can be considered as a scaffold for nerve regeneration and for stem cell differentiation toward the nerve cells. Tzu-I Chao et al. observed that surfaces coated with poly(methacrylic acid) (PMAA)-grafted CNTs offers a desirable environment with physical and chemical cues allowing direct attachment of human embryonic stem cells (hESC) and differentiation of these ESC colonies into neuronal cells. This proves that relatively mature nerve cells can be obtained directly from hESCs using polymer-grafted CNT scaffolds [108]. Similar results were observed by Chi-Shuo Chen et al. who showed that hESCs cultured on the silk-CNT scaffold exhibited higher maturity along with dense axonal projections [109]. Such scaffolds have great potential in nerve damage repair, nerve tissue engineering as well as neuron prosthesis [108]. Jin et al. observed the influence of MWCNT-coated electrospun poly (L-lactic acid-co-caprolactone) (PLCL) nanofibers on rat dorsal root ganglia (DRG) neurons. It was found that carbon nanotubes successfully modified the surfaces of the nano-fibrous polymer scaffolds while contributing to the stimulation of neurite outgrowth [110]. 5.1.1. Influence of carbon nanotube properties on nerve tissue regeneration and stimulation—selected examples The dimensions, excellent electrical properties and high specific surface area of carbon nanotubes favor their interaction with the distal dendrites of nerve cells by creating new opportunities for their use in the construction of novel nanoengineered neural devices with remarkable properties [111–113]. The electronic properties of the nanostructured carbon can be tailored to match the charge transport required for electrical cellular interfacing. In addition, chemically stable properties of the CNT facilitate integration with neural tissues [84,114]. SWCNTs and MWCNTs are of great interest as a potential scaffold for the restoration of interconnections between neurons

[100,101,111,115–117]; they are an ideal material for long-term neural implants, as they enhance the electrical recording of neurons in culture and in animals [96,113,118], thereby, reducing the impedance between the device and the cell membrane [113,114]. Carbon nanotubes are obtained by various methods using different processing conditions (temperature, pressure, atmosphere). Several post-synthetic approaches may indirectly mediate the biological effects of carbon nanotubes, together with their variable dimensions, lengths, number of walls, degrees of purity, and metal content [113]. Different variables in the synthesis process, purification and functionalization of carbon nanotubes, should always be taken into account before proceeding to biological research. Hence, before biological assessment detailed studies of the physicochemical properties of the material should be carried out. In carbon nanotube–neuron hybrid networks, the cultured neurons always display a boost in signal transmission, which is detected as an increase in the frequency of synaptic events [101,113,119]. Indeed, the electrophysiological properties of neurons grown on CNTs and the functional status of their synaptic connections were addressed by Lovat et al. [121]. The authors reported for the first time the effects of CNT substrates on the electrical behavior of neurons and neuronal networks in culture. They compared neuronal cell survival on glass surfaces (control sample) and glass surfaces covered with CNTs. MWCNT substrates boosted neuronal network activity under culturing conditions in comparison with control plain glass surfaces [101]. Similar effects were found for SWCNTs [119]. The scanning electron microscopy micrographs show that healthy hippocampal neurons adhered and grew on SWCNT substrates demonstrating typical cell body morphology and size (Fig. 3). As shown in Fig. 3, neuronal growth was accompanied by a variable degree of neurite extension on the SWCNT mat, similar to those observed for neurons grown on control, peptidefree glass surfaces and confirmed by immunocytochemical analysis [101,119]. A more detailed analysis of SEM micrographs revealed some significant details regarding the tight interactions between the cell membranes and SWCNTs at the level of neuronal processes and cell surfaces [119]. Silva and Cellot et al. indicates a significant impact of the substrate roughness on this interaction. The mean roughness of the MWCNT film, measured by AFM, was 30.7 ± 4.0 nm. This value allows for tight contacts between the substrate and the neuronal cell membrane [111,113,120]. These contacts can be a physical conduit for the electrical coupling between SWCNT and neurons because stimulation was reliably caused by SWCNT postsynaptic responses in neurons [121]. Moreover, neurons on the carbon nanotube surface are characterized by increased excitability, resulting from an increase in the frequency of action potential discharge (Fig. 4). Growth of neurons on the surface coated with the nanotube accompanied a significant increase in the network activity. Since there is no evidence of a chemical interaction between the nonfunctionalized CNTs and neurons, it is likely that the electrical conductivity of the CNT substrate may be the underlying cause of this specific electrophysiological effect [101,121]. A prerequisite for the application of CNTs as elements of biomedical devices for nerve stimulation is an appropriate understanding of their activities on neurons, in particular in relation to neuronal excitability, changes in ionic conductivity and intercellular signaling through synaptic transmission. This knowledge would certainly be helpful in the design and construction of CNT-based neuronal interfaces while minimizing unwanted interactions. The studies of Lovat et al. and Mazzatenta et al. have shown the impact of the electrical conductivity of carbon nanotubes on neuron growth. These results strongly suggested that growing neurons on conductive CNT platforms promoted a distinct increase in network operation. However, the number of surviving neurons on the CNT substrate was not increased compared to the unmodified substrate (glass coverslip) [101,119]. The authors speculated that CNTs may provide a bi-directional electronic current flow, causing a redistribution of charge along the membrane surface, ultimately increasing neuronal excitability

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Fig. 3. Scanning electron microscopy images of cultured hippocampal neurons on SWCNTs. A—High-magnification micrograph showing SWCNT details. B–D—Subsequent micrographs at higher magnifications of neurons grown on SWCNTs. E, F—Details of the framed area in D. At higher magnifications, the intimate contacts between bundles of SWCNT and a neuronal membrane are clearly shown. Scale bar: A, 1 μm; B, 200 μm; C, 25 μm; D, 10 μm; E, 2 μm; F, 450 nm [119].

[101]. It is interesting to note that several electrophysiological membrane properties measured on the cultured hippocampal neurons did not indicate the occurrence in these cells of changes in ionic conductance brought about by the CNT layers directly [101,119,121]. Cellot et al. determined how nanoscaffolds influence synapses. For this purpose, they examined and compared neuronal networks sustained by nanotube meshworks with those grown on a control substrate [113]. The electrophysiological properties of the synapses were measured using simultaneous whole-cell recordings from pairs of interconnected neurons (Fig. 5A). Paired recordings allow a detailed examination of the evoked transmissions of isolated synaptically coupled neurons of a known type [113]. Action potentials (APs) were triggered in the presynaptic cell and the resulting GABAA receptor-mediated unitary postsynaptic current was analyzed in a postsynaptic element. The GABAA receptor is an ionotropic receptor and ligand-gated ion

Fig. 4. The carbon nanotube substrate affects synaptic activity and neuronal excitability. Neurons grown on an MWCNT-based substrate display an increase in the frequency of spontaneous post-synaptic currents when compared to neurons grown on glass coverslips [101,121].

channel. Its endogenous ligand is γ-aminobutyric acid (GABA), a major neurotransmitter in the central nervous system. Fig. 5B reports the percentage of cell-coupled pairs for CNT networks and controls. The presence of coupling increased from about 33 to 60% for carbon nanotubes, with a connective ratio of 1.8, while the density of neurons was unchanged. These results have been confirmed in previous studies, in which both the SWCNT and MWCNT substrates significantly increased the frequency of synaptic events, and it suggests that an enhanced number of synaptic contacts are formed under the guidance of a nanotube layer in a network of similar size [101,111,113,119]. Moreover, the presence of CNT indicating an enhancement of GABAergic innervation through the increase of the average number of colocalized clusters (presynaptic VGAT (vesicular GABA transporter) and postsynaptic γ2) from 252.0 ± 20.1 to 401.3 ± 31.5 in comparison with a control sample (glass) [113]. This indicates that the number of synaptic connections for nerve cells on the CNT surface increases compared to control samples. The same authors also observed that carbon nanotube scaffolds remove GABAergic short-term depression. In order to verify that nanotube scaffolds affect GABAA-mediated short-term plasticity, in subsequent experiments trains of six spikes at 20 Hz were induced in presynaptic neurons and repeated 10 times at 10-s intervals. In controls, the amplitude of GABAergic currents became progressively smaller, pointing to short-term synaptic depression [113,122]. This depression was connected with 17 ± 7% of damage in subsequent postsynaptic currents (PSCs) during the train. In contrast, in CNT neurons the amplitude of GABAA-mediated PCS was stable or slightly intensified without PSC failures during the train response. Comparing the amplitude of the sixth PSC for a control was 40% of the first one, whereas for CNTs was about 86% of the first one (Fig. 6A,B). According

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Fig. 5. Bright-field image of the experimental setting for dual electrophysiological recordings: the presynaptic neuron is stimulated (under current-clamp configuration, left) to fire APs. The presence of monosynaptic connections to the postsynaptic cell (right) is assessed by voltage-clamp detection of unitary synaptic currents (A). Carbon nanotube substrates increase synaptic connectivity in cultured hippocampal networks. In the plot, the coupling probability, measured as the percentage of monosynaptically coupled pairs, is shown for five different culture series. In each culture, the coupling probability is higher for neurons grown on MWCNTs when compared to that of controls (B) [113].

to the authors, changes in short-term synaptic depression between the control sample and the CNT arise from the difference in GABA release [113]. The morphological similarity of carbon nanotubes to the extracellular matrix and nerve fibers is another factor stimulating neural tissue cells to grow. Micropatterning of cell adhesion substrates allows for precise control of localization and patterns of cellular growth. This approach is useful in the generation and investigation of neural networks in vitro [6]. The template was prepared using photolithography, micro-contact printing and chemical vapor deposition techniques (CVD). The CVD processes are suitable to synthesize carbon nanotubes on the substrate creating micropatterning [123]. Gabay et al. proposed quartz as a substrate for CNT deposition. These samples were incubated with neurons for several days to investigate the effectiveness of the method; then the samples were observed in order to localize neurons. The results show that neurons have aggregated and accumulated mainly on the CNT regions while the remaining spaces (free from nanotubes) are almost without cells (Fig. 7) [123]. Similar results were also reported by Gabay et al., and Sorkin et al., who demonstrated that neurons bind extremely well to carbon nanotube surfaces but do not adhere to SiO2 surfaces [123–125]. This observation strongly suggests that engineering the connectivity between

Fig. 6. MWCNT growth substrates affect short-term synaptic plasticity. A—Train of six current pulses at 20 Hz evoked precisely timed APs in the presynaptic neuron (top traces). Bottom, Representative averaged traces of monosynaptic PSCs recorded during the train, from control and CNT postsynaptic neurons. B—The plot summarizes results from different neuronal pairs. Recordings are from a sample of 10 pairs of control neurons (red dots) and from 14 pairs of CNT neurons (black dots) [113].

neurons is not necessary as these connections can be self-formed according to natural physical mechanisms. Moreover, the data clearly demonstrate that neuronal cells have a tendency to surface reflectivity as well as to bend and to curl in the presence of a rough topography, provided that the roughness is compatible with the length scale of the neuronal process (Fig. 8) [125]. Zhang et al., found the capability of functionalized patterned vertical carbon nanotube arrays as support platforms for guiding neurite growth and forming a synoptically communicative network. The study indicated that neurons in in vitro conditions created communicative synaptic bridges between two nanotube patterns with a distance of separation of 20 μm (Fig. 9) [46]. Telford et al., observed growth of nerve cells from the brain's hippocampal region on substrates coated with a network of carbon nanotubes and found a large increase in neural signal transfer between cells. The data provided information on the supportive devices for bridging and integrating functional neuronal networks in vitro [126]. These capabilities of carbon nanotubes make them potentially successful candidates to form scaffolds to guide neurite outgrowth. The results will impact on new tissue engineering strategies, where functional reconnections among injured neurons or the improvement in neural signal transfer is the main target. 5.1.2. Functionalization of CNT surfaces for nerve growth stimulation As mentioned earlier, carbon nanotubes are able to stimulate tissue regeneration due to their electrical conductivity, however, as has been shown in numerous publications other agents modifying CNTs may promote controlled neurite outgrowth and branching. In this respect, several research groups modified CNT scaffolds by conjugating them with biologically active compounds or with molecules yielding various charges at the surface of modified CNTs [121]. Matson et al., observed on the example of rat hippocampal neurons, the presence of multiple neurites with extensive branching when they were grown on the MWCNT substrates modified with 4-hydroxynonenal (4-HNE) bioactive molecule [100,121]. 4-HNE is a lipid peroxidation product that covalently modifies proteins and can induce an increase in intracellular Ca2+ concentration levels and modify cytoskeletal proteins and signaling mechanisms that regulate neurite outgrowth in many types of neurons [100,127]. MWCNTs were pre-coated via physiosorption, with the bioactive molecule 4-hydroxynonenal, a lipid peroxidation product that in a biological environment controls neurite outgrowth. Other substances used for nanotube modification were type IV collagen and extracellular matrix proteins [86]. These results indicate that modifications of CNTs by physiosorption of functional groups can be applied to affect the interaction between neurons and nanomaterials. It is worth noting that the molecules attached to the nanotubes as a result of physiosorption are not stable and do not have a long lasting retention to the CNTs [121]. This problem can be solved by chemical modification of CNTs by covalent attachment of functional groups to the CNTs [45]. However, the negative side of the covalent methods used for CNT modification of bioactive substances could be the possibility of losing their specific activity.

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Fig. 7. Inverted microscope images of interconnected neuronal systems formed with CNT islands. A—One hour after cell deposition cells are randomly distributed. B—After four days, neurons form clusters at the CNT sites, which form connections between these islands. Some islands are not yet connected at this stage and several short and unconnected dendrites and axons are apparent. C—Neuron cluster at a CNT island [123].

Neurotrophin is a main protein that induces the survival, development, and function of neurons, and it can be attached to carbon nanotubes covalently. Matsumoto et al. [128] demonstrated that this protein remains biologically active, supporting the neurite outgrowth of chick dorsal root ganglion neurons, despite its covalent attachment to CNTs. Covalent modifications of CNTs have also been used to manipulate the charge carried by functionalized CNTs [121]. There is a known beneficial effect of positively charged polymers, such as polylysine or polyornithine, on the surface of the glass or plastic on neuronal growth. Hu et al., functionalized MWCNT chemically with the carboxyl group, poly-m-aminobenzene sulfonic acid (PABS) or ethylenediamine to obtain CNTs that exhibited negative, neutral or positive surface charges, respectively [45,121]. They observed that positively charged MWCNT growth of rat hippocampal neurons was most intensive with more extensive branching and a larger number of growth cones. In the case of neutral or negatively charged CNTs, neuronal growth was slower [45]. The same authors observed similar results for neuronal growth on chemically modified SWCNT using the positively charged polyethyleneimine (PEI), which promoted neurite branching and outgrowth in comparison with neurites on native CNTs [115]. Liopo et al., also confirmed the adverse impact of negatively charged and neutral SWCNTs functionalized with 4-benzoic acid or 4tertbutylphenyl, respectively, on the attachment and survival of cells grown on these modified substrates [129]. Chemical modification of carbon nanotubes is one of the useful tools that allow the regulation of the nerve cell response. 5.2. Carbon nanofibers (CNFs) in nerve system regeneration and stimulation CNFs have become increasingly attractive in creating interfaces between electrodes and local neural tissues in electrical stimulation applications, such as deep brain stimulation (DBS). The main problem with

application of aligned CNFs as electrodes for nerve stimulation is their tendency for agglomeration creating bundles with bigger sizes and micron-sized fibers. The advantages of CNFs are their small sizes (10nm diameters) that enable the probing 3D neural networks, extracting and modulating neural signals more precisely with less damage to the tissue than micron electrode arrays (whose electrode diameters are 100 μm or larger) [2,86]. Yu et al., developed a CNF-based neural chip and proved its in vitro capability of both stimulating and recording electrophysiological signals from brain tissues. In this study, long-term potentiation was induced and detected through CNF arrays [83,130,131]. Good biocompatibility combined with excellent electrical and mechanical properties make such a system useful for use as nerve prostheses (electrodes) and in the electrical stimulation of nerves in the CNS and PNS [2,86]. Currently, most traditional microelectrodes are fabricated with rigid metals and semiconductors [130,132,133]. CNFs have two superior characteristics to be employed in the neural–electrical interfaces compared to metal-based microelectrode arrays. One of them is the high resolution which is difficult to obtain for conventional metalbased electrodes since decreasing their size increases the electrode impedance and thermal noise; this reduces the sensitivity of the electrode in the detection of electrical signals in the nervous system [134,135]. Secondly, CNFs not only can work at the extracellular level but also may penetrate into neurons and then work at the intracellular level (Fig. 10A) [130]. Thus, the carbon-based electrodes may be potentially superior to conventional metal electrodes. Another potential application of carbon nanofibers is electroconductive nanofiber scaffolds for neural tissue regeneration. Such scaffolds are able to enhance and direct nerve growth providing physical support for cell growth as well as impulses stimulating the nerve (Fig. 10B) [22,130]. Similar direct growth of rat hippocampal

Fig. 8. Rat neurons on CNT islands. A—The high-resolution scanning electron microscopy image of a 20-μm CNT island, with one neuron on top of the island and two cells adjacent to the circumference. B—A three-dimensional rendering of an immunofluorescent confocal laser scanning microscope (CLSM) image of a neuron on top of a CNT island. C—Large-scale view of cell nuclei demonstrating conspicuous cellular selectivity to CNT surfaces [125].

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Fig. 9. A—SEM micrograph of a communicative synaptic bridge formed between two nanotube patterns with a distance of separation of 20 μm. The axon extends over the pattern without guidance along the silicon substrate. The synapse is also formed at the pattern edge. The direction of process outgrowth is represented by the arrow. B—Optical micrograph of a synaptically communicative neural network on an MWCNT-patterned substrate [46].

neurons was observed on CNFs manufactured from single-wall carbon nanotubes (SWCNTs) using a particle-coagulation spinning (PCS) process [136,137]. The scaffold for nerve regeneration should not only stimulate nerve cell growth and their interaction but also influence glial cells, such as astrocytes, in an appropriate way. One of the main reasons leading to neural implant failures is glial scar tissue formation around implanted neural probes. McKenzie et al., and Webster et al., investigated astrocyte function on CNF-polycarbonate urethane (PCU) composites [138,139]. They observed that astrocyte adhesion evidently decreased with increasing numbers of CNFs in a PCU matrix (Fig. 11) [139]. The presence of CNFs in the polymer matrix increases surface energy and the electrical conductivity of nanocomposites in comparison with pure PCU samples. CNFs can also be used as neural–chemical interfaces for delivering medical substances, DNA or neurotransmitters to nerve cells. CNFs possess many features that determine their application as a chemical carrier. Their elongated shape, small diameter, wellordered graphite structure, large surface development and chemical inertness and stability in a physiological environment make them good candidates for interfaces. Moreover, graphene layers in CNFs can be chemically functionalized causing the formation of active sites on their surface. On the one hand, the existence of these sites allows the anchoring of chemicals (such as drug, proteins and genes), which then are introduced into the cell; on the other hand, they can operate as sensors of the substances released from the cell (such as neurotransmitters) (Fig. 12) [130]. Peckys et al., presented their results on the introduction of dsDNA (double-stranded DNA) into the cell using carbon nanofibers. The dsDNA was immobilized onto vertically aligned carbon nanofibers and subsequently this dsDNA was released following penetration and residence of these high

aspect ratio structures within cells [132]. CNFs were multistage functionalized using gold coatings and different chemical groups such as mercaptoundecanoic acid (11-MUA), (3-[2-aminoethyl]-dithio) propionic acid (AEDP) and 6-aminocaproic acid (EACA) employed to covalently bind the dsDNA [140].

5.3. Graphene in nervous system regeneration and stimulation Besides CNTs and CNFs other types of carbon nanoforms such as graphene, are investigated as materials for neuronal stimulation and monitoring. Unique electrical properties of graphene offer a significant advantage for a variety of clinical diagnostics and treatments in the nervous system. Graphene, like carbon nanotubes, was also cultured in contact with hippocampal neurons chosen to be the model of investigation due to their well-known plasticity and regeneration properties. The hippocampus is a major component of the human brain and other vertebrates. It belongs to the limbic system and plays important roles in the consolidation of several forms of learning and memory and especially during the formation of declarative memories [84,141]. Humans and other mammals have two hippocampi, one in each side of the brain. The authors indicate the non-cytotoxic effects of graphene in contact with nerve cells derived from the hippocampus in comparison with tissue culture polystyrene (TCPS). Moreover, the length and number of neurites during the developing period (7 days) on graphene were increased as compared to control samples. They also observed that the greatest statistical differences of average neurite number and length between graphene and TCPS were observed on the second day when compared with a period from 3 to 7 days (Fig. 13). It means that graphene substrates may have a stronger impact on the early developing neurons. This may be due to the fact that neurons are more sensitive to the

Fig. 10. Schematics of CNFs as neural interfaces. Neural-electrical interfaces: A—as bidirectional interfaces for electrical stimulation and recording; and B—as electroconductive nanofibrous scaffolds [130].

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Fig. 11. Decreased astrocyte cell adhesion density with increased numbers of CNFs in PCU. Representative SEM images of PCU:CNF composites. A—100:0 (PCU:CNFs wt.%), B—0:100 (PCU: CNFs wt.%); Scale bar = 1 μm [139].

physicochemical properties of the surrounding environment during the early developing stage [84]. Graphene/PET film stimulator was created by a group of researchers from South Korea in order to evaluate its potential to stimulate nerve tissue cells in vitro [142]. Graphene obtained using the CVD method was connected with polyethylene terephthalate (PET) and cultured with SHSY5Y human neuroblastoma cells. They observed that weak non-contact electric field stimulation as low as 4.5 mV/mm for 32 min was particularly effective in shaping cell-to-cell interactions and in the number of cells straightening existing cell-to-cell couplings. A possible mechanism for the interaction between the cells in contact with the graphene/PET may be the appropriate stimulation of cytoskeleton proteins such as fibronectin, actin and vinculin. The findings from this study provide growth opportunities for new techniques for the treatment of diseases of the central nervous system [142]. Deng et al., also observed interesting results for nerve regeneration using graphene oxide (GO)-polypyrrole (PPy) composite films on the Pt electrodes [143]. The PPy/GO composite films on Pt neural microelectrodes were deposited using a simple electrochemical codeposition

method by exploiting the electrostatic interaction between negatively charged GO sheets and positively charged pyrrole cation radicals and PPy. Results from the electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) show that the Pt electrodes coated with GO-PPy films exhibit lower impedance and higher capacity than those of pure PPy coated and bare Pt microelectrodes [143]. The findings from these studies provide new growth opportunities for research techniques in the treatment of neurological diseases in the CNS. 6. Questions relating to the use of carbon nanomaterials in the nervous system The high electrical conductivity of carbon nanomaterials is an important property for the functional recovery in the central nervous system of learning processes, neuronal plasticity and adaptation. These forms of plasticity might also depend upon the presence of proper chronic electrical instructions. For this reason, many important questions should be taken into consideration. Giugliano et al. [91] formulates them as

Fig. 12. Neural–chemical interfaces: as bidirectional interfaces for delivering therapeutic chemicals and biosensing neurotransmitters [130].

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Fig. 13. Average number and length of neurites during the developing period on TCPS and graphene. A—Average number of neurites per neuron on TCPS and graphene during the developing period (D2–D7—from 2 to 7 days). B—Average neurite length of neuron on TCPS and graphene during the developing period (D2–D7—from 2 to 7 days) [84].

follows: Is the nanomaterial able to transfer induced or spontaneous electrical signals generated by neurons, consisting of small amplitude voltage variations (from hundreds of mV to tenths of mV)? If transferred, can these signals evoke a response in a physically separated neuronal population? Can the design of the material be optimized to propagate electrical signals only in a chosen direction? How crucial is neuronal signaling in promoting axonal growth? What is the mechanism of the electrical interaction between carbon nanomaterials and neurons? If carbon nanomaterials are able to increase the rate of firing and of synaptic activity in a neural network, would they be suited for re-establishing communication when, e.g., neural communications are breaking down? Many questions also appear in terms of biocompatibility of carbon nanoparticles, and in particular what happens to them when they are released into the body. How to interact with the surrounding cells and tissues? Whether and how are they released from the body? What are the factors associated with carbon nanomaterials that affect cellular responses? To answer for these questions, in particular referring to the biocompatibility of carbon nanoparticles, further research is needed. This is mainly due to a number of factors that may affect this response. Opinions on the biocompatibility of carbon nanomaterials both in vitro and in vivo environments are not, however, unequivocal. Some authors indicate that CNTs are biocompatible in contact with cells and tissue, that is, they stimulate osteoblast and nerve cells to grow, proliferate and induce muscle and blood vessels to regenerate [54,128,144]. Additionally, due to their mobility potential in living systems, they may be successfully used as novel drug delivery systems for therapy and diagnosis [145,146]. Some researchers have compared them to the negative effects of asbestos fibers, ordering special care during handling or disqualifying them completely from further use [147,148]. Particular caution is advised during contact of CNTs with skin and the respiratory tract [149]. Contrary to these outcomes, many critical results point to the cytotoxicity of CNTs. Others scientists indicate that CNTs may lead to dermal toxicity due to accelerated oxidative stress in the skin and pulmonary toxicity through the induced lung lesions characterized by the presence of granulomas [55,150–152]. Analysis of the available literature shows both the beneficial and adverse impact of carbon nanomaterials on a living organism and suggests that both parties are right. Moreover, taking into account the diversity of CNTs, CNFs and graphene resulting primarily from methods of their manufacture, the catalysts used, the synthesis conditions and a variety of methods used for evaluation of their toxicity, it is difficult to agree with either view. Many studies indicate that the biocompatibility of carbon nanomaterials in both in vivo and in vitro studies may be attributed to various factors, including their lengths, functionality, their concentration, duration in the living body, catalyst impurity, agglomeration and even the dispersants used to dissolve the nanotubes [153–162].

Carbon materials and nanomaterials are biostable in the body and they are not absorptive. Some researchers suggested that the behavior of carbon nanomaterial in the body strongly depend on their functionalization, i.e. the presence of chemical groups on nanomaterials surface. For example carboxyl or hydroxyl groups on carbon nanomaterials surface change their character from hydrophobic to hydrophilic. The hydrophilic materials are better dispersed in aqueous solution and could be easier transport in the body and consequently eliminated from the body [163–165]. Liu et al. experiments have shown that more than 95% of CNTs are released in the urine from the body within a few hours [163]. Moreover, some scientists indicated that functionalized CNTs are degraded in a phagolysosomal simulant [166]. It is also important to develop and validate methods to evaluate the toxicity of nanoparticles to compare the experimental results between research institutions properly. Most aspects of carbon nanomaterial toxicity still remain inadequately identified and further long-term research is required [152]. Apart from large number of results referring to the biocompatibility of carbon nanomaterials and their great influence on the stimulation and regeneration of the nervous system, their adverse effect on this system was also observed [162]. Depending on the agglomeration degree of SWCNTs different influence on primary cultures derived from chicken embryonic spinal cords (SPCs) or dorsal root ganglia (DRG) was observed. As measured by the Hoechst assay, treatment of mixed neuro-glial cultures with SWCNTs significantly reduced the overall DNA content. This effect was significant when the cells interacted with SWCNT in the form of agglomerates as compared to the more dispersed SWCNTs. Moreover, SWCNTs reduce the amount of glial cells in both the PNSand CNS-derived cultures. For DRG-derived cultures in contact with SWCNTs, a reduction in the number of sensory neurons was observed. An intensification of acute toxicity was also observed in primary cultures from both the CNS and PNS of chicken embryos in contact with nanotubes. The level of toxicity is partially dependent on the degree of agglomeration of the carbon nanotubes [162]. Wu et al. also observed the negative impact of CNTs on axon growth in vitro. They showed that exposure of dorsal root ganglia (DRG) cultures to MWCNT significantly impaired regenerative axonogenesis without concomitant cell death. The authors indicated that the lowest dose of MWCNTs (0.1 μg/ml) did not significantly inhibit growth of neuritis, while doses of 1, 5 and 10 μg/ml revealed a dose-dependent compromise of regenerative axon growth both in length and extent of branching. The obtained results indicated a significant dose-dependent disruption of regenerative axon growth after exposure to MWCNT [167]. Carbon nanotubes may also lead to a change in ionic conductivity in cells. The first evidence testifying that the nanotubes can affect the ionic conductivity was observed by Park et al. who showed that SWCNTs

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block the flow of ions through potassium ion channels expressed in a cell line [168]. The authors speculated that the CNTs blocked the channel pore and interrupted ion permeability [121]. A similar behavior was observed by Ni et al. who showed that SWCNTs caused a significant impairment in cytoplasmic Ca2+ elevation when neurons were depolarized [116,121]. This behavior may be due to the CNTs interfering with the functioning of the Ca2+ channels. These results suggest that due to the complexity of the nervous system anatomy and function, repairing damaged nerve carbon nanotubes may inadvertently affect the activity of the cells with which they contact [121]. 7. Conclusions This chapter is a literature review regarding the application of various nanostructured carbon materials, from carbon nanofibers and nanotubes to graphene, in the reparative and regenerative processes of diseased nerve tissues as well as the stimulation of the growth of nerve cells. In the paper, attention was also drawn to which factors associated with carbon nanoparticles have a decisive impact on the response of neurons and glial cells within the central and peripheral nervous systems. Carbon nanomaterials have gained a special importance in recent years, when it was observed that their properties (particularly their electrical and mechanical characteristics, high specific surface area, their appropriate dimensions, close to the dimensions of single axons) may constitute a kind of matrix, scaffold and a factor that stimulates their growth and the regeneration of synaptic connections. In spite of the existence of many positive reports concerning the possibility of applying carbon nanomaterials for the treatment of the central and peripheral nervous systems, one should not forget about a number of issues related to the toxicity of nanomaterials as well as the incompletely understood mechanisms that have an influence on the stimulation of nerve tissue cells to grow. Therefore, for better understanding of this extremely interesting and complex area of medicine further research needs to be explored. Acknowledgment This work has been supported by the Marie Curie Actions—IndustryAcademia Partnerships and Pathways (IAPP), FP7-PEOPLE-IAPP-2008, project number 230766. This study has been supported by the Polish Ministry of Science and Higher Education, project no NN 507402039. References [1] National Spinal Cord Injury Statistical Center, Annual Statistical Report, University of Alabama, Birmingham, December 2007. [2] P.A. Tran, L. Zhang, T.J. Webster, Carbon nanofibers and carbon nanotubes in regenerative medicine, Adv. Drug Deliv. Rev. 61 (2009) 1097–1114. [3] K. Bogdan, R. Rutowski, S. Pielka, J. Gosk, D. Szarek, W. Urbanski, Evaluation of results in microsurgical operations of upper extremity peripheral nerves lesions, Adv. Clin. Exp. Med. 14 (2005) 1199–1209. [4] J.T. Seil, T.J. Webster, Electrically active nanomaterials as improved neural tissue regeneration scaffolds, WIREs Nanomed. Nanobiotechnol. 2 (2010) 635–647. [5] N. Zhang, H. Yan, X. Wen, Tissue-engineering approaches for axonal guidance, Brain Res. Rev. 49 (2005) 48–64. [6] W. Lee, V. Parpura, Carbon nanotubes as substrates/scaffolds for neural cell growth, in: H.S. Sharma (Ed.), SECTION III Nanoparticles Therapy and Neuroregeneration, Progress in Brain Research, vol. 180, 2009, pp. 110–125, (Chapter 6). [7] D. Szarek, W. Jarmundowicz, A. Fraczek, S. Blazewicz, Biomaterials in the treatment of peripheral nerve injuries—an overview of methods and materials, Eng. Biomater. 9 (2006) 40–53. [8] S. Hall, Nerve repair: a neurobiologist’s view, J. Hand Surg. 26b (2001) 129–136. [9] S.J. Archibald, J. Shefner, C. Krarup, R.D. Madison, Monkey median nerve repaired by nerve graft or collagen nerve guide tube, J. Neurosci. 15 (1995) 4109–4123. [10] M.E. Schwab, Repairing the injured spinal cord, Science 295 (2002) 1029–1031. [11] G.H. Borschel, K.F. Kia, W.M. Kuzon, R.G. Dennis, Mechanical properties of acellular peripheral nerve, J. Surg. Res. 114 (2003) 133–139. [12] X. Gu, F. Ding, Y. Yang, J. Liu, Construction of tissue engineered nerve grafts and their application in peripheral nerve regeneration, Prog. Neurobiol. 93 (2011) 204–230. [13] X. Jiang, S.H. Lim, H.-Q. Mao, S.Y. Chew, Current applications and future perspectives of artificial nerve conduits, Exp. Neurol. 223 (2010) 86–101.

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