The effects of poly(3, 4-ethylenedioxythiophene) (PEDOT) coating on magnesium degradation and cytocompatibility with human embryonic stem cells for potential neural applications.

Abstract

Magnesium (Mg) is a promising conductive metallic biomaterial due to its desirable mechanical properties for load bearing and biodegradability in human body. Controlling the rapid degradation of Mg in physiological environment continues to be the key challenge toward clinical translation. In this study, we investigated the effects of conductive poly(3, 4-ethylenedioxythiophene) (PEDOT) coating on the degradation behavior of Mg substrates and their cytocompatibility. Human embryonic stem cells (hESCs) were used as the in vitro model system to study cellular responses to Mg degradation because they are sensitive and can potentially differentiate into many cell types of interest (e.g. neurons) for regenerative medicine. The PEDOT was deposited on Mg substrates using electrochemical deposition. The greater number of cyclic voltammetry cycles yielded thicker PEDOT coatings on Mg substrates. Specifically, the coatings produced by 2, 5, and 10 CV cycles (denoted as 2x-PEDOT-Mg, 5x-PEDOT-Mg, and 10x-PEDOT-Mg) had an average thickness of 31, 63, and 78 µm, respectively. Compared with non-coated Mg samples, all PEDOT coated Mg samples showed slower degradation rates, as indicated by Tafel test results and Mg ion concentrations in the post-culture media. The 5x-PEDOT-Mg showed the best coating adhesion and slowest Mg degradation among the tested samples. Moreover, hESCs survived for the longest period when cultured with the 5x-PEDOT-Mg samples compared with the non-coated Mg and 2x-PEDOT-Mg. Overall, the results of this study showed promise in using PEDOT coating on biodegradable Mg-based implants for potential neural recording, stimulation and tissue engineering applications, thus encouraging further research.

Full text

The effects of poly(3,4-ethylenedioxythiophene) coating on
magnesium degradation and cytocompatibility with human
embryonic stem cells for potential neural applications
Meriam Sebaa,
1
Thanh Yen Nguyen,
1
Shan Dhillon,
1
Salvador Garcia,
2,3
Huinan Liu
1,3,4
1
Department of Bioengineering, University of California, Riverside, California 92521
2
Department of Biology, California State University, San Bernardino, California 92407
3
Stem Cell Center, University of California, Riverside, California 92521
4
Materials Science and Engineering Program, University of California, Riverside, California 92521
Received 26 November 2013; revised 29 January 2014; accepted 18 February 2014
Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35142
Abstract: Magnesium (Mg) is a promising conductive metal-
lic biomaterial due to its desirable mechanical properties for
load bearing and biodegradability in human body. Control-
ling the rapid degradation of Mg in physiological environ-
ment continues to be the key challenge toward clinical
translation. In this study, we investigated the effects of con-
ductive poly(3,4-ethylenedioxythiophene) (PEDOT) coating
on the degradation behavior of Mg substrates and their
cytocompatibility. Human embryonic stem cells (hESCs)
were used as the in vitro model system to study cellular
responses to Mg degradation because they are sensitive
and can potentially differentiate into many cell types of
interest (e.g., neurons) for regenerative medicine. The
PEDOT was deposited on Mg substrates using electrochemi-
cal deposition. The greater number of cyclic voltammetry
(CV) cycles yielded thicker PEDOT coatings on Mg sub-
strates. Specifically, the coatings produced by 2, 5, and 10
CV cycles (denoted as 23-PEDOT-Mg, 53-PEDOT-Mg, and
103-PEDOT-Mg) had an average thickness of 31, 63, and
78 mm, respectively. Compared with non-coated Mg sam-
ples, all PEDOT coated Mg samples showed slower degrada-
tion rates, as indicated by Tafel test results and Mg ion
concentrations in the post-culture media. The 53-PEDOT-Mg
showed the best coating adhesion and slowest Mg degrada-
tion among the tested samples. Moreover, hESCs survived
for the longest period when cultured with the 53-PEDOT-Mg
samples compared with the non-coated Mg and 23-PEDOT-
Mg. Overall, the results of this study showed promise in
using PEDOT coating on biodegradable Mg-based implants
for potential neural recording, stimulation and tissue
engineering applications, thus encouraging further research.
V
C2014 Wiley Periodicals, Inc . J Biomed Mater Res Part A: 00A:000–
000, 2014.
Key Words: magnesium degradation, biomaterials, poly(3,4-
ethylenedioxythiophene), conductive polymer, electrochemi-
cal deposition, coatings, human embryonic stem cells, cell
viability and proliferation, cytocompatibility
How to cite this article: Sebaa M, Nguyen TY, Dhillon S, Garcia S, Liu H. 2014. The effects of poly(3,4-ethylenedioxythiophene)
coating on magnesium degradation and cytocompatibility with human embryonic stem cells for potential neural applications.
J Biomed Mater Res Part A 2014:00A:000–000.
INTRODUCTION
Advantages of magnesium for medical applications
Magnesium (Mg) has attracted much attention as a potential
biomaterial for medical implant applications due to its
excellent mechanical properties, conductivity, and biode-
gradability. Specifically, Mg has a tensile strength of 180–
220 MPa and a conductivity of 22 MS/m.
1–3
Mg alloys, such
as Mg–Zn–Sr alloys, showed a further increase in the tensile
strength to 250–270 MPa.
4,5
Mg is biodegradable in physio-
logical fluids, mainly through reactions with water, as shown
subsequently:
Mg 12H 2O!Mg 2112OH 21H2"(1a)
Mg 2112OH 2
!Mg OHðÞ
2#(1b)
Since magnesium sulfate (MgSO
4
) solutions have long been
used clinically as a neuroprotective agent,
6–9
Mg ions released
during Mg degradation may be beneficial for neural stimulation
and regeneration. The rise of Mg ion concentrations in cerebro-
spinal fluid and brain has been shown to protect neural tissue
against further damages from acute stroke
8,9
and cerebral
ischemia.
10
Mg ions have also been shown to prevent atrial
fibrillation after coronary artery bypass surgeries.
11,12
Correspondence to: H. Liu; e-mail: huinan.liu@ucr.edu
Contract grant sponsor: U.S. National Science Foundation (NSF Graduate Research Fellowship for supporting Thanh Yen Nguyen); contract
grant number: NSF BRIGE award CBET 1125801
Contract grant sponsors: The Burroughs Wellcome Fund, the Hellman Faculty Fellowship, and the University of California (UC) Regents Faculty
Fellowship
V
C2014 WILEY PERIODICALS, INC. 1

The key challenge facing Mg-based medical implants lies
in the control of its degradation in the physiological envi-
ronment. Mg degrades too rapidly to meet specific require-
ments for clinical applications
13–15
; and thus the uses of
polymer coatings have been actively explored.
2,16,17
For neu-
ral applications, it is important to consider a polymer that
is conductive because the conductivity of neural implants
has been shown to play a critical role in supporting neuro-
nal growth and reducing glial scar tissue formation.
18
Rationale for poly(3,4-ethylenedioxythiophene) coating
Poly(3,4-ethylenedioxythiophene) (PEDOT) was chosen as the
coating material because it is conductive and biocompatible,
and it has shown promising results for energy and neural
electrode applications.
19–25
For example, PEDOT was electro-
deposited onto titanium (Ti) meshes and improved light to
electric energy conversion efficiency and maximum power
output for solar cell applications.
22
For neural application s,
PEDOT was coated onto gold (Au) electrodes to lower the
impedance and reduce the noise for chronic recording of
neural activity.
23
Compared with conductive polypyrrole,
PEDOT has been proven to be more electrochemically stable
with a higher resistance to biological reducing agents in liv-
ing tissue.
21
Although PEDOT coatings have been previously
deposited on different electrodes, such as titanium (Ti), gold
(Au), platinum (Pt), and indium tin oxide (ITO), for solar cell
application
22
or neural recording,
23–25
buthavenotyetbeen
extensively studied on alkaline metals. There are only a few
recent studies in which PEDOT coatings were deposited on
Mg substrates to slow down the degradation rate of Mg for
biomedical applications.
2,26
It has been reported that PEDOT is compatible with cul-
tured cells and brain tissue, and does not release any sub-
stance that elicits toxicity.
19–21,27,28
No inflammatory
reactions were observed after PEDOT coated glass sub-
strates were subcutaneously implanted into mice for a
week.
20
When PEDOT was polymerized around living neural
cells, the cells embedded within the polymer matrix were
viable for up to 120 h after polymerization.
19
When fibro-
blasts were cultured on PEDOT coated ITO electrodes, the
cells attached and spread onto the PEDOT coating with no
morphological changes, and cell viability was greater than
the ITO electrodes alone.
20
Moreover, high quality acute
neural recordings have been obtained when PEDOT (with a
bioactive peptide) coated ITO electrodes was implanted into
the cerebellum of a guinea pig.
21
PEDOT has also been
explored for applications as nerve guidance conduits
(NGCs). Greater numbers of nerve fibers were regenerated
on PEDOT coated agarose hydrogel NGCs than polydimethyl-
siloxane coated and non-coated agarose tubes.
29
When
PEDOT was electrodeposited on ITO glass electrodes, immu-
nostaining for b-III-tubulin expression increased at day 5,
indicating improved neural stem cell differentiation and
neuronal growth.
24
Additionally, electrodeposited PEDOT
induced neuronal differentiation of P19 pluripotent embry-
onic carcinoma cells (American Type Culture Collection,
Manassas, VA) without use of additional factors such as
poly-L-ornithine or retinoic acid coating.
24
Thus, PEDOT is a
promising coating material for neural applications.
Human embryonic stem cell model for in vitro
cytocompatibility studies
Stem cells can serve as sensitive in vitro model systems to
test cytotoxicity of drugs and chemicals, and potentially to
predict toxicity in humans.
30
The validated embryonic stem
cell test (EST) utilizes mouse embryonic stem cells to iden-
tify potential embryo toxic compounds because embryos
and fetuses are more sensitive to environmental toxicants
than adult cells or tissues.
31
Since this mouse-based EST
sometime does not faithfully correlate to human
responses,
32
the human embryonic stem cell (hESC) model
was selected for this study to eliminate the concerns on
species variation. The hESCs are more sensitive to toxins
and chemicals than any other human cells commonly used
in literature, such as human adult stem cells, differentiated
human cells, and immortalized human cell lines.
33–35
More-
over, hESCs have greater capacity for proliferation than
adult cells from animals or human tissues, thus reducing
the number of animals and donors needed for tissue and
cell harvesting. More importantly, previous in vitro studies
showed cytotoxic behavior of non-coated commercially pure
Mg with a purity of 99.9% against hESCs due to its rapid
degradation rate.
36
Therefore, it is important to use the
same hESC model to determine the cytocompatibility of
PEDOT coated Mg in comparison with non-coated Mg.
In addition, hESC is an attractive therapeutic option for
cell replacement therapy, tissue engineering, and regenera-
tive medicine applications considering its capacity to prolif-
erate and differentiate into different cell types, such as
neurons, osteoblasts, or fibroblasts. Previous studies have
shown that stem cells can be combined with scaffolds or
biomaterials for more effective nerve regeneration.
37–40
Thus, using hESCs as the in vitro model system will allow
us to determine simultaneously the cytocompatibility of
PEDOT coated Mg and its potential as a bioresorbable scaf-
fold for tissue engineering applications.
Objectives of this study
The objective of this study is to investigate the degradation
and cytocompatibility of PEDOT coated Mg in comparison
with non-coated Mg using the H9-OCT4 hESCs model. The
materials of interest to this study are Mg substrates with
electrochemically deposited PEDOT using 2, 5, and 10 cycles
of cyclic voltammetry (CV). The surface microstructure,
composition, thickness, and adhesion strength of the PEDOT
coatings on Mg samples were characterized before the in
vitro degradation and cytocompatibility studies.
MATERIALS AND METHODS
Deposition of PEDOT coatings using CV
As-rolled Mg samples (MiniScience, 98% purity) with a
thickness of 250 mm were cut into the T shape with a spe-
cific dimension as shown in Figure 1. These T-shaped sam-
ples were grinded using 600, 800, and 1200 grit silicon
carbide abrasive paper (PACE Technologies) to remove the
2 SEBAA ET AL. PEDOT COATING ON MG FOR NEURAL APPLICATIONS

oxidized surface layer. The samples were then ultrasonically
cleaned in ethanol for 15 min and air dried in a laminar
flow hood for 10 min. As illustrated in the experimental
setup in Figure 1, the samples were clamped to the working
electrode of a potentiostat (model 273A; EG&G Princeton
Applied Research) operated by the Powersuite software.
Platinum and silver/silver chloride was used as the counter
and reference electrodes, respectively. The experimental
samples and electrodes were then placed in an electrolyte
solution of pure 1-ethyl-3-methylimidazolium bis(trifluoro-
methylsulfonyl)imide (ionic liquid, IL electrochemical grade,
>99.5% purity, Covalent Associates) containing 1 M3,4-eth-
ylenedioxythiophene (Sigma-Aldrich). Three types of sam-
ples were fabricated in this study using CV deposition, in
which the potential was scanned from 20.5 to 12.0 V at a
scan rate of 100 mV/s for 2, 5, and 10 cycles (denoted as
23-PEDOT-Mg, 53-PEDOT-Mg, and 103-PEDOT-Mg, respec-
tively). Once the CV deposition was complete for each sam-
ple, the sample was dried in a vacuum chamber at room
temperature for 24 h, rinsed with ethanol, and disinfected
under ultraviolet radiation for 24 h before cell culture.
Characterization of coating microstructure, elemental
composition, and thickness
The surface microstructure and coating thickness of
PEDOT coated Mg samples were characterized with a
field-emission scanning electron microscope (SEM; FEI
XL30). The samples were placed on a flat mount to obtain
images of surface microstructure. To obtain cross-section
images for coating thickness measurements, PEDOT
coated Mg samples were cleanly cut with scissors and
placed on a 90mount. SEM images were acquired at an
accelerating voltage of 15 kV. The surface composition
and elemental distribution were quantified using an
attached detector for energy-dispersive X-ray spectros-
copy (EDS; EDAX). EDS analysis was performed at a mag-
nification of 20003so that a substantial portion of the
sample surface could be analyzed to obtain the average
elemental composition.
Tape test for PEDOT coating adhesion strength
The ASTM standard D3359 tape test was used to charac-
terize the adhesion strength of PEDOT coatings on the Mg
substrates. According to the ASTM defined tape test
method B, a sharp blade was used to carve a 5 mm 35
mm grid onto the PEDOT coated surface. The carved sur-
face was then evenly covered with 3M No. 3710 tape and
subsequently removed at a 180angle. The remaining
coatings on the PEDOT-coated Mg samples were character-
ized based on the classification criteria outlined in Table I.
A classification of 5B indicated a strong adhesion between
the coating (PEDOT) and the substrate (Mg) in which no
coating detached from the substrate, while a classification
of 0B indicated a weak bonding between the coating and
the substrate in which 65% or more of the coating
detached.
41
Tafel test for determining corrosion properties
The corrosion properties of the PEDOT coated and non-
coated Mg samples were determined using the Tafel test
according to the established protocol.
2,42
The electrochemi-
cal measurements for all the samples were carried out in a
simulated body fluid (SBF) solution with a pH of 7.4 using
the same three-electrode and potentiostat setup as shown
in Figure 1. The working, reference, and counter electrodes
in this setup were the PEDOT coated or non-coated Mg sam-
ples, silver/silver chloride, and platinum, respectively. The
potentiodynamic polarization curves were recorded at a
potential range of 22.0 to 2.0 V for 40 s with a constant
scan rate of 100 mV/s using the Powersuite software. Tafel
extrapolation were then performed on these curves to
determine the corrosion current (I
CORR
) and corrosion
potential. The corrosion current values from Tafel
FIGURE 1. Experimental setup for the electrochemical deposition of
PEDOT coating onto Mg substrates. Silver/silver chloride served as
the reference electrode and platinum as the counter electrode. Mg
substrates were coated in 1M EDOT/ionic liquid solution using cyclic
voltammetry (CV) ranging from -0.5 to 2V for 2, 5, and 10 cycles at
200 seconds per cycle with a constant scan rate of 100 mV/s. [Color
figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
TABLE I. Classification Criteria for Coating Adhesion Strength According to ASTM Standard D3359 Tape Test
Coating Adhesion
Classification Description
% Area
Detached
5B Smooth edges around cuts; no detached squares 0
4B Detached small coating flakes at the carved intersections <5
3B Detached small coating flakes along the carved intersections and the edges 5–15
2B Flaked coating along the carved edges and on parts of squares 15–35
1B Flaked coating along the carved edges (large ribbons); detached whole squares 35–65
0B Flaking and detachment worse than classification 1B. >65
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A |MONTH 2014 VOL 00A, ISSUE 00 3

extrapolation were used to calculate the corrosion rates of
PEDOT coated or non-coated Mg samples based on the fol-
lowing equation
42
:
CR5ICORR KEW
dA (2)
where CR 5corrosion rate, I
CORR
5corrosion current,
K5constant for converting units, EW 5equivalent weight
of Mg, d5density of Mg, and A5area of surface immersed
in SBF.
In addition to Tafel test (i.e., potentiodynamic polariza-
tion measurement), immersion test is recommended as a
complementary testing method for biomedical applications.
Thus, the media pH and Mg
21
ion concentration in the
media were measured when the samples were immersed in
the hESC culture. Mg degradation releases OH
2
and Mg
21
ions according to the reaction (1) in Introduction, and thus
the media pH and Mg
21
ion concentration are the two key
indicators of Mg degradation in immersion test.
In vitro evaluation of cytocompatibility using hESC
culture
The hESC culture with PEDOT-coated and non-coated Mg
samples. H9 hESCs (WiCell) were stably transfected with an
OCT4-eGFP reporter plasmid as previously described.
43
These H9 OCT4 hESCs were maintained in mTeSR
V
R
1 media
(Stem Cell Technologies) in Geltrex
V
R
(Invitrogen) coated T-
25 flasks without using feeder layers. Once the hESCs
reached 80–90% confluency, they were passaged and split
at a ratio of 1:5 onto a Geltrex
V
R
coated 12-well tissue cul-
ture plate (BD Falcon). Accutase (Innovative Cell Technolo-
gies) and glass beads were used to detach the cells from
the surface of the flask during passaging. The passaged cells
were then cultured under standard cell culture conditions
(that is, a sterile, 37C with 5% CO
2
/95% air, humidified
environment) for 24 h to allow the cells to attach. After 24
h, the sterilized non-coated Mg, 23-PEDOT-Mg, 53-PEDOT-
Mg, and 103-PEDOT-Mg samples were placed into the
Transwell
V
R
inserts (Corning, Union City, CA) and introduced
into the wells with hESC culture. All experimental samples
were run in triplicates and three wells were left as positive
controls without any Mg-based samples. The hESCs were
then incubated for 72 h under standard cell culture condi-
tions in a dynamic cell observation system (Nikon BioSta-
tion CT, Melville, NY) where phase contrast and
fluorescence images were taken every 6 h. The mTeSR
V
R
1
media were collected and replenished every 24 h for post-
culture media analysis. The Mg-based samples were col-
lected at the end point (72 h) for post-culture material
analysis.
Image-based analysis for normalized coverage area of
viable cell colonies. Phase contrast and fluorescence images
recorded by the Biostation CT were used to quantify the
percentage of area covered by viable H9-OCT4 hESC colo-
nies. This image-based analysis was used for cell viability
and proliferation studies instead of the commonly used tet-
razolium based assays (e.g., MTT assay) because Mg ion con-
verts tetrazolium salts to formazan and thus interferes with
the results.
44
That is, MTT assay produces false positive
data that indicate greater cell viability than the actual val-
ues.
44
Each type of sample (non-coated Mg, 23-PEDOT-Mg,
53-PEDOT-Mg, and 103-PEDOT-Mg) was run in triplicates
and imaged at two different areas on each well using the
Biostation CT. Total 6 images were analyzed for each type of
sample and the data were averaged. ImageJ software was
used to manually outline the area of viable cell colonies that
expressed the OCT4-GFP, a fluorescence marker indicating
viable undifferentiated hESCs. The percentage of area cov-
ered by viable cell colonies was quantified using the out-
lined area divided by the total image area, and then
normalized over the values from the first recorded time
point. Normalized data can be directly compared later
among different samples at the specific time points of inter-
est. The standard deviation was calculated and used to gen-
erate error bars.
Post-culture media analysis of pH and Mg ion
concentrations. A precalibrated pH meter (VWR, Model
SB70P) was used to measure pH values of the post-culture
media collected every 24 h. Inductively coupled plasma-
atomic emission spectroscopy (ICP-AES; Perkin Elmer
Optima 2000 DV) was used to measure Mg ion concentra-
tions in the post-culture media. The ICP-AES data were ana-
lyzed using a standard calibration curve that was generated
on a set of serially diluted MgCl
2
6H
2
O solutions with the
concentrations of 250, 125, 62.5, 31.25, and 15.63 mg/L (or
10.29, 5.14, 2.57, 1.29, and 0.64 mM). The collected post-
culture media were diluted using deionized water from a
water purification system (Milli-Q; Millipore) to ensure the
measured values of experimental samples were within the
linear range of the standard curve.
Statistical analysis
A script was written in the program R for the statistical
data analyses, including the Shapiro–Wilk normality test, the
Ftest to compare variance, and the standard analysis of var-
iance (ANOVA) followed by standard post hoc tests with the
Holm–Bonferroni correction. Values of p<0.05 for the Sha-
piro–Wilk test and Ftests verified the normality and the
variance of the dataset. Values of p<0.05 for ANOVA indi-
cated that the groups of data showed a statistically signifi-
cant difference.
RESULTS
Microstructure, elemental composition, and thickness of
PEDOT coatings
The SEM images and EDS spectra in Figure 2 showed that
PEDOT coatings were successfully deposited onto Mg sub-
strates using CV for 2, 5, and 10 cycles. Non-coated Mg sub-
strates showed a typical appearance of polished Mg
substrates with traces of polishing marks. As expected, the
EDS analysis of the non-coated Mg substrates showed that
Mg was the dominant element on the surface and a small
amount of oxygen (O) was observed because Mg substrates
4 SEBAA ET AL. PEDOT COATING ON MG FOR NEURAL APPLICATIONS

are susceptible to oxidation when exposed to air. In contrast
to the non-coated Mg, all the PEDOT coated samples showed
the typical morphology and elemental composition of
PEDOT on the surface, indicating successful deposition of
PEDOT. When comparing the surface microstructures of the
PEDOT coated Mg samples, the PEDOT coating on the 103-
PEDOT-Mg sample appeared to have more particulate fea-
tures [Fig. 2(D)] than the 23-PEDOT-Mg [Fig. 2(B)] and 53-
PEDOT-Mg samples [Fig. 2(C)]. When comparing the ele-
mental compositions of the PEDOT coated Mg samples, the
types of elements detected were the same, but their percen-
tages varied with the number of CV cycles. PEDOT backbone
contains carbon (C), sulfur (S), and O and the ionic liquid
contains nitrogen (N) and fluorine (F) which could be trans-
ferred as dopants into the PEDOT coating. Specifically, Mg
content decreased while C and S contents increased with
the increase in CV cycles. Interestingly, N and F contents
decreased with the increase in CV cycles. Decreased Mg con-
tent on the surface indicated thicker coating, which was in
agreement with the SEM cross-section images (Fig. 3).
Figure 3 shows the cross-sections of the PEDOT coated Mg
samples for coating thickness measurements. As the number
of CV cycles increased, the PEDOT coating thickness increased.
Specifically, the coating thicknesses of the 23-PEDOT-Mg, 53-
PEDOT-Mg, and 103-PEDOT-Mg samples were determined to
be of 31 66, 63 66, and 78 64mm, respectively.
Adhesion strength of PEDOT coatings
ASTM D3359 tape test results revealed how strongly the
PEDOT coatings adhered to the Mg substrates, as shown in
Figure 4. Figure 4 images are representative for all the
tested samples. The PEDOT coated Mg samples were
FIGURE 2. SEM images and EDS analyses of (A) non-coated Mg, (B) 2x-PEDOT-Mg, (C) 5x-PEDOT-Mg, and (D) 10x-PEDOT-Mg. Scale bar = 20
lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A |MONTH 2014 VOL 00A, ISSUE 00 5

visually inspected after the tape removal and the areas of
detachment for each sample were highlighted in Figure 4 to
provide the evidence for the classification of coating adhe-
sion strength. The adhesion strength of the PEDOT coatings
on the Mg substrates was determined to be between the clas-
sifications of 3B and 4B according to the ASTM D3359 stand-
ard classifications listed in Table I. As the number of CV
cycles increased from 2 to 10, the coating adhesion strength
initially increased and then decreased. Specifically, the 53-
PEDOT-Mg samples showed the strongest coating adhesion
with a classification of 4B, while the 23-PEDOT-Mg and
103-PEDOT-Mg samples showed decreased coating adhesion
strength with a classification of 3B. On the surface of 53-
PEDOT-Mg samples with class 4B adhesion strength, only a
couple of very small flakes of coating detached from the
intersections of carved grids. On the surface of 23-PEDOT-
Mg samples with class 3B adhesion strength, detachment
mainly occurred at the edges of carved grids and was less
than 15%. On the surface of 103-PEDOT-Mg samples with
class 3B adhesion strength, obvious detachment was
observed at the top right corners of the carved grids.
Although the tape test provided useful results for this initial
study, we understand that the ASTM D3359 standard tape
test is a qualitative method with intrinsic limitations because
it is mostly based on visual inspections. Thus, it is necessary
to conduct quantitative tests to determine adhesion strength
using a micro scratch tester in the future studies.
PEDOT coatings decreased Mg degradation rate
The Tafel test results showed that the PEDOT coatings
decreased the corrosion current density and corrosion rate,
as shown in Figure 5. Figure 5(A) shows the representative
curves of potentiodynamic polarization for the non-coated
and PEDOT coated Mg in SBF, which were used to extrapo-
late the corrosion currents (I
CORR
) and corrosion potentials.
The corrosion currents were used to calculate corrosion
rates according to Eq. (2). According to this equation, when
the surface area immersed in SBF is fixed, corrosion rate is
directly proportional to corrosion current since the other
factors are constant. Therefore, the greater corrosion cur-
rents resulted in greater corrosion rates. The extrapolated
and calculated corrosion properties of the non-coated and
FIGURE 3. SEM cross-sectional images of PEDOT coated Mg samples placed on a 90o mount. SEM images of (A) 2x-PEDOT-Mg and (B) 10x-
PEDOT-Mg samples were at a low original magnification (200x). SEM images of (C) 2x-PEDOT-Mg, (D) 5x-PEDOT-Mg, and (E) 10x-PEDOT-Mg
samples were at a higher original magnification (2000x). The PEDOT coating is highlighted in the red boxes. The PEDOT coatings deposited by
2, 5, and 10 cycles of CV had an average coating thickness of 3166lm, 6366lm, and 7864lm, respectively. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
6 SEBAA ET AL. PEDOT COATING ON MG FOR NEURAL APPLICATIONS

PEDOT-coated Mg samples were summarized in Figure 5(B).
Interestingly, as the number of CV cycles increased from 2
to 10, the corrosion rates of the PEDOT coated Mg samples
initially decreased and then increased. Specifically, the 53-
PEDOT-Mg sample showed the slowest corrosion rate of
1.48 mm/year, while the 23-PEDOT-Mg sample showed a
corrosion rate of 4.53 mm/year and the 103-PEDOT-Mg
sample showed a corrosion rate of 9.73 mm/year. The non-
coated Mg samples showed the greatest corrosion rate of
31.63 mm/year, which confirmed that the PEDOT coatings
did decrease the degradation rate of Mg.
53-PEDOT coating improved hESC viability in vitro
When the hESCs were cultured with the 103-PEDOT-Mg
samples, dark-colored particulate fragments were found in
the cell culture media, as shown in Figure 6. These particles
were most likely from the PEDOT coating based on its color
since degradation precipitates of Mg have a whitish color.
These PEDOT particles appeared in the hESC culture within
24 h and interfered cell imaging and analysis. Thus, the
103-PEDOT-Mg samples were considered undesirable for
intended medical applications, and thus excluded from fur-
ther cell culture analyses.
The time-lapse images of hESCs cultured with non-
coated and PEDOT coated Mg samples in Figure 7 showed
that the PEDOT coatings prolonged the viability of hESCs
when compared with non-coated Mg. The 53-PEDOT-Mg
FIGURE 4. Representative images of tape test and classification results of adhesion strength for 2x-PEDOT-Mg, 5x-PEDOT-Mg, and 10x-PEDOT-
Mg samples. Images in the left column showed the PEDOT coated Mg samples before they were carved by a cutter. Images in the next column
showed the PEDOT coated Mg samples after the carved grids were created. Images in the 3rd column from the left showed the PEDOT coated
Mg samples after the tape was applied and removed, which were used to determ ine the classification of coating adhesion strength based on
the visual inspection of coating detachment following the ASTM standard D3359 guideline. The areas of detachment were highlighted in yellow
ovals. Images in the right column showed the remnants on the tape after tape removal. [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
FIGURE 5. (A) Potentiodynamic polarization curves of the non-coated
and PEDOT coated Mg samples. (B) Summary of corrosion properties
of the non-coated and PEDOT coated Mg samples in an order of
increasing coating thickness. Corrosion current (I
CORR
) and corrosion
potential values were extrapolated from polarization curves. Corro-
sion current density and corrosion rates were calculated according to
the established equations. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
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maintained the hESC viability in culture for the longest
period of time, when compared with the 23-PEDOT-Mg and
non-coated Mg samples. However, when compared with the
control wells without any Mg samples, all the tested Mg
samples induced a change in hESC morphology that eventu-
ally led to cell death. The hESCs in the wells with the non-
coated and PEDOT coated Mg samples lost their tightly
packed colony morphology at different time points of cul-
ture and became more dispersed with greater cellular
extensions at the boundary of the colonies. Specifically, the
change in hESC morphology occurred at 18 and 24 h for the
23-PEDOT-Mg and 53-PEDOT-Mg, while that occurred at
12 h for the non-coated Mg. As the morphology of hESCs
changed during their cultures with the coated or non-coated
Mg samples, round floating cells and cell debris started to
appear above the attached cells that were on the bottom of
the culture plate, indicating the progression of cell death. In
contrast, the control hESCs maintained their normal tightly
packed colony morphology throughout all time points and
eventually reached confluency.
The coverage area of viable hESC colonies were quantified
and normalized using the image-based analysis technique and
plotted in Figure 8. The hESCs cultured with the 53-PEDOT
Mg samples showed the longest viability for up to 54 h. At
the time points of 24 and 30 h, the hESCs cultured with the
53-PEDOT Mg samples continued to show viable cells that
expressed green fluorescence, but predominantly dead cells
and cell debris that lost their green fluorescence signals were
observed for the non-coated and 23-PEDOT Mg samples. The
hESCs cultured with the PEDOT coated Mg samples showed a
similar trend in coverage area. That is, the coverage areas
were stable at the initial 12 h and then decreased toward
eventual cell death. Notably, the coverage area of viable hESCs
cultured with the 53-PEDOT-Mg decreased at a slower rate
than non-coated Mg and 23-PEDOT Mg, with significantly
greater colony coverage area values at 24, 30, and 42 h.
pH values and Mg ion concentrations of post-culture
media
Mg degradation releases Mg ions (Mg
21
) and hydroxide
ions (OH
2
) when immersed in aqueous media according to
the degradation reactions. The release of OH
2
ions leads to
an increase in media pH. Thus, the media pH and Mg ion
concentrations in the media were measured as the two key
indicators of Mg degradation.
Figure 9 shows the pH of the post-culture media col-
lected at every 24 h. The post-culture media with the 53-
PEDOT-Mg samples had significantly lower pH values than
that with the non-coated Mg at 48 and 72 h. In contrast, no
statistically significant difference was detected between the
pH values of post-culture media with the non-coated Mg
and the 23-PEDOT-Mg samples at 24, 48, and 72 h. All the
post-culture media with either non-coated or PEDOT coated
Mg samples at 24, 48, and 72 h showed an elevated pH to
FIGURE 6. (A) Time-lapse images of H9-OCT4 hESCs co-cultured with the 10x-PEDOT-Mg sample under standard cell culture conditions over 18
hours. Scale bars = 200 lm. (B) Images of the culture media after co-culture of hESCs with the 10x-PEDOT-Mg and non-coated Mg samples for
24 hours. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
8 SEBAA ET AL. PEDOT COATING ON MG FOR NEURAL APPLICATIONS

alkaline region compared with the control culture that had
no Mg-based samples.
Figure 10 shows the Mg ion concentrations in the post
culture media collected at every 24 h. The post-culture
media with the 53-PEDOT-Mg samples showed significantly
lower Mg ion concentrations (8 mM) than that with the 23-
PEDOT-Mg samples (12 mM) at the end point of 72 h, indi-
cating slower degradation. The Mg ion concentrations in the
post-culture media with the non-coated Mg samples were
the highest (23–34 mM) among all the samples and showed
a decreasing trend over time, which was also reported pre-
viously.
45
When compared with the non-coated Mg, both the
23-PEDOT-Mg and the 53-PEDOT-Mg significantly
decreased the amount of Mg ions released into the media;
FIGURE 7. Time-lapse phase contrast optical images of H9-OCT4 hESCs co-cultured with the non-coated and PEDOT coated Mg samples under
standard cell culture conditions over 30 hours. The control was H9-OCT4 hESCs cultured under standard cell culture conditions without Mg-
based samples. Scale bars = 50 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A |MONTH 2014 VOL 00A, ISSUE 00 9

that is, 11–13 mMfor the 23-PEDOT-Mg and 8–11 mMfor
the 53-PEDOT-Mg, respectively. All the post-culture media
with either non-coated or PEDOT coated Mg samples at 24,
48 and 72 h showed an elevated Mg ion concentrations in
comparison with the basal amount of Mg ions in the control
culture that had no Mg-based samples.
DISCUSSION
Both coating thickness and adhesion are important for
controlling Mg degradation
The results of this study demonstrated for the first time
that both coating thickness and adhesion strength played
important roles in the efficacy of PEDOT coatings for con-
trolling Mg degradation. As the number of CV cycles
increased from 2 to 10, the PEDOT coating thickness
increased from 31 to 78 mm while the coating adhesion
strength increased from 2 to 5 cycles and decreased from 5
to 10 cycles. The 53-PEDOT-Mg provided the best coating
adhesion and the slowest degradation among the tested
samples according to the Tafel test and quantification of the
degradation products (i.e., hydroxide and Mg ions) released
into the culture media. In comparison with the non-coated
Mg, the corrosion rate of the 53-PEDOT-Mg decreased by
95%, while the corrosion rate of 23-PEDOT-Mg decreased
by 86% and the corrosion rate of 103-PEDOT-Mg sample
decreased by 69%. These results indicated that among all
the PEDOT coatings tested, the 53-PEDOT-Mg had the opti-
mal combination of coating thickness and adhesion strength
to provide the most effective degradation protection for Mg
substrates.
We understand that the 103-PEDOT-Mg sample should
have the slowest corrosion rate considering that it had the
thickest coating. However, the coating might become more
susceptible to detachment as the coating thickness
increased. It was observed that 103-PEDOT-Mg sample
released particulate PEDOT when exposed to the media
(Fig. 6), whereas the 23-PEDOT-Mg and 53-PEDOT-Mg sam-
ples did not. Even though the 103-PEDOT-Mg sample had
the thickest coating, the coating might not provide the best
protection against corrosion if the coating partially delami-
nated into the media. There are very few reports in litera-
ture related to PEDOT coatings on Mg. This study is the first
to report the relationships of the PEDOT coating thickness
and adhesion strength with Mg degradation. We did not find
any other studies on PEDOT coatings deposited on compara-
ble alkaline metals. However, the basic concept reported on
other coatings was in agreement with our results. For exam-
ple, when TiN coatings were deposited on steel substrates
via chemical vapor deposition, the thickest coating detached
from the substrate during the wear test because of its lower
adhesion strength.
46
Thus, it is likely that the thickest
PEDOT coating was not optimal for degradation protection.
FIGURE 8. Percentage of area covered by viable H9-OCT4 hESC colo-
nies over time. The data was normalized over the first time point of
respective samples at 6 hours of cultures with hESCs. *p<0.05 com-
pared to non-coated Mg and 2x-PEDOT-Mg. Values are mean 6stand-
ard deviation; n=6. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
FIGURE 9. Average pH of post-culture media at each 24-hour collection
when the hESCs were co-cultured with the non-coated and PEDOT
coated Mg samples. *p<0.05 compared to the non-coated Mg. Values
are mean 6standard error of the mean; n=3. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.com.]
FIGURE 10. Average Mg ion concentrations in the post-culture media
at each 24-hour collection when the hESCs were co-cultured with the
non-coated and PEDOT coated Mg samples. The control wells indi-
cated the basal amount of Mg ions in the mTeSR
V
R
1 media. * p<0.05
compared to the 2x-PEDOT-Mg. Values are mean 6standard error of
the mean; n53. [Color figure can be viewed in the online issue, which
is available at wileyonlinelibrary.com.]
10 SEBAA ET AL. PEDOT COATING ON MG FOR NEURAL APPLICATIONS

It is also important to have a dense PEDOT layer under-
neath the top surface as the coating thickness increased.
However, it has been reported that the PEDOT coating
porosity increased with more CV cycles and thicker coating,
when the PEDOT was electrodeposited on custom-made ITO
glass slides surfaces.
47
Therefore, the thickest PEDOT coat-
ing deposited by 10 CV cycles might not necessarily be the
best to protect Mg substrates from fast degradation, if the
coating became more porous and more susceptible to
detachment with increasing thickness.
Slower Mg degradation yielded better hESC viability
The results of this study showed a strong correlation
between the degradation rate and hESC viability. As the deg-
radation rate of samples decreased in this order of non-
coated Mg, 23-PEDOT-Mg, and 53-PEDOT-Mg, the hESC via-
bility increased from 24 to 54 h in the same order of non-
coated Mg (24 h), the 23-PEDOT-Mg (30 h), and the 53-
PEDOT-Mg (54 h). The 53-PEDOT-Mg samples showed the
best coating adhesion, the slowest degradation rate, and
thus improved the hESC viability the most. Clearly, decreas-
ing the degradation rate was an effective approach in
improving hESC viability. However, the exact relationships
between Mg degradation and cell viability are yet to be
determined.
Two factors induced by Mg degradation were considered
important in affecting cell viability, including elevated media
pH and Mg ion concentrations in the media. In this study,
when the hESCs were cultured with the non-coated and
PEDOT coated Mg samples, the media pH increased to an
alkaline region of 8.1–8.4. The persistent alkaline pH envi-
ronment observed during the hESC culture might have
played a major role in cell viability. H€
anzi et al.
48
reported a
decrease in cell viability and metabolic activity of human
umbilical vein endothelial cells with increasing pH. However,
when the initial media pH was intentionally adjusted to 8.1
using 0.1 MNaOH, this transient alkaline pH did not seem
to affect hESC viability.
45
Thus, it is possible that only per-
sistent alkaline pH environment was harmful to the cells.
The elevated Mg ion concentrations in the culture media
might have contributed in some degree to cell behavior
observed in this study. The Mg ion concentrations in the
post-culture media with the non-coated Mg samples were in
a range of 23–34 mM. Grillo et al.
49
observed a cytotoxic
effect on UMR-106 cells (ATCC
V
R
rat osteosarcoma derived
cells) when Mg ions released by Mg particles into the cul-
ture media reached a concentration of greater than 480 mg/
mL (19.7 mM). For the PEDOT coated Mg tested in this
study, Mg ion concentrations in the post-culture media were
in a lower range of 8–13 mM, which alone might not be the
direct cause of cell death. In a previous study in which the
mTeSR
V
R
1 hESC culture media were intentionally supple-
mented with 10–40 mMof Mg ions in the form of MgCl
2
salt, no obvious death of hESCs was observed with the Mg
ion concentrations of 7–25 mMin the post-culture media
and the hESCs grew to confluency and retained pluripotency
at 72 h as indicated by the expression of OCT4, SSEA3, and
SOX2.
45
The elevated levels of Mg ions might not be the
direct cause of cell death, but might be the cause for the
cell morphology change observed in this study. The hESCs
lost their tightly packed morphology when cultured with
the non-coated and PEDOT coated Mg samples. This same
morphology change was also observed when the hESCs
were cultured in the media in which 10 mMand more sup-
plemental Mg ions were added in the form of MgCl
2
salt.
45
Therefore, it is possible that the combined effects of persis-
tent alkaline pH and elevated Mg ion concentrations were
responsible for the observed hESC morphology change and
subsequent cell death.
We understand that the purity of Mg substrates may play
aroleonthein vitro hESC viability. The 99.9% pure Mg sam-
ple may produce a different cytocompatibility result as com-
pared with 98% pure Mg sample. Thus, we compared hESC
survival on 98% pure Mg samples used in this study versus
99.9% pure Mg sample, the hESC viability results were very
similar. That is, in both cases, the hESCs died at 30 h of cul-
ture. The death of hESCs after induction of 99.9% pure Mg
sample for 30 h was reported previously.
36
Although the dif-
ferent purity of these two groups of Mg samples may result
in different degradation rates, these rates in both cases were
still too rapid for the survival of hESCs. This confirmed the
necessity for a coating on Mg substrates despite the purity of
Mg substrates. Moreover, since the objective of this study
was to investigate the effects of PEDOT coating on the degra-
dation and hESC compatibility of Mg, the choice of Mg sub-
strates (whether pure Mg or Mg alloys) would not affect our
ability to compare the PEDOT coated Mg substrates versus
non-coated substrates as long as these substrates were con-
sistent. Therefore, in this study, we chose to use 98% pure
Mg sample, considering that it is a more affordable choice as
compared with 99.9% pure sample that costs 50–100 times
more. Nevertheless, we still plan to deposit PEDOT on Mg
substrates with different purities, and on different Mg alloys
to determine the best coating–substrate combination in the
future studies.
Future directions
Clearly, the degradation rate of 53-PEDOT-Mg is still faster
than the optimal rate in which hESCs can survive. Thus, it is
necessary to further decrease the sample degradation rate
for future long-term studies. There are two possible ways to
decrease the degradation rate of PEDOT-coated Mg further.
One is to design new Mg alloys that have slower degrada-
tion rates than pure Mg as the substrates. Another is to
optimize the PEDOT coating process to reduce the porosity
and water permeability of the coating. We believe that the
best combination of coating properties and Mg alloy sub-
strates will be needed for future in vivo studies.
Although it is still not clear whether or how PEDOT
coating would degrade in biological systems exactly, our
results encourage further research to study PEDOT on Mg-
based biodegradable implants and its metabolic pathway for
potential clinical use. We speculated that PEDOT coating on
Mg might release small PEDOT particles when Mg degraded
and the released PEDOT particles might not cause any harm
to local tissue if they were released slowly (i.e., small
ORIGINAL ARTICLE
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A |MONTH 2014 VOL 00A, ISSUE 00 11

dosage) with small particle size. Cheng et al.
50
intravenously
injected PEGylated PEDOT:polystyrenesulfonate nanopar-
ticles of 130 nm in diameter to mice at a dosage of 10 mg/
kg and showed no apparent toxicity after 40 days, according
to comprehensive blood tests and careful histological exami-
nation. This speculation is sound based on our observations
and other studies reported in the literature. However, to
confirm this, further investigations on the long-term excre-
tion, metabolism, and toxicology of PEDOT coating on Mg
are still needed in future studies.
It is important to point out that this study showed prom-
ising results in using PEDOT coating on Mg substrates. These
results encourage further studies to optimize PEDOT coated
Mg substrates for potential neural applications. Among many
possible neural applications, two of them are the most
appealing for future studies. One is for potential neural tissue
engineering applications and another is for neural recording
and stimulation applications. For the purpose of neural tissue
regeneration, we plan to differentiate hESCs to neural cells
directly on the optimized coating-substrate combination in
the future studies. For neural recording and stimulation
applications, further long-term in vitro studies with neural
stem cells, differentiated neurons, astrocytes, and oligoden-
drocytes should be conducted. Last but not least, to advance
the potential clinical translation of the PEDOT-Mg composites
for neural applications, it is necessary to assess their per-
formance in vivo using relevant animal models.
CONCLUSIONS
Mg substrates were successfully coated with the conductive
PEDOT polymer using the CV electrochemical deposition
method. All the PEDOT coated Mg samples exhibited slower
degradation rates when compared with the non-coated Mg
sample, indicating the PEDOT coatings were effective in
reducing Mg degradation. The PEDOT coating thickness on
the Mg samples increased from 31 to 78 mm as the number
of CV cycles increased from 2 to 10. Both coating thickness
and adhesion strength are crucial for the efficacy of PEDOT
coatings for controlling Mg degradation. The 5 CV cycles
was determined to be the best in terms of coating adhesion
strength and the most effective in reducing Mg degradation
rate, considering that the 53-PEDOT-Mg had the best com-
bination of coating thickness and adhesion strength. The Mg
samples coated with the PEDOT at the 5 CV cycles improved
the in vitro hESC viability the most and released the least
amount of hydroxide and Mg ions. Further studies are still
needed to determine the exact relationships between Mg
degradation rate and hESC viability and to reveal the exact
mechanisms that led to hESC death.
ACKNOWLEDGMENTS
The authors thank California Institute for Regenerative Medi-
cine (CIRM) Bridges to Stem Cell Research for the undergradu-
ate research internship for Salvador Garcia. The authors
appreciate the Central Facility for Advanced Microscopy and
Microanalysis (CFAMM) for the use of SEM FEI XL30 and the
Stem Cell Core Facility (especially Drs. Prue Talbot and Duncan
Liew) for the use of Nikon Biostation CT at the University of
California at Riverside. The authors also thank the undergrad-
uate student researchers (Chinh Nguyen, Lauren Wong, and
Lauren Richards) for their assistance with ImageJ analysis.
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