Growth and Follow-Up of Primary Cortical Neuron Cells on Nonfunctionalized Graphene Nanosheet Film

Abstract

Background

Conductive biomaterials are an ideal biosubstrate for modifying cellular behaviors by conducting either internal or external electrical signals. In this study, based on a simple-preparation graphite exfoliation method in organic reagent, a nonfunctionalized graphene nanosheet film (NGNF) with high conductivity and large size was simply fabricated through spraying coating. The biocompatibility of the NGNF was carefully tested with primary cortical neuron cells, and its biocompatibility properties were compared with a chemical vapor deposition (CVD) graphene film.

Methods

Nonfunctionalized graphene nanosheet (NGN) was first exfoliated from graphite with a flat-tip ultrasonicator probe, and then spray-coated onto glass slide substrate to form the film. The morphology of NGNF was observed with light microscopy and SEM. The morphology and neuronal network formation of primary cortical neuron cells onto NGNF, as shown by DAPI and Alexa Fluor^® 488 staining, were observed with fluorescent microscopy. Cell viability and proliferation were measured with MTT.

Results

NGNF had better cell biocompatibility than CVD graphene film. MTT test showed that NGNF exhibited no cytotoxicity. According to neuronal network formation at 7 days of cell culture, primary neuron cells aggregated into 50-μm “nuclei”; the average neurite number and length were 3 and 100 μm, respectively. However, these values were almost doubled after 14 days of cell culture.

Conclusions

These results may improve the use of NGNF as a conductive scaffold for nerve regeneration.

Keywords

Cytotoxicity Graphene Neuron Regeneration

Introduction

Graphene is a planar aromatic macromolecular structure with unique chemical and physical properties such as high planar surface (calculated value, 2,630 m²/g) (1), excellent mechanical strength (Young's modulus ∼1,100 GPa) (2), unparalleled thermal conductivity (∼5,000 W/m/K) (3) and remarkable electronic properties (4). In the last few years, graphene or graphene-based composite materials have been highlighted in biological and biomedical fields (5) because graphene enhances cell adhesion (6) and has shown excellent sensitivity to biomolecules such as glucose, DNA and proteins (7) through strong polyaromatic π-π stacking interactions. Moreover, graphene might have the ability to alter neuron behavior through improving their collective electrical performance – i.e., spontaneous synaptic activity and action potential firing frequencies (8) – and then to develop the neural interfaces (9). However, like its allotropes such as fullerene and carbon nanotubes (CNTs), the possible cytotoxicity of graphene needs to be carefully addressed both in vitro and in vivo. For instance, Sasidharan et al (10) reported that ∼50% Vero cells died in the presence of 100 µg/mL of graphene, and this toxic effect increased with the graphene concentration. The authors attributed the cytotoxic effect to the accumulation of graphene on the cell membrane, causing oxidative stress and following apoptosis. In another report (11) where a pheochromocytoma-derived PC12 cell line was cultured on chemical vapor deposition (CVD)–produced graphene film, the authors found that graphene activated an apoptosis marker (caspase 3) and enhanced lactate dehydrogenase and reactive oxygen species (ROS). To reduce its cytotoxicity, a strategy such as hydrophilic modification (carboxylation, amination) of graphene has been used (12). As a result, graphene oxide (GO) showed no obvious cytotoxicity in a comprehensive study of the influence of GO on A549 cell morphology, viability, mortality and membrane integrity (13). Nevertheless, dose-dependent GO cytotoxicity, through the induction of oxidative stress, resulting in a slight loss of cell viability, was reported at high GO concentrations (85 µg/mL) (7). Grafting with biocompatible polyethylene glycol (PEG) is another strategy to reduce graphene cytotoxicity. PEG-GO showed good biocompatibility (14), with enhanced aqueous solubility and stability in physiological solutions including serum. However, the sp² structure of graphene was mostly destroyed in the oxidation to GO, reducing its conductivity. The reduction of GO by hydrazine hydrate partially restored the graphitic structure, but its cytotoxicity was significantly increased (7).

Electrical excitability is believed to govern signaling in the nervous system, by changing phosphorylation or proteolysis of regulatory and structural proteins through voltage-dependent and transmitter-activated channels (15). The message signal is simply converted from an electrical to chemical form and then back again between neurons crossing a synapse with the formation of spike/wave-like impulses, “electrical stimulation” (16). At the electrophysiological level, this type of excitability is responsible for neurite outgrowth and guides neurons in the intact spinal cord, by controlling the migration of growth cones and axons to the appropriate target regions, using local guidance cues like substrate pattern morphology (17). Therefore, external electrical signals can result in cellular physiological functions being modified. Several groups have demonstrated that electrical stimulation (ES) enhanced neurite outgrowths in vitro (18-19-20-21). For example, with ES, PC12 neurites were 1.5- to 2-fold longer compared with no-ES control (21). In 2003, Gordon et al (22) found that ES could promote peripheral nerve regeneration and functional recovery. In addition, the same group found ES promoted successful regeneration in cases of chronic peripheral nerve injury (23). ES increased the expression of growth-associated genes in injured motor and sensory neurons and further promoted the speed and accuracy of motor and sensory axon regeneration (24, 25). It has been demonstrated, in the past 20 years, that ES assisted functional recovery after cerebral injury including cortical ablation, cortical ischemia and head trauma in laboratory animals (26). For example, the direct ES of the brain modulated local neuronal activity in enhancing behavioral recovery by stimulating peri-infarcted cortex facilitates to recruit the neurons into a functional neural network (27). With electrodes positioned over peri-infarct areas, the rat's efficacy of rehabilitation of motor functions was improved by coupling it with cortical ES (28). By neuromodulation, ES was used to modulate central nervous system structures to evoke its excitability, release neurotransmitters and alleviate the effects of many neurological diseases, such as depression and obsessive-compulsive disorder (29). Biocompatible conductive polymers, polypyrrole (PPy) and polyaniline (PAn), can deliver localized electrical stimuli at a specific site and provide a physical surface; therefore, these polymers have been utilized as a potential scaffold for the neuron regrowth. For example, in vitro, Kotwal and Schmidt (30) found that ES through conductive PPy enhanced the adsorption of fibronectin. It is clear that if the nerve damage between cut ends of a nerve is greater than 2 cm, a suitable scaffold which enhances and guides the nerve growth becomes essential (31). In vivo, a recent paper even showed that ES enhanced regeneration across a rat sciatic nerve gap (13 mm) which exceeded distances that allow spontaneous regeneration (32). However, those conducting polymers normally are nonsoluble and nonfusible fragile particles, which makes them hard to process. Thus, conductive polymers have to be blended with other polymers to cast substrate membrane which reduces the conductive membrane conductivity and electrical sensitivity because of the diluting process (33). Moreover, another problem with these polymers is that their conductivity decays in the aqueous phase, because of de-dopant processing (33, 34).

Some members of the carbon allotropes, such as CNTs and graphene, present higher conductivity than polymer. Also, their electrical conductive properties can be utilized to modify neuron cellular behaviors. However, carbon nanotube is hard to form into a large-size solid substrate by itself, and graphene either from the CVD method or from GO has been reported to show controversial biocompatibility results by different groups (35). Besides the disadvantages mentioned above, the CVD method suffers from heavy metal contamination, and the GO redox method involves chemical reagent cleaning (36).

In this study, the exfoliation method did not destroy the sp² structure of graphene, and the spray-coating method was an easy way to manipulate large-size substrate. Since, the nonfunctionalized graphene nanosheet film (NGNF) prepared with chemical reagent, its biocompatibility will be carefully addressed. Immortal cell lines (such as PC-12) have been extensively used to evaluate the neurotoxic potential of biosubstrate (37). Nevertheless, the transformed cells simultaneously also present characteristics which are different from primary cells. As far as the primary neuron cells, their culture conditions are much tougher than those of cell lines, and as long as successfully plated, neurons can form synapses and become electrically active. Therefore, primary cortical neuron cells were used to analyze the neurotoxic effect of graphene chemicals.

Materials and Methods

Graphene nanosheet exfoliation

The procedure used was based on a reported ortho-dichlorobenzene (ODCB)–graphene exfoliation method (21) but with a little modification. Briefly, 1-g graphite flakes (Sigma-Aldrich, St. Louis, MO, USA) used as received, was mixed with 100 mL of ODCB in a 250-mL Schott laboratory glass bottle, and then a flat-tip ultrasonicator probe (Sonicator^® 3000; Misorix, Newtown, CT, USA) was placed halfway into the bottle. The ultrasonication operation was performed by program for 4 hours with 15-second sonication and 45-second intervals. After exfoliating, the ODCB media were settled for 24 hours, and the supernatant was collected for NGNF preparation.

Graphene nanosheet film

Medical-class polyurethane (PU) (Tecoflex 80; Lubrizol Corp., Wickliffe, OH, USA) was used as the adhesive between NGNF and glass substrate. PU spin coating was run at 2,000 rpm for 30 seconds with a PU concentration of 40 mg/mL in CHCl₃ (P-6000 spin coater; Spin Coating Speciality Coating Systems, Inc., Indianapolis, IN, USA). Then, the graphene nanosheets in ODCB (5 mg/mL) were spray-coated onto PU by airbrush (Iwata Eclipse HP-BCS) at 250°C on a digital hot plate (Fig. 1). The graphene-coated samples were stored with 50/50 methanol/H₂O for at least 2 weeks before sterilization. The chemical reagent used for storage was changed every 2 days.

Fig. 1

Spray-coating a nonfunctionalized graphene nanosheet (NGN) on a glass slide. To fix the NGN onto the glass slide, polyurethane (PU) was firstly spincoated onto glass slides as a polymer matrix, then NGN particles were spray-coated on by airbrush.

The low-magnification photography of NGNF was performed with a Nikon Eclipse ME600 microscope with a Nikon digital camera DXM1200f (Nikon Instruments Inc., Melville, NY, USA). The morphology of NGNF was observed with a FEI Quanta 400 high-resolution field emission scanning electron microscope (SEM); the accelerating voltage employed was 15 kV. The thickness of the NGNF was also calculated with SEM photos.

The conductivity of NGNF used in this study was measured by a 4-point probe (Cascade™; Microtech, Beaverton, OR, USA; coupled with a Keithley 2010 multimeter; Keithley Instruments, Cleveland, OH, USA) as 125 ± 24 Ω/square. Twelve graphene nanosheet film slides were randomly selected, and at least 6 sets of different points on each piece were measured. The data were analyzed with SigmaPlot 10.0 software.

CVD graphene film

High-quality monolayer CVD graphene was grown on Cu foil (99.8% purity, 0.025-mm thick; Alfa Aesar) using CH₄ as carbon source gas. Firstly, the flow rate of the CVD system was heated to 1035°C at 10 mTorr in 2 hour, and the Cu foil was annealed in the oven in 10 minutes with an H₂ flow of 500 cm³ (STP)/min before graphene growth. Secondly, the CH₄ flow was brought into the oven at a rate of 4 cm³ (STP)/ min for 45 minutes to form monolayer graphene on the Cu foil. After growth, and finally, graphene was transferred to the glass slide or Si wafer through polymethylmethacrylate (PMMA) film attachment, and Cu etching.

The CVD graphene film was provided by Dr. Gedeng Ruan of Rice University.

Sterilization

The samples were sterilized by exposure to ethylene oxide in an Anprolene AN74i EtO box (Andersen Products Inc., Haw River, NC, USA) for 24 hours according to the manufacturer's instructions.

Primary neuron cell culture and staining

Before cell culture, the graphene films were first coated with a 1/50 dilution of Matrigel™ (BD Biosciences, Franklin Lakes, NJ, USA) in Gibco^® F-12 Nutrient Mixture (Life Technologies, Grand Island, NY, USA) and incubated for 30 minutes at 37°C. Then, primary cortical neuron cells (Neuromics, Edina, MN, USA) were carefully plated onto the graphene film according to the protocol described by the manufacturer. After 1 or 2 weeks in cell culture, primary cortical neuron cells were directly fixed in -20°C methanol for 3 minutes, and then 1/500 MAP-2 (H-300; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in phosphate-buffered saline (PBS) was added to the cells for 2 hours, and 1/500 Alexa Fluor^® 488 anti-Rabbit (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in PBS was sequentially added to the samples and settled for 2 hours. Then the sample slides were slipped (covered) using Vectashield^® Hard Set™ mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). Finally, the samples were dried at room temperature for 24 hours, and their morphology was observed using a Nikon Eclipse 80i fluorescence microscope (Nikon Instruments Inc., Melville, NY, USA) with a DAPI-FITC-Texas Red filter.

To give a comprehensive view of neurite growth for different cell culture times, relatively low magnification images (scale bar = 500 μm) were chosen. For each measurement, as least 20 separated cells for each cell culture interval were employed and measured. However, the size of neuron nuclei aggregation and neurite cord were measured from the images at high magnifications (scale bar = 50 μm). The data were analyzed with SigmaPlot 10.0 software.

MTT test

The NGNF slide specimens were cut into 1.0 × 1.0 cm² sections and placed into a 24-well plate; primary cortical neuron cells were plated onto the NGNF specimen accordingly. The 24-well plates were then incubated for 7 days and 14 days under 5% CO₂ at 37°C before methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) assay. The culture medium was refreshed every 2 days. When the specimens were chosen for biocompatibility analysis, they were removed into new 24-well plates for the MTT assay. Culture medium (2 mL) was first added to each well, followed by 200 μL of MTT reagent, then reincubated for 4 hours. HCl (2 mL) in isopropanol (0.04 M) was added to each well, and incubation continued for an additional 15 minutes. Finally, 200 μL of solution was transferred into the wells of a 96-well plate. Optical densities were read at 570 nm with a model Varioskan™ Flash Multimode Reader (Thermo Fisher Scientific Inc, Waltham, MA, USA).

Results

Conductivity

The surface conductivity of NGNF was measured as 125 ± 24 Ω/square.

Morphology of NGNF

The low-magnification photography of NGNF with the digital camera showed that a black and smooth layer of NGNF was spray-coated onto the glass slide. In the SEM photos, an irregular shape of NGNF sheets was randomly piled and connected into membrane. On light microscopy, because of the light reflection of the grapheme sheets, shining sheets were observed with thicknesses in the range of 4-7 µm. Moreover, light microscopy photography confirmed that those grapheme sheets varied in micrometer size range (Fig. 2).

Fig. 2

Low-magnification photography of nonfunctionalized graphene nanosheet film (NGNF) coated on a glass slide (2.54 × 7.62 cm.), showing a black and smooth surface after the coating, with the uncoated glass slide still transparent (top image). Morphology of NGNF coated on glass slide was observed by SEM photograph (lower left) and light microscopy photograph (lower right), showing that the NGNF was formed with irregular-sized NGNs which were embedded onto polyurethane (PU), and some part of the sheets were still exposed to air.

Primary neuron cells adhesion/growth/morphology on NGNF

After 7 days of cell culture, the primary neuron cells successfully adhered to the graphene film. The photos taken from relatively lower magnification (compared with the higher magnification ones shown below) were chosen (Fig. 3) for low-magnification photography in which, the visible aggregated neuron was observed, and their axon bundle clusters are spraying and traveling outside from the nuclei. According to the statistical analysis (Fig. 4), the mean neurite length of the neurons was around 100 μm, and the mean neurite number per neuron was around 3. However, when the cell culture time was increased to 14 days, these values were significantly increased to 300 μm and 7 (p<0.01), respectively.

Fig. 3

A comprehensive view of neurite number and length of primary neuron cells with a relatively low magnification (scale bar = 500 μm). Cells were cultured onto nonfunctionalized graphene nanosheet film (NGNF) for 1 week and 2 weeks. Alexa Fluor^® 488 anti-Rabbit green staining showed that both the neurite length and number were increased after an additional 7 days of growth.

Fig. 4

Neurite length and number were 100 μm and 3 after 7 days of cell culture; however, those values were almost doubled after 14 days of cell culture, showing significantly higher proliferation for the additional 7 days of cell culture on nonfunctionalized graphene nanosheet film (NGNF; p<0.01).

The photos taken from higher magnification presented clearer cellular growth and morphology information such as cell viability, nuclei size and axon cluster, as well as the network formed through the axon cluster. DAPI was used to blue-fluorescent DNA stain the fixed primary neuron cells on NGNF. Alexa Fluor^® 488 Phalloidin, for green-fluorescent F-actin staining, was used to dye the protruding axon clusters. TThe size of nuclei, showing as the aggregates, were measured to be approximately 100 μm in diameter, which fractionalized as the knots, and the protruded clusters, showing as cables in the neuron network, had diameters close to 5 µm (Fig. 5). However, as the cell culture time increased to 14 days, both of the values were increased to significantly twice as much (p<0.01) (Fig. 6).

Fig. 5

Nuclei formed in 7 and 14 days of cell culture are shown at a relatively high magnification (scale bar = 50 μm): 1/500 MAP-2 (H-300) and 1/500 Alexa Fluor^® 488 anti-Rabbit stained green for neuron microtubules and DAPI stained blue neuron nuclei.

Fig. 6

Diameter of neuron nuclei aggregation was around 50 μm on day 7, and 100 μm on day 14. CVD = chemical vapor deposition; NGNF = nonfunctionalized graphene nanosheet film.

To confirm this observation and assess the effect of NGNF on cell proliferation/growth, an MTT assay, assessing cell viability, was performed. As shown in Figure 7, the MTT values were significantly increased from 0.41 (day 7) to 1.25 (day 14), and the results showed that NGNF conductive membranes were not toxic to primary neuron cells.

Fig. 7

Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay results for primary cortical neuron cells cultured on nonfunctionalized graphene nanosheet film (NGNF) for 7 and 14 days, showing significantly higher proliferation for the additional 7 days of cell culture on NGNF (p<0.01).

Primary neuron cells adhesion/growth/morphology on CVD graphene film

There were few cells on the CVD graphene film. Less than 15 per slide were observed on both glass and silicon substrates after 7 days of cell culture. It should be mentioned that even the cell culture time was increased twofold to 14 days. The primary neuron cells number still did not change a great deal; it almost kept to the same level (less than 15). However, those cells that survived also extended their slim and short neurites, but no integrated circuit was formed (Fig. 8).

Fig. 8

Primary neuron cells grown on chemical vapor deposition (CVD) graphene, showing that few cells grew on the CVD graphene. The neural microtubes (green) were stained with MAP-2 (H-300) and Alexa Fluor^® 488 anti-Rabbit, and the nuclei (blue) were stained with DAPI (scale bar = 50 μm).

Mean neurite length and number were also calculated, and the results are presented in Figure 9. It is clear that both the neurite length increased from 25 μm (day 7) to 50 μm (day 14; p<0.05) and the neurite number per cell was increased from 2 (day 7) to 3 (day 14; p>0.05).

Fig. 9

After 14 days of cell culture, mean neurite numbers per cell showed no statistically significant difference compared with those for 7-day cell culture. However, neurite length increased with statistical significance after another 7 days.

Discussion

NGNF preparation

Graphene, with its high surface area and remarkable electrical conductivity, is an excellent conductive scaffold, assuming its biocompatibility can be improved. In a previous study (38), NGNF were prepared with a high yield in ODCB using a tip-ultrasonicator. This novel exfoliation method avoids damaging the graphene sp² structure, and thereafter the ODCB-dispersed graphene can remain highly surface conductive (i.e., 125 ± 24 Ω/square).

Cells, such as fibroblasts, osteoblasts, neuron and so on, specialize in the integration and propagation of electrical events, as well as communicating with each other. Through such electrical activity, cellular behavior (adhesion, proliferation, differentiation etc.) can be modified. In our previous publications, we demonstrated that the fibroblasts and osteoblasts showed their effective electrical sensitivity at approximately 50-100mV/mm (39-40-41). Neurons are inherently regular and highly reproducible fire feature rather than a stochastic one, their electrical activities are related to both frequency (a wide range was tested, e.g., 1-50 Hz) and intensity, which in cells is approximately -60 to -80 mV based on the resting membrane potential of nerves (42). Obversely, conductive biomaterials could become the electrical sensor to guide cellular activities.

In this study, we prepared highly conductive NGNF by spray-coating the ODCB supernatant containing graphene sheets onto PU precoated glass slides. After drying to remove the ODCB solvent (Fig. 1), the NGNF was prepared. In Figure 2, the low-magnification photography from digital photographs showed “smooth and black” NGNF. On light microscopy and SEM, the surface of the NGNF was seen to be roughly formed with irregular-sized NGN sheets, which were partially embedded onto PU and exposed to air. The NGN did not detach from the PU membrane for 3 weeks. PU worked as an adhesive to the glass slides and graphene, it also used as a matrix-like for the graphene sheets beacuse the exposure graphene sheets did not enbeded into PU, therefore the exposure graphene sheets could contribut the high conductivity. However, NGN formed on PU membrane was not integrated and uniform, which might be due to defects in spray-coating skill (Fig. 2) in the lab. If a fine-cyber-control spray machine could be used, the nonintegrated and nonuniform shortcomings could be overcome. CVD graphene was shown to be highly uniform and was regarded as the highest quality layered graphene in many reports (43). But, CVD graphene is not yet an easy-to-control process, because the chemical reactions are so complicated, and there are many parameters involved, such as carbon source, temperature, gas flow of rate, catalysts, metal foil etching etc.

Neuronal networks formation

Aggregation is a natural behavior of neurons. When neurons regroup and reconstitute, the grouped cellular protrusions from neurons will form interconnections with those constitutive bundles, so that the grouping of neurons used to induce the neuronal 3-dimensional networks, by which the signaling between the health functional nerve cells communicates. Depends on how fast an individual bare axon can conduct an action potential and how rapidly the communication is between circuits of neurons. Thus, large axon bundles and highly connected neuronal network formation are needed for nerve regeneration. From the fluorescent stained photos in Figure 3, which were the relatively lower magnification images, a more comprehensive view of neuron growth could be clearly observed. It was obvious that the nerve cells had successfully attached onto the graphene film, and nerve cords formatted with the axon bundle clusters. And after 7 and 14 days of cell culture, the neurite number per cell and the neurite length of cell significantly increased (Fig. 4).

With higher magnification photos, the neurofilament fibers already traveled along with the bundles, producing collaterals connection in the aggregates some of them formed the nuclei which are around 50 μm in diameter, showing as cellular knots (Fig. 5), and those bundles of protrusions showing as cables in the network with their diameter close to 5 μm. However, some nuclei were not interconnected. The individual axon bundle pathways and neuronal cell body aggregates were clearly distinguished and shown with Alexa Fluor^® 488 staining green and DAPI staining blue. And for twice the time of cell culture, after 14 days, the bundled axons significantly grew up to 10 μm in diameter, as well as connecting with more peripheral nuclei. And the size of their nuclei was doubled (Fig. 6).

DAPI staining, which stained double-stranded DNA of fixed neuron cells, presented a significant different in the size of neuron nuclei aggregation. For the cell proliferation test, an MTT assay showed a statistically significant difference in cell viability after a 7-day cell culture (Fig. 7). It was clear that the primary neuron cells could adhere and grow on NGNF.

Neuron growth on CVD graphene film

Few cells survived on both glass and silicon substrate CVD graphene film (Fig. 8) in 1 or 2 weeks of cell culture. Due to their easy mounting features, glass slides and silicon substrate are the 2 popular solid supports (substrates) for CVD graphene. Normally, the cultured healthy neurons in biofriendly surroundings can create outgrowing sprouts to adhere onto substrates. However, the few surviving cells extended their slim and short neurites, and they could not form any integrated circuit. The reasons for CVD graphene cytotoxicity are very complicated. In current reviews (44, 45), the authors list some explains, such as molecular interactions of graphene with proteins, DNAs, lipid molecules and cell membranes (both bacteria and mammalian cells). We understand that various elemental metals such as Cu and Ni are typically used as catalysts in CVD methods to lower the energy barrier of the chemical reaction to achieve heterogeneous grapheme deposition on precursor surfaces. We hypothesize that cytotoxicity from the catalyst contamination in CVD graphene could be due to iron and copper having the ability to gain and lose electrons very easily, which makes them common catalysts of oxidation reactions that produce ROS (46). As a comparison, the highly “purified” single-wall CNTs which are also synthesized by the CVD method still contain significant quantities (1.2%-14.3%) of residual metals after it has been postprocessed to reduce metals (47). Small amounts of catalytic metals might still remain after the acid-etching and scooping steps in CVD graphene preparation, which consequently result in ROS production, and then cytotoxicity. The statistical results showed that the neurite length was significantly different, but the neurite number per cell was low (Fig. 9).

NGN shows no cytotoxicity to primary cortical neuron cells. The highly conductive NGN might be an idea for a promising scaffold using neuron cellular activity modification, as well as being biosensors for electrophysiology study.

Conclusion

NGN was simply created by spray coating onto PU membranes, and the primary cortical neuron cells were able to grow on this film. This strategy presented a simple method to explore graphene biomaterial application as a highly conductive scaffold for tissue engineering and as an ES sensor for monitoring cellular electrophysiology.

Footnotes

Acknowledgement

The author is grateful to William Dameida for his technical assistance.

Financial support: Financial support for this work was provided by the Natural Science Foundation of Chongqing (CSTC2012JJA1718) and the Scientific Research Start-up Capital Project of Chongqing Technology and Business University (CTBU: 2011-56-06).

Conflict of interest: None of the authors has any financial interest related to this study to disclose.

References

Stoller

Park

Zhu

Ruoff

Graphene-based ultracapacitors.

Nano Lett. 2008; 8(10):3498–3502.

Lee

Wei

Kysar

Hone

Measurement of the elastic properties and intrinsic strength of monolayer graphene.

Science. 2008; 321(5887):385–388.

Balandin

Ghosh

Bao

et al.

Superior thermal conductivity of single-layer graphene.

Nano Lett. 2008; 8(3):902–907.

Chen

Tang

Graphene-based materials in electrochemistry.

Chem Soc Rev. 2010; 39(8):3157–3180.

Wang

Lin

Graphene and graphene oxide: biofunctionalization and applications in biotechnology.

Trends Biotechnol. 2011; 29(5):205–212.

Stankovich

Dikin

Dommett

et al.

Graphene-based composite materials.

Nature. 2006; 442(7100):282–286.

Feng

Liu

Graphene in biomedicine: opportunities and challenges.

Nanomedicine (Lond). 2011; 6(2):317–324.

Cellot

Cilia

Cipollone

et al.

Carbon nanotubes might improve neuronal performance by favouring electrical shortcuts.

Nat Nanotechnol. 2009; 4(2):126–133.

Sanchez

Jachak

Hurt

Kane

Biological interactions of graphene-family nanomaterials: an interdisciplinary review.

Chem Res Toxicol. 2012; 25(1):15–34.

10.

Sasidharan

Panchakarla

Chandran

et al.

Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene.

Nanoscale. 2011; 3(6):2461–2464.

11.

Zhang

Ali

Dervishi

et al.

Cytotoxicity effects of graphene and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells.

ACS Nano. 2010; 4(6):3181–3186.

12.

Berlin

Leonard

Pham

et al.

Effective drug delivery, in vitro and in vivo, by carbon-based nanovectors noncovalently loaded with unmodified Paclitaxel.

ACS Nano. 2010; 4(8):4621–4636.

13.

Chang

Yang

Liu

et al.

In vitro toxicity evaluation of graphene oxide on A549 cells.

Toxicol Lett. 2011; 200(3):201–210.

14.

Sun

Liu

Welsher

et al.

Nano-graphene oxide for cellular imaging and drug delivery.

Nano Res. 2008; 1(3):203–212.

15.

Meng

Rouabhia

Zhang

Electrical stimulation in tissue regeneration.

In: Gargiulo GD, McEwan A, eds. Applied Biomedical Engineering. Rijeka, Croatia: InTech. 201137–62.

16.

Moroder

Runge

Wang

et al.

Material properties and electrical stimulation regimens of polycaprolactone fumarate-polypyrrole scaffolds as potential conductive nerve conduits.

Acta Biomater. 2011; 7(3):944–953.

17.

Spitzer

Lautermilch

Smith

Gomez

Coding of neuronal differentiation by calcium transients.

BioEssays. 2000; 22(9): 811–817.

18.

Aebischer

Valentini

Dario

Domenici

Galletti

Piezoelectric guidance channels enhance regeneration in the mouse sciatic nerve after axotomy.

Brain Res. 1987; 436(1):165–168.

19.

Patel

Poo

Orientation of neurite growth by extracellular electric fields.

J Neurosci. 1982; 2(4):483–496.

20.

Schmidt

Shastri

Vacanti

Langer

Stimulation of neurite outgrowth using an electrically conducting polymer.

Proc Natl Acad Sci USA. 1997; 94(17):8948–8953.

21.

Meng

Nerve cell differentiation using constant and programmed electrical stimulation through conductive non-functional graphene nanosheets film.

Tissue Engineering and Regenerative Medicine. 2014; 11(4):274–283.

22.

Gordon

Sulaiman

Boyd

Experimental strategies to promote functional recovery after peripheral nerve injuries.

J Peripher Nerv Syst. 2003; 8(4):236–250.

23.

Al-Majed

Neumann

Brushart

Gordon

Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration.

J Neurosci. 2000; 20(7):2602–2608.

24.

Geremia

Gordon

Brushart

Al-Majed

Verge

Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression.

Exp Neurol. 2007; 205(2):347–359.

25.

Brushart

Jari

Verge

Rohde

Gordon

Electrical stimulation restores the specificity of sensory axon regeneration.

Exp Neurol. 2005; 194(1):221–229.

26.

Kolb

Teskey

Gibb

Factors influencing cerebral plasticity in the normal and injured brain.

Frontiers in Human Neuroscience. 2010; 4Article no. 204.

27.

Brown

Lutsep

Cramer

Weinand

Motor cortex stimulation for enhancement of recovery after stroke: case report.

Neurol Res. 2003; 25(8):815–818.

28.

Adkins

Campos

Quach

Borromeo

Schallert

Jones

Epidural cortical stimulation enhances motor function after sensorimotor cortical infarcts in rats.

Exp Neurol. 2006; 200(2):356–370.

29.

Hassler

Boretius

Stieglitz

Polymers for neural implants.

J Polym Sci B Polym Phys. 2011; 49(1):18–33.

30.

Kotwal

Schmidt

Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials.

Biomaterials. 2001; 22(10):1055–1064.

31.

Prabhakaran

Venugopal

Chyan

et al.

Electrospun biocomposite nanofibrous scaffolds for neural tissue engineering.

Tissue Eng Part A. 2008; 14(11):1787–1797.

32.

Haastert-Talini

Schmitte

Korte

Klode

Ratzka

Grothe

Electrical stimulation accelerates axonal and functional peripheral nerve regeneration across long gaps.

J Neurotrauma. 2011; 28(4):661–674.

33.

Meng

Rouabhia

Shi

Zhang

Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,L-lactide) composites.

J Biomed Mater Res A. 2008; 87(2):332–344.

34.

Zhang

Meng

Zhang

Electroactivity and stability of polylactide/polypyrrole composites.

J Biomater Sci Polym Ed. 2011; 22(14):1931–1946.

35.

Caffo

Merlo

Marino

Caruso

Graphene in neurosurgery: the beginning of a new era.

Nanomedicine (Lond). 2015; 10(4):615–625.

36.

Ambrosi

Pumera

The CVD graphene transfer procedure introduces metallic impurities which alter the graphene electrochemical properties.

Nanoscale. 2014; 6(1):472–476.

37.

Karmakar

Zhang

Neurotoxicity of nanoscale materials.

J Food Drug Anal. 2014; 22(1):147–160.

38.

Hamilton

Lomeda

Sun

Tour

Barron

High-yield organic dispersions of unfunctionalized graphene.

Nano Lett. 2009; 9(10):3460–3462.

39.

Meng

Zhang

Rouabhia

Accelerated osteoblast mineralization on a conductive substrate by multiple electrical stimulation.

J Bone Miner Metab. 2011; 29(5):535–544.

40.

Meng

Rouabhia

Zhang

Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin-bioactivated conductive scaffolds.

Bioelectromagnetics. 2013; 34(3):189–199.

41.

Rouabhia

Park

Meng

Derbali

Zhang

Electrical stimulation promotes wound healing by enhancing dermal fibroblast activity and promoting myofibroblast transdifferentiation.

PLoS ONE. 2013; 8(8):e71660.

42.

Veech

Kashiwaya

King

The resting membrane potential of cells are measures of electrical work, not of ionic currents.

Integr Physiol Behav Sci. 1995; 30(4):283–307.

43.

Sun

Yan

Yao

Beitler

Zhu

Tour

Growth of graphene from solid carbon sources.

Nature. 2010; 468(7323):549–552.

44.

Zhou

Gao

Cytotoxicity of graphene: recent advances and future perspective.

Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2014; 6(5):452–474.

45.

Ryu

Kim

Culture of neural cells and stem cells on graphene.

Tissue Engineering and Regenerative Medicine. 2013; 10(2): 39–46.

46.

Wypijewska

Galazka-Friedman

Bauminger

et al.

Iron and reactive oxygen species activity in parkinsonian substantia nigra.

Parkinsonism Relat Disord. 2010; 16(5):329–333.

47.

Liu

Guo

Morris

Kane

Hurt

Targeted removal of bioavailable metal as a detoxification strategy for carbon nanotubes.

Carbon N Y. 2008; 46(3):489–500.