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Neuroscience Letters 501 (2011) 10–14

Contents lists available at ScienceDirect

Neuroscience Letters

 journa l homepage: www.elsevier .com/ locate /neulet

Neurite outgrowth of dorsal root ganglia neurons is enhanced on aligned

nanofibrous biopolymer scaffold with carbon nanotube coating

Guang-Zhen Jin a, Meeju Kim a,b, Ueon Sang Shin a, Hae-Won Kim a,b,c,∗

a Institute of Tissue Regeneration Engineering (ITREN), Dankook University, SouthKoreab Department of Nanobiomedical Science andWCU Research Center, Dankook UniversityGraduate School, SouthKoreac Department of Biomaterials Science, School of Dentistry, Dankook University, SouthKorea

a r t i c l e i n f o

 Article history:

Received26 April 2011

Receivedin revised form 2 June 2011

Accepted 11 June 2011

Keywords:

Carbon nanotube

Aligned nanofiber

PLCL 

Neurite outgrowth

FAK

Dorsal root ganglia

a b s t r a c t

Nerve regeneration and functional recovery have been a major issue following injury of  nerve tissues.

Electrospun nanofibers are known to be suitable scaffolds for neural tissue engineering applications. In

addition, modified substrates often provide better environments for neurite outgrowth. This study was

conducted to determine if multi-walled carbon nanotubes (MWCNTs)-coated electrospun poly (l-lactic

acid-co-caprolactone) (PLCL) nanofibers improved the neurite outgrowth of rat dorsal root ganglia (DRG)

neurons and focal adhesion kinase (FAK) expression of PC-12 cells. To accomplish this, the DRG neurons

in either uncoated PLCL scaffolds (PLCL group) or MWCNTs-coated PLCL scaffolds (PLCL/CNT group) were

cultured for nine days. MWCNTs-coated PLCL  scaffolds showed improved neurite outgrowth of  DRG

neurons. Moreover, FAK expression was up-regulated in the PLCL/CNT group when compared to the

PLCL group in a non-time-dependent manner. These findings suggest that MWCNTs-coated nanofibrous

scaffolds may be alternative materials for nerve regeneration and functional recovery in neural tissue

engineering.

© 2011 Elsevier Ireland Ltd. All rights reserved.

Nerveregeneration and functionalrecovery have been major issues

in the therapeutic field of injured neurons following accidents pri-

marily due to either misdirection of regenerating axons toward an

inappropriate targetor gaps between thecut ends of a nerve greater

than 1 cm [1,19]. Although autografts, allografts, and xenografts

have been used for strategies of nerve regeneration, they possess

the disadvantages of limited availability and immunological rejec-

tion, respectively [5,25]. Therefore, the development of artificial

nerve grafts is required.

Electrospun nanofibers have been investigated as scaffolds

for neural tissue regeneration applications. Specifically, aligned

nanofibers are better suited for neurite outgrowth than randomly

oriented nanofibers because the alignment provides guidance cues

and the anatomical features of natural nerves are highly organized

and aligned [8,15]. Moreover, some studies have shown that fiberswith diameters comparable to cell or smaller than that produce

faster neurite outgrowth than fibers 100m in diameter or larger

[2,3,12,21].

 Abbreviations: PLCL, poly (l-lactic acid-co-caprolactone); CNTs, carbon nan-

otubes; MWCNTs,multi-walledCNTs; DRG,dorsal rootganglia; FAK,focal adhesion

kinase; SEM, scanning electron microscopy; PBS, phosphate buffered saline; DAPI,

4 ,6-diamidino-2-phenylindole; DIV, days in vitro.∗ Correspondingauthor at: Institute of Tissue Regeneration Engineering (ITREN),

Dankook University, SouthKorea. Tel.: +8241 550 3081; fax: +8241 550 3085.

E-mail addresses: kimhw@dku.edu, kimhw@dankook.ac.kr (H.-W. Kim).

Carbon nanotubes (CNTs) are allotropes of carbon with a

cylindrical nanostructure and have unique structural, electrical,

and mechanical properties, which makes them potentially use-

ful for applications in many fields, including biomedical materials

[1,16,18]. In particular, the feasibility of using CNTs as a sub-

strate for neuronal growth has been demonstrated. Moreover,

several groups have suggested that neurons grown on physically

or chemically modified CNTs are more suitable than those grown

on unmodifiedmultiwalled carbon nanotubes (MWCNTs)[4,9]. Our

previous study also showed that chemically modified MWCNTs-

coated PLCL scaffolds improved neurite outgrowth of PC-12 cells

[6]. However, the mechanism responsible for improved neurite

outgrowth observed for CNTs is poorly understood.

In this study, rat dorsal root ganglia (DRG) neurons were cul-

tured on MWCNTs-coated electrospun polymer nanofibers fornine days. As the nanofiber composition, poly (l-lactic acid-co-

caprolactone) (PLCL) was used because this polymer is known

to be biocompatible and degradable and has been widely used

in tissue regenerative scaffolds. The neurite changes in the cells

under the influence of the MWCNT coating from the electrospun

fibers were then evaluated. Because focal adhesion kinase (FAK)

is a dominant protein involved in neurite outgrowth regulation

factors that play an important role in neurite extension and elon-

gation [11,13,20,22], we further examined the effects of CNTs

coated on the PLCL nanofibrous scaffolds on the FAK expression

of cells.

0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.neulet.2011.06.023

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G.-Z. Jinet al./ Neuroscience Letters 501 (2011) 10–14 11

The fabrication of aligned PLCL scaffolds has been described

previously [6]. Briefly, the aligned nanofibers were formed by elec-

trospinning PLCL (12.5% w/v) solution dissolved in a co-solvent

(20% ethanol + 80% dichloromethane). The polymer solution was

fed into a 10 m l syringe attached to a 21 gauge blunted stainless

steel needle using a syringe pump at a flow rate of 0.4ml h−1 with

an applied voltage of 13kV. A rotating cylindrical metal collector

was set at a speed of 4000rpm. Thenanofibers were then collected

onto the collector, which was kept 15cm from the needle tip.

Pristine MWCNTs purchased from ILJIN Nanotech Co., Ltd.

(Seoul, Korea) were subjected to ionic modification for approxi-

mately 30min to produce [MWCNT]SbF6 [17]. Anion exchange of 

the first derivative of [MWCNT]SbF6 was achieved by dissolving it

in potassium sodium tartrate and then stirring it for 3 h in an aque-

oussuspension of thederivative,which resulted in theformation of 

[MWCNT]tartrate. [MWCNT]tartrate (0.1mg/ml) solution was then

prepared in ethanol by ultrasonic treatment for 3 min. Next, the

PLCL scaffolds were placed in the modified MWCNTs solution for

about 1s, which led to formation of a MWCNT monolayer on the

scaffolds.

After dryingundervacuum, thesamples were coatedwith a thin

layer of gold andexamined by scanning electron microscopy (SEM;

Hitachi 3000, Japan) at an accelerating voltage of 15kV.

To assess the neurite outgrowth, the scaffolds (PLCL and PLCL-

CNT group) were placed in a 4-well plate and coated overnight

using poly-d-lysine (Sigma) with laminin (Sigma). Rat DRG neu-

rons (Cat. # R-DRG-505; Lonza Inc.) were then platedat a density of 

1×103 cells per scaffold and cultured in PNBM Basal Medium (Cat.

# CC-3256; LonzaInc.) supplemented with PNGMSingleQuots (Cat.

# CC-4462; Lonza Inc.) in a 37◦C, 5% (v/v) CO2 incubator for three,

six and nine days. To inhibit Schwann cell proliferation, the mitotic

inhibitors,uridine (17.5g/ml; Cat. # U-3003; Sigma) and5-fluoro-

2-deoxyuridine (7.5g/ml; Cat. # F0503; Sigma), were added to

the medium. During culture, the medium was changed every three

days by replacing 50% with fresh medium containing the mitotic

inhibitors.

PC-12cellswere also used todetect FAKexpression in this study.

The scaffolds were placed in a 24-well plate and the cells wereplated at a density of 5×104 cells per scaffold in Dulbecco’s Modi-

fiedEagle Medium(Gibco, USA) containing 0.5% fetal bovine serum

(low serum-containing medium). The cells were then treated with

50ng/ml of nerve growth factor (NGF; Cat. # GF028; Chemicon) in

low serum-containing medium for three, six and nine days.

An inverted microscope (IX-71; Olympus, Tokyo, Japan) was

used to observe the morphology of the DRG neurons. For Alexa

Fluor 546-conjugated phalloidin staining, the cells were fixed with

4% paraformaldehyde for 30min, treated with 0.2% Triton X-100

for 5min, blocked with PBS containing 1% (w/v) bovine serum

albumin for 30min, and then incubated with 20nM Alexa Fluor

546-conjugated phalloidin diluted in PBS for 30min. The nuclei of 

thecellswere counterstained using 4,6-diamidino-2-phenylindole

(DAPI, Sigma). A fluorescenceimage wasobtainedusing an inverted

microscope equipped with a DP-72 digital camera.

Ten images were taken from randomly selected areas in each of 

at least three scaffolds for each condition. A minimum of 250 cells

was evaluated in each experiment. To quantify the neurite exten-

sion, the longest neurite on each neurite bearing cell wasmeasured

using image analysis software (DP2-BSW, Olympus Co.) and the

mean values were then calculated.

Western blot analysis was conducted to assess the quantity

of FAK in PC-12 cells at days 3, 6, and 9 on both the PLCL and

the PLCL/CNT groups. Briefly, samples were homogenized in RIPA

buffer, after which the lysates were centrifuged at 12,000rpm for

10min at 4 ◦C. Next, the protein concentrationswere determinedby

the Bradford’s method. Subsequently, the proteins were separated

by 8% sodium dodecylsulfate polyacrylamide gel electrophoresis

and transferred electrophoretically to polyvinylidene difluoride

membrane. Nonspecific binding was blocked by immersing the

membrane in 5% non-fat dry milk for 1 h at room tempera-

ture. The membrane was then incubated with primary antibody

against FAK (1:1000; polyclonal, Santa Cruz Biotechnology, USA)

and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000;

polyclonal, Santa Cruz Biotechnology, USA) at 4 ◦C overnight.

The membrane was then washed and further incubated with

horseradish peroxidase-conjugated anti-rabbit IgG (1:5000; Santa

Cruz Biotechnology, USA). The blots were then developed using

the enhanced chemiluminescence method (Amersham Pharmacia

Biotech).

All results shown are expressed as the mean±S.E.M. Statistical

comparisons between the PLCL/CNTgroup and the PLCLgroup were

assessed by a Student’s t -test. A P < 0.05 or P < 0.01 was considered

to be significant.

Fig. 1 shows the SEM morphologies of aligned PLCL (A) and

CNT-coated PLCL (B) scaffolds. The surface of the CNT-coated PLCL scaffold was generally rough when compared to the PLCL scaffold.

The CNT molecules were uniformly coated on the PLCL nanofibers,

which were shown to preserve the nanofibrous aligned morphol-

ogyof thePLCLscaffold. Thediameters of thenanofibers as deduced

from the SEM images were about 1.3–1.5m.

The CNTs used in this study were modified to be water-soluble

by an ionic modification process [17]. In fact, CNTs are extremely

insoluble under aqueous conditions, which limits their utility for

Fig. 1. SEM examination of the structural morphology of PLCL (A) and CNT-coated PLCLaligned nanofibrous scaffolds (B).

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12 G.-Z. Jin et al. / Neuroscience Letters 501 (2011) 10–14

Fig. 2. Fluorescence images of DRG neurons cultured on PLCL scaffolds for three days (A and B), six days (C and D), and nine days (E and F). (A, C, and E) PLCL alone and (B,

D, and F) CNT-coated PLCL. Yellow, actin cytoskeleton stained with Alexa Fluor 546-conjugated phalloidin; blue, nuclei counterstained with DAPI. (For interpretation of the

references to color in this figure legend, thereader is referred to theweb version of thearticle.)

the modification of biomaterial substrates. However, the CNTs

newly developed here by an ionic modification could be homog-

enized in water and consequently applied to uniformly coat the

nanostructured biopolymer surface. A uniform coating of the sur-

face with CNTs isthus mainlydue tothe homogeneousdispersionof 

CNTs in an aqueous solvent where the biopolymer was also intact

without degradation. The coating method used herein is consid-

ered to be simple andrapidly performed, a versatile process to coatthe surface of complex shaped biomaterials. Although many CNT-

related studies of nerveregeneration have focused on the behaviors

of neural cells directly on CNTs that were supported on flat glass

substrates [4,9], the present study exploited the use of nanofibrous

scaffolds and their modifications with CNTs, proposing a system

more relevant to the applications for neural tissue engineering.

The DRG neurons were grown on the PLCL and CNT-coated PLCL 

nanofibers for three, six and nine days, after which they were fixed

and stained with an Alexa Fluor 546 phalloidin to visualize their

neuritic growth. Consistent with our previous study [6], we found

that theneurite outgrowthof theDRG neurons occurred after three

days of incubation and continued during the following nine days.

The direction of the neurite elongation was largely parallel to the

artificial nanofibers (Fig. 2). To determine if the CNT coating on thenanofibers influenced the extent of neurite growth, the lengths of 

allof therepresentativeneuriteswere measured.Comparison of the

mean neurite lengths of the DRG neurons cultured on CNT-coated

PLCL with those of neurons cultured on uncoated PLCL revealed

that the neurites on the CNT-coated PLCL were significantly longer

at all time points: 3 days in vitro (DIV): 204.12±28.40m; 6

DIV: 376.76±37.24m; 9 DIV: 576.75±36.35m. The lengths of 

neurites on PLCL alone were: 3 DIV: 117.39±25.35m; 6 DIV:

273.72±20.70m; 9 DIV: 425.67±25.36m. These lengths also

increased significantly with time (Fig. 3).

Fig. 3. Average lengths of neurite outgrowth from DRG neurons. Neurites on CNT-

coated PLCL fibers grew farther than those on uncoated PLCL fibers. *P <0.05 and

**P < 0.01, by Student’s t -test.

To determine if coating with MWCNTs affected FAK expression

of the neural cells, FAK protein was detected by Western blotting

(Fig. 4). The results showed a stronger signal at 125 kDa in the

PLCL/CNTgroup than in the PLCL group with statistical significance

at all time points (*P < 0.05), and this up-regulation occurred in a

non-time-dependent manner.

We previously demonstrated that CNTs-coated PLCL nanofi-

brous scaffolds promoted adhesion, proliferation and neuriteoutgrowth of PC-12 cells [6]. In the present study,we extended our

work to theneurite outgrowthbehaviorof therat DRGneurons and

observed the stimulation of neurite outgrowth by the CNTs coated

on the nanofiber scaffolds. We also examined the molecular basis

of the effect of the CNTs coating on neurite outgrowth in terms

of FAK expression. The results revealed that FAK expression was

significantly improved in the CNTs-coated scaffolds.

Modification of CNTs should alter the surface properties of the

PLCL biopolymer. The smooth PLCL surface became rough at the

nanoscale level because the CNT molecules have diameters of only

a few nanometers. Moreover, as the CNTs were ionically modified,

the coverage of CNTs would make the hydrophobic PLCL surface

more hydrophilic. Furthermore, the ultrahigh electrical conduc-

tivity of CNTs should alter the nonconducting nanofiber into anelectrically conducting substrate. Although we could not elucidate

which effect was greater in stimulation of the neurite outgrowth of 

the DRG neurons, the application of CNTs on the polymer nanofi-

brous substrate was clearly demonstrated to have positive effects.

More in-depth evaluations on these physical and electrical proper-

ties and the relationship between those factors and the neuronal

cell behaviors are considered as an important and interesting area

needs future study.

We focused on FAKas oneof themolecules that maybe involved

in the CNTs-related neuronal outgrowth. FAK is a cytoplasmic

tyrosine kinase that has been found to play a key role in integrin-

mediated signal transduction pathways [14,23]. FAK activation

mediated by the integrin signaling pathway has been shown to

be essential for the induction of neurite outgrowth in neuronsand MSCs [10,11,20]. Therefore, we assumed that CNTs might acti-

vate the extracellular receptor, which may then up-regulate FAK

expression and neurite outgrowth via the aforementioned path-

way. Previously, Zhang et al. showed that hydrophilic CNTs were

beneficial to cell viability and growth, and able to induce up-

regulation of the expression of cellular FAK [26]. In our system,

the CNTs-tethered surface nanostructure may have influenced the

expression of the key adhesion molecule, FAK.

Another possible mechanism of the neurite outgrowth by CNTs

in the cells used in this study might be the influx of Ca ions and

associated plasma membrane/vesicular recycling [24], which in

turn regulate the growth cone motility and neurite outgrowth

[7,9]. Therefore, the enhanced neurite outgrowth observed on

the CNTs-coated PLCL nanofibers may also be attributed, at least

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G.-Z. Jinet al./ Neuroscience Letters 501 (2011) 10–14 13

Fig. 4. FAK protein of PC-12 cells on PLCL and CNT-coated PLCL nanofibrous scaffolds was measured by Western blotting. Data shown are the ratios of the FAK to GAPDH

densities. *P <0.05forn= 3, by Student’s t -test.

in part, to the changes in the intracellular Ca2+ level induced

by the CNTs, although a more in-depth investigation of this

is necessary. Specifically, a detailed mechanism for the role of 

the CNTs in the promotion of FAK expression and neurite out-

growth still needs to be determined, as well as the method by

which the nanofibers interact with other intra- and extracellularproteins.

As demonstrated, the PLCL nanofibrous scaffolds coated with

CNTs proved to improve neuronal outgrowth in vitro. This result,

together with the properties of the matrices such as biodegrad-

ability, tissue compatibility and the aligned morphology, supports

the possible usefulness as implantable nerve guidance in sci-

atic or spinal cord injury site. However, in vivo animal tests

will be needed to confirm the feasibility of the scaffolds for the

applications.

In conclusion, the CNTs-coating of PLCL nanofibrous scaf-

folds showed a remarkable increase in the neurite outgrowth

of the rat DRG neurons and up-regulation of the FAK expres-

sion. Based on the present experimental results, the CNTs-coated

nanofibrous scaffolds may be alternative substrates for nerve

regeneration and functional recovery in neural tissue engineer-

ing.

 Acknowledgements

This work was supported by Priority Research Centers Pro-

gram through the National Research Foundation of Korea (NRF)

funded by the Ministry of Education, Science and Technol-

ogy (grant no. 2009-0093829). We deeply thank Dr. J.K. Hyun

(Department of Nanobiomedical Science and WCU Research Cen-

ter, Dankook University) for the kind donation of rat DRG

neurons. Authors appreciate the support of IBST, Dankook Univer-

sity.

 Appendix A. Supplementary data

Supplementary data associated with this article can be found,in

the online version, at doi:10.1016/j.neulet.2011.06.023.

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