ARTICLE IN PRESS

Biomaterials 26 (2005) 26032610

www.elsevier.com/locate/biomaterials

Electrospinning of nano/micro scale poly(L-lactic acid) aligned bers

and their potential in neural tissue engineering
F. Yanga, R. Muruganb, S. Wangc, S. Ramakrishnaa,b,d,
a
Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576
b
NUS Nanoscience and Nanotechnology Initiative, National University of Singapore, 9 Engineering Drive 1, Singapore 117576
c
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos #04-01, Singapore 138669
d
Division of Bioengineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576

Received 8 March 2004; accepted 17 June 2004

Available online 2 November 2004

Abstract

Efcacy of aligned poly(L-lactic acid) (PLLA) nano/micro brous scaffolds for neural tissue engineering is described and their
performance with random PLLA scaffolds is compared as well in this study. Perfectly aligned PLLA brous scaffolds were
fabricated by an electrospinning technique under optimum condition and the diameter of the electrospun bers can easily be tailored
by adjusting the concentration of polymer solution. As the structure of PLLA scaffold was intended for neural tissue engineering, its
suitability was evaluated in vitro using neural stem cells (NSCs) as a model cell line. Cell morphology, differentiation and neurite
outgrowth were studied by various microscopic techniques. The results show that the direction of NSC elongation and its neurite
outgrowth is parallel to the direction of PLLA bers for aligned scaffolds. No signicant changes were observed on the cell
orientation with respect to the ber diameters. However, the rate of NSC differentiation was higher for PLLA nanobers than that
of micro bers and it was independent of the ber alignment. Based on the experimental results, the aligned nanobrous PLLA
scaffold could be used as a potential cell carrier in neural tissue engineering.
r 2004 Elsevier Ltd. All rights reserved.

Keywords: Electrospinning; Poly(L-lactic acid); Nanober; Alignment; Contact guidance; Neural stem cell; Neural tissue engineering

1. Introduction provide optimism by creating a permissive environment

Tissue engineering is an emerging area in the con-
temporary human health care administration, in which
the basic understanding of cellular biology and the
application of bioengineering are harnessed together for
developing feasible substitutes to aid in the clinical
treatment associated with human nervous system. More
precisely, the treatment of nerve tissue repair arising from
the spinal cord injury requires an additional care or new
medical therapy, because axons do not regenerate
appreciably in their native environment. Fortunately,
recent advances in the neural tissue engineering (NTE)
Corresponding author. National University of Singapore, 9
Engineering Drive 1, Singapore 117576. Tel.: +65 68742142; fax:
+65 68742162.
E-mail address: seeram@nus.edu.sg (S. Ramakrishna).
for nerve regeneration [14]. A variety of biomaterials, in
particular polymers, have been investigated for their
suitability in tissue engineering application. Among them,
PLLA is a promising scaffold material due to its
biocompatibility and biodegradability.
Recent studies focus on the use of scaffolds with
various advanced techniques in order to create complex
guidance channels, which precisely mimic a natural
repairing process in the human body [58]. Our previous
investigation also demonstrated an elegant way
to produce nanobrous scaffolds using PLLA by
a liquidliquid phase separation method quite similar
to natural extracellular matrix (ECM) and its efcacy
in supporting the NSC differentiation and neurite
outgrowth [9]. Though this method provided scaffolds
for NTE, there were however difculties encountered in

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biomaterials.2004.06.051
 ARTICLE IN PRESS
2604 F. Yang et al. / Biomaterials 26 (2005) 26032610
controlling ber diameter and alignment. As an alter-
native, a ber spinning technique, the so-called electro-
spinning, has been developed and succeeded in
fabricating brous scaffolds with required sizes and
dimensions depending upon the eld of application [10].
In general, the process of electrospinning is chiey
affected by (i) system parameters, such as polymer
molecular weight, molecular weight distribution and
solution properties (e.g. viscosity, surface tension,
conductivity); (ii) process parameters, such as ow rate,
electric potential, distance between capillary and collec-
tor, motion of collector, etc. [1113]. Therefore, these
parameters should be carefully optimized while control-
ling ber diameter and its alignment. Our previous
studies clearly demonstrated the feasibility of ber
spinning with controllable ber diameter and alignment
by varying polymer concentration and motion of the
collector [10,14]. However, as per literature survey, there
is no report on the design of aligned synthetic polymeric
scaffolds using an electrospinning technique, especially
in the nano or submicron scale range, resembling
natural ECM for purpose of NTE. As it is anticipated
that the aligned bers could provide better contact
guidance effects on the neurite outgrowth, the present
study of electrospinning of aligned PLLA brous
scaffold was undertaken and its suitability in NTE was
evaluated using NSC as a model cell line.
There is a growing research interest in NSC, in
particular C17.2, which generates hope for the develop-
ment of biomimetic cell therapy. This kind of cell line is
the primordial, multipotent self renewing cells, which
can be used as neuron precursors since they involve in
the normal development of cerebellum, embryonic
neocortex and other structures upon implantation
[8,15,16]. Furthermore, C17.2 cells are capable of
differentiating without the interaction with adhesion
molecules (without coating), such as laminin, bronec-
tin, collagen, poly-L-lysine or MatrigelTM, which are
generally required as permissive substrates in neurite
outgrowth. This offers the convenience of investigating
the physical effects of PLLA brous scaffolds because
the coating of adhesive molecules will affect the surface
topography of the scaffold. Based on the above reasons,
we have made an attempt to fabricate an aligned PLLA
nano brous scaffold by electrospinning and use C17.2
cells to assess its efcacy in promoting neuron differ-
entiation and guiding neurite outgrowth in vitro.
2. Experimental section
2.1. Fabrication of PLLA scaffolds
Both aligned and random PLLA bers were fabri-
cated by electrospinning technique under optimum
conditions [10,14,17]. Polymer solution was prepared
by dissolving the PLLA (Mw=300,000, Polysciences,
USA) into dichloromethane (DCM)/n,n-dimethyl-for-
mamid (DMF) (70:30) at the concentrations of 1%, 2%,
3% and 5% w/w. From each concentration of the
polymer solution, 10 g was fed into a 10-ml plastic
syringe (B-D, Singapore), which was controlled by a
syringe pump (KDS100, KD Scientic Inc., USA) at a
feeding rate of 1.0 ml/h. A Teon tube was used to
connect the syringe and a needle, which was set up
vertically. The needles of 18-G with an inner diameter of
1.2 mm were used for 5% w/w PLLA solution and the
needles of 22-G with inner diameter of 0.7 mm was used
for other PLLA solutions. The needle tips were cut into
a length of 1 cm in advance. The distance between the
syringe needle tip and the collector was adjusted to
10 cm. For collecting aligned bers, a rotating disk was
used, whereas a at aluminum plate was used for
collecting random bers. The linear rate of the rotating
disk at the edge was set to 1000 rpm. A high voltage of
12 kV was applied by a voltage regulated DC power
supply (ES30P/0.2/230/M826, Gamma High Voltage
Research, USA) to generate the polymer jet. The
resulting PLLA bers were collected on the 15-mm
coverslips placed at the respective collectors and used
for cell culture study after sterilization under the UV
lamp (30 W, 25 nm), overnight.
2.2. Structural morphology of PLLA scaffolds
The morphology of electrospun PLLA scaffolds was
studied by a scanning electron microscopy (SEM) (JSM
5600, JEOL, Japan) with an accelerating voltage of
15 kV. Before the observation, the scaffolds were coated
with gold using a sputter coater (Jeol JFC-1200 ne
coater, Japan). The diameter of the ber was measured
from the SEM photographs using an image analysis
software (Image J, National Institutes of Health, USA).
2.3. Cell culture and seeding
Neonatal mouse cerebellum C17.2 stem cells were
cultured in Dulbeccos Modied Eagle Medium (DMEM,
Sigma, USA) supplemented with 10% fetal calf serum and
5% horse serum as well. After reaching about 70%
conuence, the cells were detached by 0.05% trypsin and
viable cells were counted by trypan blue assay. Then the
cells were seeded onto PLLA scaffolds, placed in a 24-well
plate with the density of 2.8  104 cells/cm2 and cultured
with DMEM/F12 1:1 mixture (Gibco, USA) containing
N-2 supplement (Gibco, USA).
2.4. Cell morphology and neurite outgrowth studies
2.4.1. Phase contrast light microscopy (PCLM)
Live cell morphology was studied using an in-
verted phase contrast light microscope (Leica DMIRB,
 ARTICLE IN PRESS
F. Yang et al. / Biomaterials 26 (2005) 26032610
Germany). The phase contrast micrographs were taken
on cells attached to the scaffolds after rst day of cell
seeding at random locations of each scaffolds.
2.4.2. Laser scanning confocal microscopy (LSCM)
Immunouorescent labeling for neurolament 200 kD
(NF200) was performed to observe and compare the
phenotypes and the neurite outgrowth of differentiated
C17.2 cells seeded onto PLLA scaffolds. The scaffolds
were treated with a special care before LSCM observa-
tion. Briey, after 2 days of culture, the scaffolds were
rinsed with phosphate buffered saline (PBS) twice and
xed in 4% paraformaldehyde for 15 min at room
temperature followed by permeation with 0.5% Triton
X-100 for 5 min. Non-specic labeling was blocked by
incubating with goat serum (diluted in PBS at 1:20
ratio). Then the samples were incubated in rabbit anti-
NF200 (diluted as 1:200, Sigma, USA) overnight and
then kept in uorescein isothiocyanate (FITC) conju-
gated goat anti-rabbit secondary antibody (diluted as
1:50, Sigma, USA) for 4060 min. Finally they were
mounted on to glass slides and viewed under LSCM
(Leica TCS SP2, Germany) using the 20  and 40 
objectives. Fluorochromes were excited using an argon
laser at the wavelength of 488 nm for FITC.
The neurite orientation and outgrowth was studied
from the LSCM micrographs using the same image
analysis software as mentioned in the Section 2.2. Data
were collected from the ten LSCM micrographs at
random locations for each sample. Triplicate samples
were used for each scaffold. For the cells grown on
aligned PLLA scaffolds, the angle of neurite orientation
relative to the PLLA ber direction (y) was determined
as described by the earlier report [18]. Angles were
considered as parallel to the ber direction if y was
between 01 and 101 and as perpendicular if y was
between 801 and 901. The length of neurite was also
measured from the LSCM micrographs. Neurite length
was dened as a straight-line distance from the tip of the
neurite to the junction between the cell body and neurite
base. In the case of non-straight neurites, the length of
the real neurite curve was measured. Single-factor
analysis of variance (ANOVA) was employed to assess
statistical signicance of the results and the difference
was considered statistically signicant at po0.05.
2.4.3. Scanning electron microscopy
The SEM was employed to study the morphology of
cells grown on the scaffolds. The scaffolds were taken
out after 2 days of cell seeding and treated with standard
xation procedure. Samples were xed in 2% glutar-
aldehyde for 90 min and post-xed with 1% osmium
tetroxide (OsO4) for 20 min. After that, the samples were
treated with 1% tannic acid and 1% OsO4 for 30 min
each for conductive staining and then dehydrated with a
graded concentration (50100% v/v) of ethanol. Subse-
2605
quently, the samples were treated with hexamethyldisi-
lazane (HMDS) twice each for 30 min and kept in a
fume hood for air drying. Finally, the samples were
mounted onto a stub and coated with gold using sputter
coating for the observation of cell morphology.
3. Results and discussion
3.1. Electrospun PLLA fibrous scaffolds
The processing condition of fabricating aligned
nanobrous PLLA scaffold was optimized and it was
found that their properties were strongly inuenced by
the concentration of the polymeric solution. The
morphological structure of aligned electrospun PLLA
bers obtained from the 2% and 5% polymer solution
are shown in Fig. 1. Both the brous scaffolds exhibited
a high order of alignment, which was independent on
the polymer concentration. The ber diameter was
determined for both the cases, which ranged from 150
to 500 nm for 2% of PLLA concentration and
8003000 nm for 5% of PLLA concentration. The
average was about 300 nm (nanometer scale) for the
rst case, which was named as aligned nanobers (ANF)
and 1.5 mm (submicron scale) for the second case which
was named as aligned micro bers (AMF). The results
indicate that the ber diameters are directly propor-
tional to the polymer concentration and they increase
with increasing the solution concentration, which is
consistent with our previous reports of random PLLA
bers fabricated by electrospinning [10]. It was also
noticed that the average diameter of aligned bers was
smaller than that of the random bers obtained from the
same processing conditions. For example, the average
diameters of random bers obtained from the 2% and
5% w/w of PLLA concentration was found to be about
700 nm and 3.5 mm, respectively. The reason behind this
phenomenon may be explained by considering the high-
speed rotation of the wheel collector, which exerted a
tangential force on the polymer jet to stretch the
resulting bers.
It is anticipated that the aligned brous scaffold can
be fabricated to any thickness by adjusting the collecting
time. But we found that the ber orientation became
disordered at the top layer of the electrospun mesh when
the collecting time was longer than 30 min, which may
be due to the residual charges on the collecting bers. It
is known that an electrically charged polymer jet can be
created when the applied electrical force overcomes the
surface tension of the polymer solution. After the
polymer jet dries, it forms charged nano or micro size
bers. These residual charges seemed to be sufcient to
exert a repel force on the next depositing ber by
changing its orientation or pushing it aside, leading to
pores or voids between the aligned bers.
 ARTICLE IN PRESS
2606 F. Yang et al. / Biomaterials 26 (2005) 26032610
Fig. 1. SEM micrographs of PLLA (a) ANF; (b) AMF; (c) RNF and (d) RMF.
In order to investigate the effects of ber alignment on
NSC behavior, the random PLLA brous scaffolds with
similar ber diameters were chosen as their counterparts
in the following cell culture studies. These two scaffolds
were fabricated by PLLA solution with the concentra-
tion of 1% and 3% and the average ber diameter was
found to be about 250 nm, which is named as random
nanobers (RNF), and the average diameter of 1.25 mm
was named as random micro bers (RMF), shown in
Figs. 1c and d, respectively.
3.2. Cell morphological studies
As the NSCs play a unique and obligatory role in
nerve repair, their morphology, phenotype, neurite
outgrowth and cell interaction with the PLLA scaffolds
were studied by various microscopic techniques, such as
PCLM, LSCM and SEM.
Fig. 2 represents the PCLM micrographs of C17.2
cells cultured for 1 day on different scaffolds, namely
ANF, AMF and RNF. The overall results indicated that
the NSCs were well attached on all the scaffolds and the
cells changed their original round shape to elongated
and spindle-like shape. Further, it showed an extensive
neurite-like outgrowth on all the scaffolds, which
provided a morphological evidence of NSC differentia-
tion. As an important aspect, it can be seen from the
results that the direction of NSC elongation and neurite
outgrowth was exactly parallel to the direction of ber
alignment for ANF and AMF (Figs. 2a and b,
respectively), whereas it was random for RNF as
expected (Fig. 2c).
To observe the expression of the cytoskeleton proteins
of NSCs as well as the relationship between the
cytoskeleton proteins and topographies of the scaffolds,
such as ber alignment and diameter range, neurola-
ment immunocytochemistry staining and LSCM obser-
vation was performed on the cell cultured scaffolds at day
2. The orientation and outgrowth of neurites were
assessed from the ten LSCM micrographs taken at
random locations. Fig. 3 shows the LSCM micrographs
of NSCs cultured on the aligned PLLA bers with
different diameters. It can be seen that the NSCs cultured
on the aligned brous scaffolds exhibited a classical
contact guidance by growing parallel to the PLLA bers,
which supports and cooperates with the results of PCLM.
The majority of differentiated NSCs exhibit bipolar
shape with two extended neurites, which emerged from
the regions of the somas parallel to aligned PLLA bers
and were symmetrically distributed around the soma.
However, some exceptional cases were also found
occasionally (Fig. 3b, arrows). Although neurites out-
growth was not affected by the ber alignment at the
initial period, they all turned through large angles in
order to grow parallel to the ber alignment, which
suggests that the favorite growing direction of NSC
neurites is parallel to the PLLA nano and micro bers
and the process is dynamically directed over time.
 ARTICLE IN PRESS
F. Yang et al. / Biomaterials 26 (2005) 26032610
Fig. 2. PCLM micrographs showing NSCs attachment on (a) ANF; (b) AMF and (c) RNF, after 1 day of culture.
It was estimated that the frequency of parallel was
94% for ANF and the same was 88% for AMF. At the
same time, 4% neurites on ANF and AMF showed the
perpendicular response and the remaining 2% neurites
on the ANF and 8% neurites on AMF were in the
intermediate state, whose angles were between 101 and
801. These results indicate that the PLLA ber direction
is the most preferred orientation for NSCs and, further,
they did not show any signicant response on the
orientation of neurites outgrowth that was affected by
the ber dimensions.
Fig. 4 shows the LSCM images of neurolament
expression of NSCs cultured on the RNF and RMF
scaffolds. The results indicated that the random bers
had signicant changes in the phenotypes of NSCs
cultured for 2 days as compared to aligned bers. The
most signicant changes obtained from the results are (i)
the neurites were randomly orientated; (ii) the cells
extended multiple processes unlike the NSCs on aligned
bers; (iii) some of the neurites branched randomly
during the development and connected with the other
neurites and somas to form synaptic junctions. These
results suggest that the neurite outgrowth might be
mainly guided by the chemical or cellular cues since
there was no obvious oriented topographical guidance
provided by the random bers.
The effects of PLLA ber dimension and alignment
on the rate of differentiated NSCs, which changed their
shape from round to elongate, were also studied by
2607
counting the cell numbers from LSCM images. It was
found that more cells differentiated on ANF than those
on AMF and their quantitative differentiation rates
were 80% and 40%, respectively. These results were also
consistent with the results obtained from the cells
cultured on the RNF and RMF, i.e. about 80% of cells
had elongated morphology on the RNF whereas only
about 40% on the RMF. These results indicate that the
NSC differentiation rate seems to be independent of the
ber alignment, but depends on the ber dimension,
which suggest that the nanobers strongly support or
stimulate the cell differentiation as compared to the
micro bers.
The successful nerve regeneration relies on the
extensive growth of axonal processes. For this reason,
the present study also focused on the effect of PLLA
ber alignment and dimension on the neurite length.
Fig. 5 shows the neurite length of the NSCs cultured for
2 days on the scaffolds. The average neurite length of the
NSCs on ANF was calculated as 100 mm, which was
signicantly longer than that of other scaffolds. There
were no signicant differences among the neurite lengths
between AMF and random bers and statistically their
average values were between 75 and 80 mm. These
ndings suggest that the nanobers help to improve
the neurite outgrowth when they are in a highly
orientated status as compared to the results of ANF
and AMF, whereas PLLA ber alignment has strong
effects on the neurite outgrowth as compared to the
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2608 F. Yang et al. / Biomaterials 26 (2005) 26032610

Fig. 3. LSCM micrographs of immunostained neurolament 200 kD in NSCs after 2 days of culture; (a) on ANF, low magnication (  200); (b) on
ANF, high magnication (  400); (c) on AMF; low magnication (  200) and (d) on AMF, high magnication (  400).

Fig. 4. LSCM micrographs of immunostained neurolament 200 kD in NSCs after 2 days of culture; (a) on RNF and (b) on RMF.

results of ANF and RNF. However, the results showed
that there was no signicant effect on the neurite length
when the bers are in micron scale.
Guidance by substratum contours has been widely
investigated due to the importance of topographical
guidance in the tissue development, including establish-
ing precursor migratory patterns and guiding axonal
trajectories in the developing nervous system [3,7,18,19].
The results of this investigation showed that the aligned
nanobers (300 nm) could improve the NSC differentia-
tion and support the neurite outgrowth as compared to
other electrospun bers. As per literature survey, there is
no report available on the contact guidance of NSCs
cultured on the electrospun scaffolds. For the rst time,
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F. Yang et al. / Biomaterials 26 (2005) 26032610 2609

this study provides necessary information for the
understanding of the contact guidance of NSCs cultured
on PLLA scaffolds. However, the cellular mechanism
behind the directional effects of substratum contours on
the behavior of NSCs need to be studied further, which
is currently under progress.
The representative SEM micrographs of NSCs
cultured for 2 days on the PLLA scaffolds are shown
in Fig. 6. The SEM image of a single NSC cultured on
ANF can easily be seen in Fig. 6a and indicates that the
cell body had an apparent bipolar elongated morphol-
ogy with the outgrowing neurites. Both cell elongation
and neurite outgrowth follow the same direction of
PLLA nanobers. It also shows the interaction between
120
* that the ber alignment may have considerable effects in
neurite length (m)
the NSCs and ANF. It is obvious that some lament-
like structures extend out from the NSC cell body and
neurites and attach to the nanobers (Fig. 6a, arrow).
These structures were suspected to be the focal contacts
or focal adhesions, which are structurally dened
adhesion sites between the cultured cells and the ECM
[14,20].
The same evidence was also shown by the NSCs
cultured onto AMF (Fig. 6b, arrow). As focal contacts
are integrin-based structures that mediate strong cell
substrate adhesion and transmit information in a
bidirectional manner between ECM molecules and the
cytoplasm, this study provides an evidence for the
strong cellmatrix interaction. However, we have not
observed any signal to show the lament-like structure
of NSCs on the random bers (Fig. 6c), which implies
mediating the interaction between the NSCs and the
scaffolds. Further, the cellmatrix interaction studies are

80 under progress to disclose the molecular composition

and organization underlying their interactions on these
electrospun bers.
40

4. Conclusion

0 This study demonstrates the possibilities of fabricat-

ANF AMF RNF RMF ing aligned PLLA nano/micro brous scaffolds
Fig. 5. The average length of the longest neurite per cell was measured by electrospinning technique under optimum condi-
for 50 randomly selected cells per scaffold (mean for n=37Standard tion. It is obvious that the ber diameter can easily be
Error of Mean). Note that 2.8  104 cells/cm2 were plated (*po0.05). tailored by manipulating the processing parameters. The

Fig. 6. SEM micrographs of NSCs seeded on (a) ANF, (b) AMF and (c) RNF for 2 days showing the cellmatrix adhesion between the NSCs and
the PLLA bers. Bar=5 mm.
 ARTICLE IN PRESS
2610 F. Yang et al. / Biomaterials 26 (2005) 26032610
suitability of PLLA scaffolds for the NSC culture was
studied in terms of their ber alignment and dimension.
It was found that the NSCs elongated and their neurite
outgrew along with the ber direction for the aligned
scaffolds, whereas the ber diameter did not show any
signicant effect on the cell orientation. On the contrary,
the NSC differentiation rate was higher for nanobers
than that of micro bers, but independent with respect
to the ber alignment. As expected, the aligned
nanobers highly supported the NSC culture and
improved the neurite outgrowth. Based on the experi-
mental results, this study recommends the aligned PLLA
nanobrous scaffold as a potential cell carrier in NTE.
Acknowledgements
This study was supported by the Ministry of
Education of Singapore, Ofce of Life Sciences,
National University of Singapore and the Agency for
Science, Technology and Research (A* STAR), Singa-
pore.
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