Proc. Natl. Acad. Sci.
USA
Vol. 94, pp. 8948–8953, August 1997
Applied Biological Sciences
Stimulation of neurite outgrowth using an electrically
conducting polymer
(conductive polymeryoxidized polypyrroleynerve regenerationyPC-12 cellsyelectrical stimulationyneuronal stimulation)
CHRISTINE E. SCHMIDT*†‡, VENKATRAM R. SHASTRI*‡, JOSEPH P. VACANTI§,
*Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139; and §Department of Surgery, Children’s Hospital,
Harvard University, Boston, MA 02115
Contributed by Robert Langer, June 13, 1997
ABSTRACT Damage to peripheral nerves often cannot be
repaired by the juxtaposition of the severed nerve ends.
Surgeons have typically used autologous nerve grafts, which
have several drawbacks including the need for multiple sur-
gical procedures and loss of function at the donor site. As an
alternative, the use of nerve guidance channels to bridge the
gap between severed nerve ends is being explored. In this
paper, the electrically conductive polymer—oxidized polypyr-
role (PP)—has been evaluated for use as a substrate to
enhance nerve cell interactions in culture as a first step toward
potentially using such polymers to stimulate in vivo nerve
regeneration. Image analysis demonstrates that PC-12 cells
and primary chicken sciatic nerve explants attached and
extended neurites equally well on both PP films and tissue
culture polystyrene in the absence of electrical stimulation. In
contrast, PC-12 cells interacted poorly with indium tin oxide
(ITO), poly(L-lactic acid) (PLA), and poly(lactic acid-co-
glycolic acid) surfaces. However, PC-12 cells cultured on PP
films and subjected to an electrical stimulus through the film
showed a significant increase in neurite lengths compared
with ones that were not subjected to electrical stimulation
through the film and tissue culture polystyrene controls. The
median neurite length for PC-12 cells grown on PP and
subjected to an electrical stimulus was 18.14 mm (n 5 5643)
compared with 9.5 mm (n 5 4440) for controls. Furthermore,
animal implantation studies reveal that PP invokes little
adverse tissue response compared with poly(lactic acid-co-
glycolic acid).
The current clinical approach to repair a peripheral nerve over
a gap involves the utilization of autologous nerve grafts (1).
However, this procedure has several disadvantages, including
loss of function at the donor nerve graft site and mismatch of
damaged nerve and graft dimensions. As an alternative to
nerve autografts, natural and synthetic tubular guidance chan-
nels have been the subject of intensive research (2). Guidance
channels help direct axons sprouting off the regenerating nerve
end, provide a conduit for diffusion of neurotrophic and
neurotropic factors secreted by the damaged nerve stump, and
minimize infiltrating fibrous tissue. Materials that have been
investigated for nerve repair range from autologous veins (3)
and muscles (4), to prosthetic tubes derived from collagen,
laminin, fibronectin (5–7), silicone (8), and various other
biodegradable and nonbiodegradable polymers (2). However,
the engineering of an ideal nerve guidance channel that
provides an attractive clinical alternative to nerve autografts
remains a challenge.
Past work has demonstrated that electrical charges play an
important role in stimulating either the proliferation or dif-
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8948
AND ROBERT L ANGER*¶
ferentiation of various cell types. For example, it has been
shown that neurite outgrowth is enhanced on electrets such as
poled polyvinylidene fluoride (9, 10) and poled polytetrafluo-
roethylene (11). Electrets are broadly defined as materials
possessing quasi-permanent surface charge because of trapped
monopolar charge carriers. In piezoelectric materials such as
poled polyvinylidene fluoride, transient surface charges are
generated as a result of minute mechanical deformations of the
material, whereas poled polytetrafluoroethylene displays a
static surface charge. Enhanced neurite outgrowth has been
attributed to the presence of these surface charges. Further-
more, extensive research both in vitro and in vivo has shown
that electromagnetic fields play an important role in neurite
extension and regeneration of transected nerve ends (12–14).
It occurred to us that a polymer scaffold or guidance channel
derived from an electrically conducting polymer could prove
potentially useful in not only providing neuronal guidance but
in localizing electromagnetic stimulation as well. The electri-
cally conducting polymer, oxidized polypyrrole (PP), was
chosen for this study because of its inherent electrical con-
ductive properties, ease of preparation, flexibility of altering
surface characteristics, and its in vitro compatibility with
mammalian cells (15, 16). In its oxidized form, polypyrrole is
a polycation with delocalized positive charges along its highly
conjugated backbone and is an electronic conductor (17).
Charge neutrality is achieved by the incorporation of nega-
tively charged ions termed ‘‘dopants.’’ PP also exhibits revers-
ible electrochemistry allowing for the control of surface-
charge density by varying the oxidation state of the polymer.
For example, the application of a reduction potential to PP
converts it from a conductive form to its neutral (insulative)
form with the release of dopant anions or the incorporation of
cations to maintain charge neutrality. Potential biomedical
applications have been developed based on the electrochem-
ical properties of PP. These include the use of PP as a matrix
for controlled delivery of dopamine (18) and as a biosensor for
detection of glucose (19) or proteins (20).
Surface characteristics such as charge density and wettabil-
ity play a key role in protein adsorption and cell–substrate
interactions. Recently, it has been shown that both cell–surface
interactions and cellular functions (e.g., DNA synthesis) on PP
thin films can be controlled by either changing the oxidation
state of the polymer (15) or by changing the wettability
(hydrophobicity) of the polymer film using appropriate do-
pants (16). Surface characteristics are critical because the
interaction of endogenous proteins with a biomaterial at the
implantation site will have a significant impact on the adhe-
Abbreviations: PP, polypyrrole; PLA, poly(L-lactic acid); PLGA, poly-
(lactic acid-co-glycolic acid); TCPS, tissue culture polystyrene; ITO,
indium tin oxide; NGF, nerve growth factor.
†Present address: Department of Chemical Engineering, University of
Texas, Austin, TX 78712.
‡C.E.S. and V.R.S. contributed equally to this work.
¶To whom reprint requests should be addressed. e-mail: rlanger@
mit.edu.
 Applied Biological Sciences: Schmidt et al.
sion, differentiation, and proliferation of surrounding cells.
Flexibility at the level of controlling protein adsorption pro-
vides a means to engineer materials that yield predictable and
desirable cell–surface interactions. Therefore, PP is an inter-
esting candidate for tissue engineering applications, by virtue
of its inherent electrical conductivity, the ease with which one
can control crucial surface properties such as wettability and
charge density, and its compatibility with mammalian cells.
In this study, the utility of the electrically conductive poly-
mer, PP, as a substrate to enhance nerve cell differentiation in
culture was evaluated with the hope of ultimately using
electrically conducting polymers to stimulate in vivo nerve
regeneration. We show that PP is a suitable material for in vitro
nerve cell culture and that application of an electric stimulus
through PP enhances neurite outgrowth. We also show that PP
does not elicit an adverse tissue response when implanted in
rats.
MATERIALS AND METHODS
Preparation of Polypyrrole Films. PP was synthesized elec-
trochemically (21). Indium tin oxide (ITO)-conductive boro-
silicate glass (Delta Technologies, Still Water, MN) was used
as the electrically conductive surface for PP film deposition.
ITO glass slides (75 3 25 mm or 50 3 25 mm, 40Vysquare)
were cleaned before use by sonication in hexane, methanol,
and methylene chloride for 5 min each. A three-electrode
setup consisting of an ITO glass working electrode, platinum
gauze counter electrode, and a AgyAgCl reference was used
for the electrochemical synthesis of PP. PP film was deposited
at a constant potential of 0.7 V versus AgyAgCl reference from
an aqueous solution of 0.1 M pyrrole (Aldrich) containing 0.1
M sodium salt of poly(styrenesulfonate) (PSS) (Aldrich). A
Pine Instruments AFRDE4 bipotentiostat (Grove City, PA)
was used as the source of constant voltage. Films of two
different thickness were synthesized: 0.1–0.15 mm (thin film)
and 1.8–2.0 mm (thick film). The film thickness was controlled
by the passage of charge (21), which was determined by
integrating current over time. Thin films were optically trans-
parent and were used for in vitro analyses, and thick films were
used for in vivo implantation studies. Polypyrrole, with PSS as
the dopant, will be referred to here simply as PP.
Due to the fragile nature of freestanding PP thick films, they
were laminated with PLGA films, a polymer that has been
approved by the Food and Drug Administration for certain
applications, to yield processible and suturable disks for in vivo
studies. In brief, the PP thick film was gently peeled off the ITO
glass and then floated in distilled deionized water and subse-
quently transferred onto a clean glass slide. The PP film was
then wetted with methylene chloride and layered with PLGA
film. PLGA 85:15 (Birmingham Polymers, Birmingham, AL)
films of 40 6 2-mm thickness were solvent cast from a
methylene chloride solution (100 mg of purified PLGA in 1 ml
of methylene chloride) at room temperature overnight. For
implantation, laminated PP films were cut into 5-mm-diameter
disks and sterilized by exposure to UV light for 30 min.
Cell Culture. Rat PC-12 cells (American Type Tissue Col-
lection) (22) were used for in vitro studies. PC-12 cells respond
reversibly to nerve growth factor (NGF) by differentiation into
the neuronal phenotype (extension of neurites). In addition,
sciatic nerve explants were isolated from 16-day chick embryos
as previously described (23).
PC-12 cells were cultured in 85% high-glucose DMEM, 10%
heat-inactivated horse serum, and 5% fetal bovine serum.
Cells were maintained in a humid, 5% CO2 incubator and
passaged using 0.25% trypsin at 1:2 dilution every other day.
PC-12 cells were ‘‘primed’’ by the addition of 25 ngyml NGF
(Boehringer Mannheim) 24 h prior to seeding cells for an
experiment. During experiments, cells were maintained in
medium supplemented with 25 ngyml NGF. Cells were seeded
Proc. Natl. Acad. Sci. USA 94 (1997) 8949
into wells formed by the attachment of sterile Plexiglas wells
(1 cm 3 1.5 cm, inner dimension) to PP films using autoclaved
vacuum grease. In all experiments, 1 ml of medium containing
2–2.5 3 104 cells was used per well (1.33–1.67 3 104 cellsycm2).
Chick sciatic nerve explants were maintained in DMEM
supplemented with 10% fetal bovine serum, glucose to a total
of 6 mgyml, 1 mM sodium pyruvate, and 25 ngyml NGF.
In Vitro Electrical Stimulation. Primed PC-12 cells were
plated into cell wells assembled onto PP thin films (see above)
at densities of 1.33 3 104 cellsycm2 and then incubated for 24 h
to permit attachment and spreading. After this initial 24-h
period, the PC-12 cells were subjected to a steady potential of
100 mV for 2 h. For electrical stimulation, the PP film served
as the anode and a gold (Au) wire placed at the opposite end
(along the length) of the well served as the cathode. A silver
(Ag) wire served as a quasi-reference electrode. A bipoten-
tiostat (Pine Instruments) was used as the source of constant
voltage. Cells were maintained in a CO2 incubator for the
duration of electrical stimulation. After electrical stimulation
(S), the cells were incubated for an additional 24 h (a total of
48 h from the start of the experiment). Images of cells were
obtained both before (24 h) and after exposure to the constant
potential (48 h). PC-12 cells plated on PP thin films that were
not subjected to any electrical stimulation (NS) served as
controls. Cells grown on tissue culture polystyrene (TCPS) and
not subjected to any electrical stimulus served as additional
controls. A seeding density of 1.33 3 104 cellsycm2 was
maintained for all experiments. The neurite lengths of cells
under stimulation were compared with controls to estimate the
extent of differentiation.
To assess any effects of possible convective ion transport or
oxidationyreduction products on neurite outgrowth, cells
grown on PP were subjected to a constant potential (100 mV)
that was applied through the medium and not the polymer film
(‘‘solution control’’). To accomplish this, two gold wires were
affixed to opposite ends of the cell well and served as anode
and cathode. A silver wire on a third side of the well was used
as a quasi-reference.
In addition to the application of a constant potential,
electrical leads were directly attached to opposite ends of the
PP film and constant current of 10 mAmp was passed through
the polymer film. For comparison, the constant potential
scenario of 100 mV corresponds to a current of about 100
mAmp, given that the resistance of the PP film is about 1 kV.
The use of this magnitude of current is justified based on
previous electrical stimulation studies in rats using currents of
0.6 mAmp (13), 10–30 mAmp (24), and 400 mAmp (25).
Neurite Length Measurements. Thin PP films permitted the
use of light microscopy and quantitative image analysis tools to
study cell–material interactions in detail. Cells were viewed
using an inverted phase contrast microscope (Diaphot-TMD;
Nikon) and a 203 objective. Images from the microscope were
acquired using a CCD video camera (HVC-20; Hitachi, Japan)
and were subsequently digitized using NIH IMAGE software and
a Scion image capture board (LG-3; Scion, Frederick, MD).
The lengths of the individual neurites for each cell were
measured using the NIH IMAGE software. Length was defined
as the straight-line distance from the tip of the neurite to the
junction between the cell body and neurite base. In the rare
case of branched neurites, the length of the longest branch was
measured from the tip of the neurite to the cell body, then each
branch was measured from the tip of the neurite to the neurite
branch point.
Neurite Data Analysis and Statistical Evaluation. For a
single experiment, each condition (S, NS, solution control, or
TCPS) was run in duplicate cell wells. For each cell well,
approximately 10–20 images were acquired randomly by scan-
ning the wells from left to right and top to bottom. Experi-
ments were repeated on at least two separate days. The total
number of neurites (N) for each condition is reported.
8950 Applied Biological Sciences: Schmidt et al. Proc. Natl. Acad. Sci. USA 94 (1997)

The measured neurite lengths were not normally distrib-

uted, and, therefore, standard statistical parameters (e.g.,
population mean and standard deviation) were not computed.
Instead, the actual distributions of neurite lengths are pre-
sented for each condition along with the population median.
In addition, the Kruskal–Wallis H test (26) was used to assess
statistical differences between the neurite populations. The
Kruskal–Wallis H test is a nonparametric statistical test anal-
ogous to ANOVA.
Scanning Electron Microscopy. Samples were fixed with 1%
gluteraldehyde for 10 min, then exposed to increasing con-
centrations of ethanol (50%, 60%, 80%, 90%) for 2 min each,
and finally allowed to dry overnight. Images of cells were
obtained using an environmental scanning electron micro-
scope (ESEM) (Electro Company, Boston, MA) equipped
with a Trecor detector set at an accelerating voltage of 15 kV
under a vacuum of 4.9 torr and 6% humidity.
In Vivo Tissue Response. To evaluate both short-term and
long-term tissue response, PLGA-laminated PP disks were
implanted in adult male Lewis rats in subcutaneous and
intramuscular locations. The PP disks along with the surround-
ing tissue were harvested at 1, 2, and 14 weeks postimplanta-
tion for histological evaluation. The tissues were fixed in
formalin, embedded in paraffin, sectioned ('4.5-mm thick-
ness), and stained using hemotoxylin and eosin.

RESULTS
In Vitro Nerve Cell Cultures. Phase-contrast optical micro-
graphs of PC-12 cells cultured for 48 h in the presence of NGF
on TCPS, PP film (without electrical stimulation), and poly(L-
lactic acid) (PLA) film are shown in Fig. 1. For these exper-
iments, cells were seeded at 25,000 cells per well. It is apparent
from the images that PC-12 cells attach and differentiate
equally well on TCPS (A) and PP (B) films and more poorly on
PLA films (C). PC-12 cells also attach poorly to films of PLGA
and ITO surfaces (data not shown). Scanning electron micros-
copy was used to verify that PC-12 cells adhered to and
extended healthy neurites on PP thick films (Fig. 2). In addition
to the PC-12 cell line, PP also supports the growth of chick
sciatic nerve explants for up to at least 1 week in culture (data
not shown). These explants contained Schwann cells, fibro-
blasts, and other neuronal support cells in addition to neurons.
Thus, PP is capable of sustaining primary nerve cells and the
necessary support cells that are critical for regeneration.
In Vitro Electrical Stimulation. To elucidate the effect of FIG. 1. Differentiation of PC-12 cells on polypyrrole compared
with controls. Cells were grown on tissue culture polystyrene (TCPS)
electrical stimulation through PP films on neuronal differen- (A), oxidized polypyrrole (PP) (B), and poly(L-lactic acid) (PLA) (C)
tiation, experiments were performed in which PC-12 cells for 48 h in the presence of NGF. Bar 5 100 mm. [Reproduced with
grown on PP were subjected to a potential of 100 mV for 2 h permission from Materials Research Society (Copyright 1996).]
through the film (S). Controls consisted of: (i) PC-12 cells
grown on PP but not subjected to any stimulation (NS), (ii) in any of the experiments, and no measurable differences in pH
cells grown on PP and subjected to a potential through the or temperature were detected. Estimates of the electrical
solution only (solution control), and (iii) cells grown on TCPS. resistance of PP thin films and DMEM (culture medium) were
Cells were cultured for 24 h on PP films prior to electrical obtained using a multimeter (electrode spacing was '2 cm)
stimulation and then cultured for an additional 24 h (48 h and were 1 kV and '180 kV, respectively.
total). Controls not requiring an electrical stimulus were Image analysis was used to quantify the effect of electrical
cultured uninterrupted for a total of 48 h. For these experi- stimulation on PC-12 differentiation. The distributions of the
ments, cells were seeded at a density of 20,000 cells per well
neurite lengths for the different conditions (S, NS, solution
(1.33 3 104 cellsycm2).
control, TCPS) are shown in Fig. 4, where frequency is the
The application of an external electrical stimulus through
the PP substrate significantly enhanced differentiation of number of neurites of a given measured length and N is the
PC-12 cells (Fig. 3). Qualitatively, cells exposed to a potential total number of neurites measured for each condition over
(S, Fig. 3B) extended longer neurites compared with cells multiple experiments. These data show that the neurite dis-
grown on PP but not exposed to any electrical stimulus (NS, tribution in the stimulated cell population is shifted to the right
Fig. 3A). In addition, cell spreading for the stimulated cell (to longer neurites) compared with controls. This becomes
population was more pronounced than for any of the control more obvious if the peak frequencies are normalized by the
cases. Similar effects were observed for cells exposed to a total number of neurites measured (N) (e.g., the normalized
constant current of 10 mAmp, in which case electrode leads peak frequency for the stimulated case is the number of peak
were directly attached to the PP at opposite ends of the cell neuritesytotal neurites, or 820y5,643). The normalized peak
well (data not shown). There were no signs of cytotoxic effects frequencies are 0.14, 0.27, 0.29, and 0.31 for the S, NS control,
 Applied Biological Sciences: Schmidt et al.
FIG. 2. Scanning electron microscopy of a PC-12 cell on polypyr-
role. PC-12 cells were cultured in NGF-supplemented medium for 48 h
on thick disks of PP, then processed for scanning electron microscopy.
Bar 5 10 mm.
solution control, and TCPS cases, respectively. This indicates
that the distribution of the neurite lengths in the ‘‘S’’ case is
‘‘flattened’’ and shifted to the right compared with the nega-
tive controls. The fact that the median neurite length (18.14
mm, n 5 5643) in the ‘‘S’’ case is about twice that of the
negative controls [9.5 mm for NS (n 5 4440), 8.66 mm for
FIG. 3. PC-12 cell differentiation on polypyrrole without (A) and
with (B) application of an electric potential. PC-12 cells were grown
on PP for 24 h in the presence of NGF, then exposed to electrical
stimulation (100 mV) across the polymer film, S (B). Images were
acquired 24 h after stimulation. Cells grown for 48 h but not subjected
to electrical stimulation, NS, are shown for comparison (A). Bar 5 100
mm. [Reproduced with permission from Materials Research Society
(Copyright 1996).]
Proc. Natl. Acad. Sci. USA 94 (1997) 8951
FIG. 4. Neurite length histograms. Shown are histograms of neurite
lengths for cells on PP with (S) (A) and without (NS) (B) potential
applied through PP, on PP with potential applied through the solution
(C), and on tissue culture polystyrene (TCPS) (D). Images are after
48-h total incubation time.
solution control (n 5 2375), and 8.23 mm for TCPS (n 5 1356)]
confirms that the neurite distribution in the ‘‘S’’ case reveals
a significant increase in the neurite lengths. Finally, a Kruskal–
Wallis H test verified that the distribution of neurite lengths for
the ‘‘S’’ case is significantly different from the distributions for
the negative controls (P , 0.0001).
In addition, constant current of 10 mAmp applied directly
through the PP film resulted in a median neurite length of 15.3
mm (n 5 273), which is greater than the median neurite length
for PP without an electrical stimulus (9.5 mm, n 5 4440).
In Vivo Tissue Response to Oxidized Polypyrrole. PLGA-
laminated PP disks were implanted in adult male Lewis rats in
subcutaneous and intramuscular sites to determine the in vivo
tissue response to PP. It was observed that the inflammation
associated with the PP film was less severe than that induced
by the PLGA film (Fig. 5). The histological section is an
intramuscular implant harvested 2 weeks after implantation.
The black material adjacent to the muscle at the left side of the
figure is PP, and the translucent film adjacent to the tissue at
the right side of the image is the PLGA film (original magni-
fication, 1003). More inflammatory cells are associated with
the Food and Drug Administration-approved PLGA film
compared with PP, suggesting that PP is a relatively inert and
FIG. 5. Histology of tissue response to polypyrrole. Shown is a
histological tissue section stained with hemotoxylin and eosin of a
PPyPLGA disk that had been implanted in rat muscular tissue for 2
weeks. The PP is seen as a black film (Left), and the PLGA is almost
transparent (Right). The PP and PLGA films separated during histo-
logical processing, explaining the gap in the center of the image.
(3100.)
8952 Applied Biological Sciences: Schmidt et al.
biocompatible material. Furthermore, at 14 weeks there was
minimal evidence of a chronic inflammatory response as a
result of PP implantation (data not shown).
DISCUSSION
This study demonstrates that the electrically conducting poly-
mer, oxidized polypyrrole, is a suitable material for in vitro
nerve cell culture and for in vivo implantation. The favorable
interaction of PC-12 cells with PP compared with PLA and
PLGA may be the result of increased adsorption of positively
charged matrix proteins onto PP’s highly negatively charged
surface (16, 17). The high surface negative charge is due to the
incorporation of the polyanionic dopant poly(styrenesulfon-
ate). PP also supports the growth andyor differentiation of
primary chick sciatic nerve explants containing Schwann cells,
fibroblasts, and other ganglion-derived cells in addition to
neurons. Thus, PP is capable of sustaining primary nerve cells
and the support cells that are critical for regeneration.
Application of an electrical stimulus to PC-12 cells cultured
on PP significantly enhanced PC-12 neurite outgrowth and
spreading. The median neurite length for PC-12 cells grown on
PP and subjected to an electrical stimulus through the film
(18.14 mm, n 5 5643) was nearly doubled compared with cells
grown on PP without the application of a constant potential
(9.5 mm, n 5 4440). This represents a 90% increase in neurite
length, which is significantly greater than the 20–40% increase
in neurite lengths observed for primary nerve cultures on
piezoelectric meterials (10). Cells grown either on TCPS or on
PP in which charge was passed through the solution and not
through the polymer film possessed neurite length distribu-
tions indistinguishable from the ‘‘NS’’ controls (Fig. 4). This
suggests that the observed effects result primarily from elec-
tronic conduction through the PP and not from ionic conduc-
tion through solution.
The precise mechanism for the observed effect in this study
is unclear and is the subject of a current investigation. Several
theories have been suggested to explain the effect of electric
stimulation on nerve regeneration, including (i) electro-
phoretic redistribution of cell membrane growth factor and
adhesion receptors or cytoskeletal proteins such as actin (27,
32), all of which are involved in growth cone migration, (ii)
favorable membrane or extracellular matrix protein confor-
mational changes (10), (iii) direct depolarization or hyperpo-
larization of nerves (14), (iv) enhancement of protein synthesis
(12), and (v) field-induced gradients of ions and molecules in
the culture medium or tissue fluid. Patel and Poo (27) used an
extracellularly applied electric field to show that neurites
preferentially orient toward the cathode. In their studies, they
found a cathodal redistribution of growth-controlling surface
glycoproteins, suggesting that a field-induced electrophoretic
redistribution of charged membrane proteins gives rise to
growth cones that migrate preferentially toward the cathode.
Results from this study also argue against the formation of
field-induced gradient of ions and molecules in the culture
medium or tissue fluid. Similarly, studies in Xenopus have
shown that cell surface receptors for neurotransmitters un-
dergo a cathodal reorientation in an electric field (28), al-
though other studies also in Xenopus demonstrate that neurites
can turn either toward the cathode or anode depending on the
identity of the substratum (29). In contrast, Davenport and
McCaig (31) found that dendrites turn cathodally while axons
do not respond, whereas Cork et al. (30) found that PC-12 cell
neurite turn toward the anode. Thus, the true effect of
electrical stimulation on neurite outgrowth is still the subject
of debate.
In the present study, electrical charges pass through the
underlying PP film on which cells are attached via an adsorbed
protein monolayer. Because the resistance of PP is significantly
lower than that for the electrolyte cell medium (1 kV and '180
Proc. Natl. Acad. Sci. USA 94 (1997)
kV, respectively), charge will pass preferentially through the
PP film until charge transfer reactions are absolutely necessary
for current to reach the gold counter electrode at the far end
of the cell well. This suggests that enhanced neurite extension
results primarily from the passage of electronic current
through the material, and not from ionic flow of current
through the electrolyte medium. Also, enhanced effects on
neurite outgrowth were observed when constant current was
passed directly through the PP film and in which electrochem-
ical reactions were absent [median neurite length 5 15.3 mm
(n 5 273) compared with 9.5 mm for controls (n 5 4440)]. This
indicates that the contribution of ionic current on neurite
outgrowth can be excluded.
Furthermore, the effects of convective ion transport and
oxidationyreduction products can be considered negligible
based on two additional results. First, the ‘‘solution control,’’
in which cells grown on PP were exposed to 100 mV that was
passed exclusively through the medium and not the polymer
film, did not resemble the ‘‘S’’ case and more closely compared
with the ‘‘NS’’ control. This suggests that products from
oxidationyreduction reactions, under these experimental con-
ditions, did not either adversely or positively affect neurite
outgrowth. Second, the inability to detect a significant pH
change during these short-term experiments also indicates that
products from the electrochemical reactions did not likely
contribute to the observed positive effect of electrical stimu-
lation. These data are consistent with the conclusion by Patel
and Poo (27) that field-induced gradients of ions or molecules
in the cell medium do not play a role in enhanced neurite
outgrowth.
In our study, upon electrical stimulation no directional bias
of neurite outgrowth toward either electrode was observed. It
was observed that neurite extension was enhanced uniformly
in all directions for cells exposed to either a constant potential
or a constant current applied through the PP film. This
observation argues against an electrophoretic redistribution of
cell matrix proteins or of cell membrane proteins, which would
likely give rise to an overall directional bias. Thus, these data
support the hypothesis that an electrical stimulus enhances
overall differentiation rather than influencing the direction of
neurite extension. Others have shown increased protein syn-
thesis in neurons with electrical stimulation (12), which may
help account for the observations presented here if proteins
essential to neurite outgrowth were up-regulated. Further-
more, Kojima et al. (33) have shown that protein production in
tumor cells plated on platinum and indium tin oxide surfaces
is stimulated by the application of a constant potential. How-
ever, the mechanism by which protein synthesis is activated is
not known. Studies are currently underway to understand the
changes in electrophysiology of the cells during and after
electrical stimulation.
The use of PP in vivo has also been evaluated in the present
investigation. Both short-term and long-term tissue compati-
bility studies in the rat reveal that PP promotes little negative
tissue or inflammatory response. In its present form, PP is
nondegradable, and relatively thin films of PP, backed with
PLGA for support, were used for in vivo implantation. Even
after 14 weeks, the PP films were found to be intact, although
the PLGA backing had completely degraded. Studies are
currently in progress to determine the effect of an electrical
stimulus applied through PP conduits on actual sciatic nerve
regeneration in rats.
There are two key advantages to considering electrically
conducting polymers for use in tissue engineering applications.
First, unlike exogenous electromagnetic fields, electrical stim-
ulation through such polymers would be predominantly fo-
cused to the area around the polymer, allowing for spatial
control of stimulation. In the use of exogenous electromagnetic
fields for neuronal stimulation, the inability to localize the
stimulation has been cited as a disadvantage (34). Although
 Applied Biological Sciences: Schmidt et al.
piezoelectric materials such as polyvinylidene difluoride can 8.
aid in localized stimulation, the external control over stimu- 9.
lation is lacking. Other electret materials such as polytetra-
fluoroethylene display a static surface charge, but the stability 10.
and distribution of charges are highly dependent on processing
11.
conditions. Although electrets may hold promise for nerve
regeneration applications (11), the possibility that electrical 12.
stimulation can be externally controlled and precisely regu-
lated with electrically conducting polymers such as PP may 13.
prove advantageous. Second, the flexibility of synthesizing
electrically conducting polymers, such as PP, with varying 14.
surface properties offers many advantages. PP is easy and 15.
inexpensive to synthesize, and different dopant ions can be
16.
incorporated into the PP chain during synthesis to provide
varied surface characteristics. For instance, we found that 17.
PC-12 cells do not adhere to PP films having the chloride ion
as a dopant, lending the possibility of patterning a surface with 18.
adhesive and nonadhesive areas. Finally, matrix analogs and
growth factors could be either physically incorporated and 19.
subsequently released or tethered to the PP film (16). This 20.
offers the possibility of enhancing neuronal regeneration using
multiple forms of stimuli. Furthermore, it may be possible that 21.
electrically conducting polymers can be used as an interactive
22.
scaffold material for stimulation of other tissue types such as
bone. 23.
24.
We thank N. Chen, I. George, R. Bellamkonda, P. Basser, D. Odde,
T.-H. Kim, R. Solomon, I. Joris, M. Frangelo, and S. Charnick for their 25.
help. This work was supported by a National Science Foundation 26.
Grant (BES-9525913) to R.L. and a National Institutes of Health
postdoctoral fellowship to C.E.S. 27.
28.
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