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Research ArticleEnhanced Neural Cell Adhesion and Neurite Outgrowth onGraphene-Based Biomimetic Substrates

Suck Won Hong,1,2 Jong Ho Lee,1 Seok Hee Kang,1 Eun Young Hwang,2 Yu-Shik Hwang,3

Mi Hee Lee,4 Dong-Wook Han,1 and Jong-Chul Park 4

Department of Cogno-Mechatronics Engineering, Pusan National University, Busan -, Republic of Korea Education Program for Samsung Advanced Integrated Circuit, Pusan National University, Busan -, Republic of Korea

Department of Maxillofacial Biomedical Engineering, School of Dentistry and Institute of Oral Biology, Kyung Hee University,Seoul -, Republic of Korea Cellbiocontrol Laboratory, Department of Medical Engineering, Yonsei University College of Medicine,

Seoul -, Republic of Korea

Correspondence should be addressed to Dong-Wook Han; [email protected] and Jong-Chul Park; [email protected]

Received November ; Accepted December ; Published January

Academic Editor: Inn-Kyu Kang

Copyright © Suck Won Hong et al. Tis is an openaccess article distributed under the CreativeCommons AttributionLicense,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Neural cell adhesion and neurite outgrowth were examined on graphene-based biomimetic substrates. Te biocompatibility o

carbon nanomaterials such as graphene and carbon nanotubes (CNs), that is, single-walled and multiwalled CNs, againstpheochromocytoma-derived PC- neural cells was also evaluated by quantiying metabolic activity (with WS- assay),intracellular oxidative stress (with ROS assay), and membrane integrity (with LDH assay). Graphene lms were grown by usingchemical vapor deposition and were then coated onto glass coverslips by using the scooping method. Graphene sheets werepatterned on SiO

2/Si substrates by using photolithography and were then covered with serum or a neural cell culture. Both types

o CNs induced signicant dose-dependent decreases in the viability o PC- cells, whereas graphene exerted adverse effectson the neural cells just at over . ppm. Tis result implies that graphene and CNs, even though they were the same carbon-based nanomaterials, show differential inuences on neural cells. Furthermore, graphene-coated or graphene-patterned substrateswere shown to substantially enhance the adhesion and neurite outgrowth o PC- cells. Tese results suggest that graphene-basedsubstrates as biomimetic cues have good biocompatibility as well as a unique surace property that can enhance the neural cells,which would open up enormous opportunities in neural regeneration and nanomedicine.

1. Introduction

Graphene is a single-atom thick and is dened as a two-dimensional sheet o hexagonally arranged carbon atomsisolated rom its three-dimensional parent material, graphite[]. As with many novel materials, applications o grapheneand its amily nanomaterials, such as graphene oxide (GO),reduced GO (rGO), and graphene nanosheets, offer many technological opportunities since they exhibit interestingelectrical, thermal, mechanical, and optical properties [].Te practical uses o graphene amily nanomaterials areextensive, covering applications as diverse as battery elec-trodes, super-capacitors, nanoelectronics (e.g., transistors

and sensors), antibacterial paper, and many biomedical usesor drug delivery, diagnosis, and therapy [–]. Tese numer-ous potential applications o graphene and related materialsmake them very attractive to boththe scientic and industrialcommunity. However, to ensure the sae development o graphene and its amily nanomaterials, their potential impacton health and environment remains unelucidated yet.

Carbon nanotubes (CNs) and graphene, despite bothbeing carbon-based, are two very distinct nanomaterials, andtheir biological applications still keep wide open. Duringthe last decade, many studies o interactions between neuralcells and carbon nanomaterials (CNMs) including CNs,

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 212149, 8 pageshttp://dx.doi.org/10.1155/2014/212149

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BioMed Research International

graphene, and their derivatives were carried out with termi-nally differentiated primary cells or cell lines [, ]. Te pri-mary ocuses o very recent studies were on establishing bio-compatibility and biounctionality o the proposed materials,revealing that by pretreating ratswith amine-modied single-walled CNs (SWCNs) neurons could be protected and the

recovery o behavioural unctions in rats with induced strokecould be enhanced [], and graphene substrates exhibitedexcellent biocompatibility and signicantly promoted neuritesprouting and outgrowth o mouse hippocampal cells [].

In the present study, the biocompatibility between neuralcells and three CNMs, namely, graphene, SWCNs, and mul-tiwalled CNs (MWCNs), was evaluated and compared by quantiying metabolic activity, intracellular oxidative stress,and membrane integrity. Neural cell adhesion and neuriteoutgrowth were examined onto graphene-based biomimeticsubstrates.

2. Experimental

.. Synthesis and Morphological Observation of CarbonNanomaterials (CNMs). Grapheneand SWCNs weregrownby using chemical vapor deposition (CVD), as previously described [, ]. MWCNs were synthesized by using spray pyrolysis combined with a subsequent thermal CVD process,as described elsewhere [, ]. Afer being synthesized,each CNM was weighed by using an electronic balance(with a readability o . mg, Adventurer Analytical Balance,Ohaus, Bradord, MA). Te surace morphology o eachCNM was observed by using scanning electron microscopy (SEM). In brie, all CMNs were coated with an ultrathinlayer o gold/platinum by an ion sputter (E, Hitachi,

okyo, Japan) and were then observed with a eld emissionscanning electron microscope (FESEM, Hitachi S-) atan accelerating voltage o kV or graphene and kV orboth CNs. A colloidal dispersive solution o each CNMwas prepared in Dulbecco’s phosphate-buffered saline(DPBS,Sigma-Aldrich Co., St Louis, MO, pH .) with a nal concen-tration o ppm and was then sonicated or homogenousdispersions under mild conditions by using a water bathsonicator with a bath temperature o ∘C overnight. Forbiocompatibility evaluations, the suspension o each CNMwas serially diluted with × Dulbecco’s modied Eagle’smedium (DMEM, Sigma-Aldrich Co.) and was then treatedto the cultured monolayer o neural cells.

.. Preparation of Graphene-Based Substrates. Graphenelms were grown on catalytic copper (Cu) surace by usinga CVD method [, ]. For the preparation o a graphene-coated substrate, the grown graphene lm on a Cu oilwas transerred onto a glass coverslip by using the scoop-ing process. In detail, wt% o poly(methyl methacrylate)(PMMA, Sigma-Aldrich Co.) was spin-casted on a Cu oilat rpm or seconds and was then placed into an Cuetchant solution (ransene Company, Inc., Danvers, MA) tocompletely remove the Cu oil. Next, graphene covered with aPMMA substrate was scooped onto a glass coverslip, ollowedby removal o the PMMA by adding acetone or min.

For the neural cell adhesion, the top surace o graphenecoated on the glass coverslip was covered with % etalbovine serum (FBS, Sigma-Aldrich Co.) or hour whileshaking at ∘C and rpm []. In the case o the graphene-patterned substrate, a patterned array o graphene lms withrectangular shapes (100m × 150 m) was abricated on a

SiO2/Si substrate by using a conventional photolithography (AZ ) as described elsewhere []. Te morphology o the patterned graphene array was observed under a scanningelectron microscope (SEM, Hitachi S-, okyo, Japan)at an accelerating voltage o . kV. o observe the neuriteoutgrowth, the top surace o the patterned graphene wascovered with FBS by using the same method as mentionedabove.

.. Cell Cultures and Conditions. PC- cells (derived rompheochromocytoma o rat adrenal medulla) were obtainedrom American ype Culture Collection (ACC, Rockville,MD). Cells were routinely maintained in RPMI- media

(Sigma-Aldrich Co.) supplemented with % horse serum,% FBS, and % antibiotic antimycotic solution (including, U penicillin, mg streptomycin, and g ampho-tericin B per mL, Sigma-Aldrich Co.) at ∘C in a humidiedatmosphere o % CO

2 in air.

.. WST- Assay for Metabolic Activity Determination.Te number o viable cells was quantied indirectly by using highly water-soluble tetrazolium salt (WS-, -(-methoxy--nitrophenyl)--(-nitrophenyl)--(,-disulo-phenyl)-H -tetrazolium, monosodium salt; Dojindo Lab.,Kumamoto, Japan), reduced to a water-soluble ormazandye by mitochondrial dehydrogenases. Te cell viability was

ound to be directly proportional to the metabolic reactionproducts obtained in WS- []. Briey, the WS- assay was conducted as ollows: PC- cells were treated withincreasing concentrations (.∼ ppm) o each CNMand were then incubated with WS- reagent or the last hours o the culture period ( hours) at ∘C in the dark.Parallel sets o wells containing reshly cultured cells, whichwere not treated with any CNMs suspended in the sameconcentration ratio o DPBS and DMEM, were regarded asnegative controls. Te absorbance was determined at nmby using an ELISA reader (SpectraMax , MolecularDevice Co., Sunnyvale, CA). Te relative cell viability wasdetermined as the percentage ratio o the optical densities in

the media (containing CNMs at each concentration) to thato the resh control medium.

.. DCF Assay for Oxidative Stress Determination. Te ,-dichlorodihydrouorescein (DCF) assay is a widely usedmethod to detect intracellular reactive oxygen species (ROS)levels in pharmacological studies [, ]. Te accumulationo intracellular ree radicals rom CNMs was quantied usinga ROS assay kit (OxiSelect, Cell Biolabs, Inc., San Diego, CA),which employs the cell-permeable uorogenic probe ,-dichlorodihydrouorescein diacetate (DCFH-DA). DCFH-DA is an ROS detector that can cross cell membranes andbe deacetylated by intracellular esterases to nonuorescent

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,-dichlorodihydrouorescein (DCFH). In the presence o ROS, DCFH is rapidly oxidized to the highly uorescentDCF, which is readily detectable. Te uorescence intensity is proportional to the ROS levels within the cell cytosol.PC- cells were exposed to increasing concentrations (.∼ppm) o each CNM or h and werethenincubated with

DCHF-DA or minutes at

∘

C in the dark. Parallel sets o wells containing reshly cultured cells, which were not treatedwith any CNMs suspended in the same concentration ratioo DPBS and DMEM, were regarded as negative controls.Te uorescence emission o DCF was monitored at regularintervals at an excitation wavelength o nm and anemission wavelength o nm in a uorescence plate reader(VICOR Multilabel Counter, PerkinElmer, Inc., Waltham,MA). Te amount o DCF ormed was calculated roma calibration curve constructed using an authentic DCFstandard. Te relative DCF intensity was determined as thepercentage ratio o the uorescence intensities in the wells(containing CNMs at each concentration) to that in the reshcontrol well.

.. LDH Assay for Membrane Integrity Determination. Cellmembrane integrity was monitored using a lactate dehydro-genase (LDH) assay kit (akara Bio Inc, Shiga, Japan) todetermine the release o LDH into the medium according tothe manuacturer instructions. In this assay, LDH releasedrom damaged cells oxidizes lactate to pyruvate, whichpromotes conversion o the tetrazolium salt IN to a water-soluble red ormazan product []. Briey, afer hoursexposure to increasing concentrations (.∼ppm) o eachCNM, the supernatant rom each well was transerred to anew -well plate. Reconstituted substrate mix was added

to each well and the plates were kept or minutes in thedark at room temperature. Stop solution was then added toeach well. Parallel sets o wells containing reshly culturedcells, which were not treated with any CNMs suspended inthe same concentration ratio o DPBS and DMEM, wereregarded as negative controls. Released LDH catalyzed theoxidation o lactate to pyruvate with simultaneous reductiono NAD+ to NADH. Te rate o NAD+ reduction wasdirectly proportional to LDH activity in the cell medium. Teintensity o red color ormed in the assay was measured ata wavelength o nm with an ELISA reader (SpectraMax, Molecular Device Co.), which was proportional to thenumber o damaged cells. Te relative LDH release was

determined as the percentage ratio o the optical densities inthe media (containing CNMs at each concentration) to thato the resh control medium.

.. Assays for Neural Cell Adhesion, Neurite Outgrowth,and Proliferation. Te adhesion o PC- cells and theirneurite outgrowth were investigated onto graphene-coatedand graphene-patterned substrates, respectively, under theconditions o the culture media without neuralgrowth actorsor neuronal differentiation. Neural cells were seeded withhigh density o 2 × 105 cells/mL onto glass coverslips withFBS-covered graphene on them lying in a -well plate andwere then incubated or days. Afer incubation, cellular

morphology adhered onto graphene-coated substrates wasobserved under an inverted microscope (IX-F, OlympusOptical, Osaka, Japan). For observing neurite outgrowth,PC-

cells (low density o initial seeding, 1 × 1 04 cells/mL) werecultured or days onto FBS-covered graphene patterned ona SiO

2/Si substrate lying in a -well plate. Afer cultivation,

neurite outgrowth onto graphene-patterned substrate was visualized by using atomic orce microscopy (AFM, Innova,Veeco Instruments Inc., Plainview, NY). In order to comparethe prolieration pattern o PC- cells, cells were seeded onglass coverslips without and with FBS-covered graphene onthem and then cultivated or , , , and days at ∘C ina CO

2 incubator. Afer incubation, the cell prolieration was

determined by the WS- assay as described above.

.. Statistical Analysis. All variables were tested in threeindependent cultures or each cytotoxicity assay, which wasrepeated twice ( = 6). Quantitative data are expressed asmean ± standard deviation (SD). Data were tested or homo-

geneity o variances using Levene’s test, prior to statisticalanalysis. Multiple comparisons to detect the dose-dependenteffects o CNMs on PC- cells were carried out using one-way analysis o variance (ANOVA, SAS Institute, Cary, NC),which was ollowed by the Bonerroni test when varianceswere homogeneous and the amhane test when varianceswere not. Statistical analysis or the prolieration study wasmade by using the Student’s t -test. A value o < 0.05 wasconsidered statistically signicant.

3. Results and Discussion

.. SEM Analysis. Figure shows FESEM images o pristine

graphene, SWCNs, and MWCNs. All the CNMs werewell dispersed in the culture medium (DMEM) with serum.Most o graphene nanoplatelets existed as single or ew layersand presented both large and small sheets. Several graphenenanoplatelets with lateral sizes o around ∼ nm havebeen observed while a ew nanoplatelets showed smaller sizeswithin the range o ∼ nm. SWCNs and MWCNsmostly ormed nanobrous bundles o ∼ nm and ∼nmin diameter, respectively, and several m in length in thesuspension.

.. Effects of CNMs on Metabolic Activity. In order toevaluate the neural cell biocompatibility o CNMs, the effects

o CNMs on the metabolic activity o PC- cells wereexamined with the WS- assay where the ormation o ormazan dye depends on the mitochondrial enzyme activity.As shown in Figure , the viability o PC- cells decreasedin a dose-dependent manner afer hours o exposure toincreasing concentrations o each CNM. Graphene started torecord signicant ( < 0.05) mitochondrial toxicity rom. ppm and showed about % loss in the cell viability even at the topconcentrationtested (ppm) in comparisonto unexposed controls. In contrast, signicant ( < 0.05)cytotoxicity was induced at . ppm o both CNs, whichresulted in approximately %∼% inhibition o the viability in comparison to untreated controls. A recent study reported

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400nm 20nm 20nm

F : FESEM images o the surace morphologies o graphene nanoplatelets, SWCNs, and MWCNs.

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F : Effects o graphene, SWCNs, and MWCNs on mito-chondrial toxicity o PC- cells. Cells were treated with differentconcentrations o CNMs or hours. At the end o the incubationperiod, the WS- assay was perormed to evaluate the cytotoxicity as described in Section . Data were expressed as mean ± standarddeviation (SD) based on at least duplicate observations rom threeindependent experiments. Te letter “a” indicates statistically signi-icant difference rom the untreated control; the letter “b” indicatesstatistically signicant difference rom cells treated with graphene atthe same concentration ( < 0.05).

that GO showed stronger hemolytic activity against red bloodcells than aggregated graphene sheets whereas compactedgraphene sheets were more damaging to mammalian brob-lasts than less densely packed GO []. Moreover, it wasrevealed that .∼ ppm o SWCNs reduced the total DNAcontent o mixed neuroglial cultures []. MWCNs havebeen shown to induce massive loss o cell viability in humandermal broblasts through cell cycle arrest in the G

1 phase,

downregulation o adhesion-related genes, DNA damage,and programmed cell death as well as cause cytoskeletondamage and disturbance o actin stress bers in the range o ∼ ppm [, ]. In addition,our previousstudy revealed

that primary-cultured broblasts were more susceptible toCNMs than the broblast cell line []. As a result, it isconsidered that the WS assay has any detection limit to ndout cytotoxic effects o CNMs at relatively low concentrations(

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F : Effects o graphene, SWCNs, and MWCNs on ROS

generation in PC- cells. Cells were treated with different con-centrations o CNMs or hours. At the end o the incubationperiod, the DCF assay was perormed to evaluate the cytotoxicity as described in Section . Data were expressed as mean ± standarddeviation (SD) based on at least duplicate observations rom threeindependent experiments. Te letter “a” indicates statistically signi-icant difference rom the untreated control; the letter “b” indicatesstatistically signicant difference rom cells treated with graphene atthe same concentration ( < 0.05).

Furthermore, it was reported that vitamin E might protectPC- cells rom the injury induced by SWCNs through

the downregulation o oxidative stress and prevention o mitochondrial-mediated apoptosis [].

.. Effects of CNMs on Cell Membrane Integrity. LDHleakage is well known as a useul index or cytotoxicity on the basis o loss o membrane integrity, a hallmark o necrosis. All the CNMs induced apparent LDH release romPC- cells, revealing the adverse effect o CNMs on cellmembrane integrity (Figure ). Signicant LDH release wasnoted only afer hours o exposure to graphene at higherconcentrations ( and ppm). At lower concentrations(.∼ ppm), graphene had no effect on the release o LDH. In contrast, both SWCNs and MWCNs began to

induce a signicant ( < 0.05) increase in LDH releaserom . ppm and resulted in % and %, respectively at the highest concentration ( ppm) in comparison tountreated controls. Some reasons can be evoked to explainthe difference in cytotoxicity between graphene and CNs.Generally, the size (namely, dimensions), shape, composition,surace charge, and surace chemistry (e.g., unctionalization)o nanomaterials as well as the target cell type are criticaldeterminants o intracellular responses,degree o cytotoxicity and potential mechanisms o toxicity []. Te chemicalcomposition and dimensions o graphene are similar tothose o CNs, but the shape o graphene is completely different rom that o CNs (planar versus cylindrical) [].

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F : Effects o graphene, SWCNs, and MWCNs on LDH

release rom PC- cells. Cells were treated with different concentra-tions o CNMs or hours. At the end othe incubationperiod, theLDH assay was perormed to evaluate the cytotoxicity as describedin Section . Data were expressed as mean ± standard deviation(SD)based on at least duplicate observations rom three independentexperiments. Te letter “a” indicates statistically signicant differ-ence rom the untreated control; the letters “b” and “c” indicatestatistically signicant differences rom cells treated with grapheneand SWCNs, respectively, at the same concentration ( < 0.05).

Tus, it is likely that the piercing, needle-like CN may bemore mobile than the sheet-like graphene and can more

readily penetrate the cell membrane, resulting in greater cellmembrane damage []. Tese dose-dependent responses o PC- cells to CNMs correlated exactly with those rom theDCF assay, implying that cell membrane damage is anothermechanism or the toxicity o CNMs. In this study, the> ppm o graphene increased the LDH release and ROSgeneration. However, lowerdoses (.∼. ppm) o graphenehadno effect on multiple endpoints such as metabolicactivity,LDH leakage, and ROS production. Tereore, lower levels o exposure (

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A1 A2

(a)

B1 B3

B2

(b)

F : Neural cell adhesion (a) and neurite outgrowth (b) on graphene-basedbiomimetic substrates. Bright-eld images o a glasscoverslip

with FBS-covered graphene on it (A, scale bar = m) and PC- cells on the boundary area between glass (lower) and graphene (upper) days afer cell culture (A). SEM images (B and B) o graphene patterned on a SiO

2/Si substrate (B, enlarged image o B) and AFM image

o neurite outgrowth rom PC- cells on patterned graphene covered with FBS afer days o incubation (B, scale bar = m).

without graphene-coated layers afer days o incubation.Moreover, PC- cells adhered on the bare glass coverslipappeared to partly take a spindle shape, while the graphene-coated substrate did not seem to have the same effect oncells. Tis pattern in the cellular adhesion was in goodagreement with our previous study showing that adhesionand prolieration o PC- cells cultured onto graphene-coated glass coverslips were superior to those onto uncoated

ones []. It has been reported that NIH- broblasts,although the cell type is different rom neural cells, show highly improved cell growth, adhesion, and gene transectionefficiency on rGO/MWCN-coated substrates []. Anotherreport has revealed that rGO is biocompatible with PC- cells, whereas the SWCN network is inhibitory tothe prolieration, viability, and neuritegenesis o PC-cells []. Tis contrasting phenomenon was explainedby the hypothesis that could be attributed to the distinctnanotopographic eatures o these two kinds o nanocarbonsubstrates. Controlling microenvironments o cells oncertain substrates makes it possible to mimic in vivosituations and consequently contributes to the differentiation

o stem cells into specic cell types []. On the otherhand, PC- cells were shown to spread with apparentneurite outgrowth on the patterned graphene covered withFBS afer days o incubation (Figure (b)). As shownin Figure , PC- cells were cultured on glass coverslipswithout and with FBS-covered graphene on them, andtheir prolieration was examined by using the WS- assay.Te time-dependant prolieration pattern o PC- cells

on the glass coverslip with FBS-covered graphene on itwas almost similar to that o the cells on the bare glasscoverslip. However, PC- cells on the glass coverslip withFBS-covered graphene on it better prolierated than on thebare glass coverslip. Te differentiation o PC- cells couldbe initiated simply by exchanging culture media withoutany neural growth actors or neuronal differentiation.Tis result suggests that graphene-patterned substratesas biomimetic cues have a specic surace property thatcan promote neural cells. Recent evidence supports thissuggestion, showing that behaviors o neural stem cells,such as attachment, prolieration, and differentiation onthe surace-unctionalized graphene with laminin, were

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F : Prolieration o PC- cells cultured on bare glasscoverslips with and without FBS-covered graphene on them afer ,

, , and days o incubation. At the end o each incubation period,the WS- assay was perormed to examine the cell prolieration asdescribed in materials andmethods. Data were expressed as mean±standard deviation (SD) based on at least duplicate observationsrom three independent experiments. Te asterick denotes signi-cant difference in the prolierationbetween bareglass coverslips withand without FBS-covered graphene, < 0.05.

signicantly better than those o the pure graphene surace[]. In addition to this evidence, it has been reported thatCNs enhance the excitability o neurons by orming tightcontacts with the cell membrane so that electrical activity is

diverted through the nanotubes [, ].

4. Conclusion

From evaluation o the biocompatibility between neuralcells and CNMs, it was demonstrated that graphene exertedmuch less adverse effects on neural cells than both types o CNs, namely, SWCNs and MWCNs, at the concentra-tions lower than . ppm. Graphene-coated or graphene-patterned substrates were shown to substantially enhance theadhesion, neurite outgrowth, and prolieration o neural cells.Tereore, it is concluded that graphene-based biomimeticsubstrates have good biocompatibility as well as a unique sur-ace property that can enhance neural cells, which would bepotentially applied to neuralregeneration and nanomedicine.

Conflict of Interests

All the authors o this study do not have any direct nancialrelationship with commercial identities.

Acknowledgments

Tis research was supported by the Basic Science ResearchProgram through the National Research Foundation o Korea

(NRF) unded by the Ministry o Education, Science andechnology (RAAA and -).

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