neurite outgrowth of dorsal root ganglia neurons is enhanced on aligned
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7/30/2019 Neurite Outgrowth of Dorsal Root Ganglia Neurons is Enhanced on Aligned
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Neuroscience Letters 501 (2011) 10–14
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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: [email protected], [email protected] (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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