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Engineering a Freestanding Biomimetic
Cardiac Patch Using Biodegradable
Poly(lactic-co-glycolic acid) (PLGA) and Human
Embryonic Stem Cell-derived Ventricular
Cardiomyocytes (hESC-VCMs)
a
Yin Chen, Junping Wang, Bo Shen, Camie W. Y. Chan, Chaoyi Wang,
Yihua Zhao, Ho N. Chan, Qian Tian, Yangfan Chen, Chunlei Yao,
I-Ming Hsing, Ronald A. Li,* Hongkai Wu*
Microgrooved thin PLGA film (30 mm) is successfully fabricated on a Teflon mold, which could
be readily peeled off and is used for the construction of a biomimetic cardiac patch. The
contraction of it is studied with optical mapping on transmembrane action potential. Our
results suggest that steady-state contraction could be easily established on it under regular
electrical stimuli. Besides, the biomimetic cardiac patch recapitulates the anisotropic
electrophysiological feature of native cardiac tissue and is
much more refractory to premature stimuli than the
random one constructed with non-grooved PLGA film, as
proved by the reduced incidence of arrhythmia. Consider-
ing the good biocompatibility of PLGA as demonstrated in
our study and the biodegradability of it, our biomimetic
cardiac patch may find applications in the treatment of
myocardial infarction. Moreover, the Teflon mold could be
applied in the fabrication of various scaffolds with fine
features for other tissues.
Y. Chen,
[+]
C. Yao, Prof. I.-M. Hsing, Prof. H. Wu
Division of Biomedical Engineering, The Hong Kong University of
Science and Technology, Hong Kong, China
E-mail: chhkwu@ust.hk
Dr. J. Wang,
[+]
Dr. C. W. Y. Chan, Prof. R. A. Li
Stem Cell & Regenerative Medicine Consortium, LKS Faculty of
Medicine, The University of Hong Kong, Hong Kong, China
E-mail: ronaldli@hku.hk
Dr. J. Wang, Prof. R. A. Li
Department of Physiology, LKS Faculty of Medicine, The
University of Hong Kong, Hong Kong, China
Dr. C. W. Y. Chan
Department of Anatomy, LKS Faculty of Medicine, The University
of Hong Kong, Hong Kong, China
B. Shen, Dr. Y. Zhao, H. N. Chan, Q. Tian, Y. Chen, Prof. H. Wu
Department of Chemistry, The Hong Kong University of Science
and Technology, Hong Kong, China
C. Wang
Department of Civil and Environmental Engineering, The Hong
Kong University of Science and Technology, Hong Kong, China
Prof. I.-M. Hsing
Department of Chemical and Biomolecular Engineering, The
Hong Kong University of Science and Technology, Hong Kong,
China
[+]
These authors contributed equally to this work.
a
Supporting Information is available online from the Wiley Online
Library or from the author.
Full Paper
426 ß2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim DOI: 10.1002/mabi.201400448
Macromol. Biosci. 2015, 15, 426–436
wileyonlinelibrary.com
1. Introduction
Heart diseases have become one of the leading causes of
morbidity and mortality all over the world in the past few
decades.
[1]
It was estimated that more than 17 million
people worldwide died from heart diseases in 2008, with
the number increasing annually.
[1]
Although enormous
amounts of resources have been invested in the fight
against heart diseases, the outcome is unsatisfactory,
mainly due to the lack of regenerative capability of
cardiovascular tissues.
[2]
For end-stage heart failure
patients, transplantation is their last resort.
[3,4]
Unfortu-
nately, this is seriously limited by the disparity between the
large number of patients in need and the small number of
donors available.
[5]
In recent years, the fast development of tissue engineer-
ing has ignited new hope for those suffering from severe
heart diseases. The great achievements in stem cell
technology make it possible that unlimited numbers of
stem cell-derived cardiac cells could be provided for
regeneration purpose.
[6–8]
Another major challenge is a
competent scaffold, which can support cell growth and
formation of an integrated tissue or even organ.
[7–9]
Ideally,
a scaffold material must have appropriate mechanical
property, good biocompatibility, and favorable degradation
rate.
[10–12]
As the first step toward the treatment of severe
heart diseases such as myocardial infarction (MI), many
artificially engineered cardiac tissues have been con-
structed in lab so far based on synthetic soft materials
such as polyurethane (PU), poly(dimethylsiloxane) (PDMS),
poly(glycerol sebacate) (PGS), and polyethylene (PE) or
protein-based materials such as collagen, tropoelastin,
gelatin, fibrinogen, and Matrigel.
[13–21]
Although the
structural, mechanical as well as the electrophysiological
properties similar to those found in native cardiac tissue
have been recapitulated in artificial ones, those materials
either lack biodegradability, require special chemical
modification, or are too expensive to be widely used,
thereby limiting their applications.
[13–21]
As a result,
construction of cardiac tissue with biodegradable materials
which are commercially available and cost-effective
becomes increasingly important due to their potential
application in the treatment of MI.
Poly(lactic-co-glycolic acid) (PLGA), a copolymer of poly-
(lactic acid) (PLA) and poly(glycolic acid) (PGA), has been
extensively employed as a scaffold material for engineering
tissues such as bone, cartilage, nerve, and skin in the past
few decades.
[12,22–25]
Various geometrical shapes such as
sphere, fiber, film, and bulky chunk have been fabricated
successfully from nanoscale to macroscale.
[22–24,26–36]
As
an FDA-approved biodegradable polymer, it has attracted
much more attention over other simililar materials like
poly(caprolactone) (PCL), poly(3-hydroxybutyrate) (PHB),
PLA, and PGA owing to its good biocompatibility, tunable
biodegradability, and versatile processability.
[12,22–24,27]
As native tissues typically possess well-aligned structures
at cellular level for their biological functions, the introduc-
tion of fine features on a substrate is crucial for the
construction of a biomimetic tissue.
[37–39]
In many cases,
soft lithography on a prefabricated mold was used for
creating micropatterns on the scaffold substrates of
various materials. However, the fabrication of thin PLGA
film (<100 mm) with fine features is very difficult since the
polymer film generated by solvent casting or melt
processing method can hardly be peeled off inorganic
substrates (such as silicon and glass) without damage or
organic solvents could swell or even destroy most organic
molds (such as PDMS and poly(methyl methacrylate)
(PMMA)), limiting its application in the construction of
biomimetic tissues.
[40,41]
One solution to this problem is to
use a sacrificial layer, but this leads to additional work and
cost.
[42]
This method could also be restricted when high
fidelity of the replicated features is needed. To overcome
these obstacles, Teflon molds seem to be the best
solution. Teflon plastics are a class of perfluorinated
paraffinic polymers whose hydrogen atoms are fully
replacedby fluorines, suchas polytetrafluoroethylene(PTFE),
fluorinated ethylenepropylene (FEP), and perfluoroalkoxy
(PFA).
[43–45]
With the inertness to almost any solvents and
antifouling property, those polymers allow easy peel-off of
the film formed on them by solvent casting.
[43,44]
In this
study, we demonstrated a facile method for the fabrication
of microgrooved PLGA film which could be used for
constructing freestanding biomimetic cardiac patch.
2. Experimental Section
2.1. Fabrication of Microgrooved PLGA Film
2.1.1. Materials
Silicon wafers (KD-03, 525 mm in thickness), negative photoresist
(SU-8 3005) and trichloro(1H,1H,2H,2H-perfluorooctyl)silane (Cat.
No.: 448931) were purchased from GRITEK (Beijing, China),
MicroChem (Newtown, MA, USA) and Sigma–Aldrich (St. Louis,
MO, USA), respectively. PDMS prepolymer kit (RTV615) was
provided by Momentive Performance Materials (Waterford, NY,
USA). FEP plates (1 mm in thickness) and PLGA solids (Mw¼40
000) were obtained from Yuyisong, Inc. (Shanghai, China) and
Daigang Biomaterial Co., Ltd. (Jinan, China), individually. Chloro-
form (Analytical grade) was bought from BDH Laboratory Supplies
(Poole, UK) and used as received.
2.1.2. Preparation of Microgrooved PDMS Master
SU-8 photoresist was spin-coated (1 500 rpm for 10 s and 4 000 rpm
for 30 s) on a clean silicon wafer. Micro-structure with desired
geometry was generated after soft bake (2 min at 95 8C), UV
exposure (7 s, 18 mW cm
2
) under a custom-made chrome mask,
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post exposure bake (1 min at 65 8C and 2 min at 958C) and develop
(1 min in SU-8 developer). The silicon wafer with patterned
photoresist was then silanized with trichloro(1H,1H,2H,2H-per-
fluorooctyl)silane vapor in a vacuum container after treatment in
an oxygen plasma cleaner (PDC-32G, Harrick Scientific Products,
Inc.) for 2 min. Microgrooved PDMS master membrane was made
by curing the degassed prepolymer mixture (10:1 elastomer to
curing agent) on the patterned wafer at 70 8C for 1 h. After that the
membrane (300 mm in thickness) was peeled off and placed on a
piece of clean glass with its patterned surface exposed.
2.1.3. Manufacture of FEP Mold
An FEP plate was sandwiched between the PDMS master
membrane and another PDMS-coated glass plate. The assembled
parts were then placed on a hot compressor (TM-101F, Taiming Co.
Ltd., Shanghai, China) and embossed (275 8C for upper clamp and
265 8C for lower clamp) for 5 min with a pressure of tens of kPa.
Subsequently, the assembled parts were removed from the
clamps and rapidly cooled between two CPU coolers. The FEP
mold (4.5 cm 3.5 cm) with negative relief was then released at
ambient temperature.
2.1.4. Fabrication of Microgrooved PLGA Film
Microgrooved PLGA film was fabricated by casting PLGA solution
(1.4 mL, 5 mg mL
1
in chloroform) on an FEP mold. After drying in a
fume hood overnight, the FEP mold was heated at 80 8C for 1 h to
thoroughly remove the remaining solvent. The film was readily
peeled off the FEP mold with sticky tape from its edge once it had
cooled down to ambient temperature. As a control, non-grooved
PLGA film was also prepared.
2.1.5. Morphological Study of PDMS Master, FEP Mold
and Microgrooved PLGA Film
To obtain intact cross-sections with minimum damage and
deformation, PDMS master was partially cut on the back and
pulled apart while microgrooved PLGA film was fractured in
liquid nitrogen. Unfortunately, we were unable to get cross-
sections of FEP mold in good condition due to its plastic nature even
in liquid nitrogen. Instead, the surface of an FEP was studied. Pieces
of PDMS master and microgrooved PLGA film were mounted with
double-sided sticky tape on the inclined plane of an angled (458)
brass stub with their cross-sections and patterns upward while a
piece of FEP mold on the flat surface of a regular brass stub with
patterns exposed. The specimens were sputter-coated (K575X,
Quorum Technologies Ltd) with gold and observed under a
scanning electron microscope (SEM, JSM 6300F, JEOL). Images
were taken at 20 kV accelerating voltage and 1 000magnification.
2.2. Biocompatibility and Cell Alignment Guidance
of PLGA Film
2.2.1. Culturing of JEG-3 Cells
JEG-3 (Human placental choriocarcinoma cell line, HTB-36
TM
,
ATCC) cells were cultured in Eagle’s minimal essential medium
(EMEM) (Cat. No.: 61100-061, Life Technologies) supplemented
with 1.5 g/L NaHCO
3
, 10% FBS and 1% penicillin/streptomycin as
recommended by ATCC. They were maintained in 10 mL medium in
culture dishes (60.1 cm
2
, Cat. No.: 93100, TPP) at 37 8C with 5% CO
2
and the medium was renewed every other day. When 90%
confluence had been reached, the medium was replaced by 5 mL
phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM
Na
2
HPO
4
, 1.8 mM KH
2
PO
4
,pH¼7.4). After 5 min in the incubator,
PBS was removed and 1 mL 0.05 wt.-% trypsin (Cat. No.: 0458,
Amresco) with 0.53 mM ethylenediaminetetraacetic acid disodium
salt (Cat. No.: LE118, EDTA.2Na, Genview) (trypsin/EDTA.2Na)
solution was added and the cells were placed back in the incubator
again until thoroughly detached with gentle shake. Subsequently,
9 mL medium was added and the cells were either used for passage
or for seeding on PLGA films.
2.2.2. Seeding of JEG-3 cells on PLGA Film
Agarose (Cat. No.: 15510-027, Gibco) was dissolved in boiling
ultrapure water (NANOpure, Thermo Scientific Barnstead) to the
concentration of 4 wt.-%. The hot solution was immediately poured
into petri dishes (Cat. No.: 10060, D
in
¼6 cm, SPL life Sciences Co.,
Ltd.) until the bottom surface was fully covered (about 6 mL).
Hydrogel was formed after the agarose solution had cooled down to
ambient temperature. PLGA film (both microgrooved and non-
grooved) was cut into smaller pieces (3 cm 2 cm) and one piece
was placed on each hydrogel slab. As PLGA film is hydrophobic in
nature, it was coated with gelatin (Cat. No.: G1890, Sigma-Aldrich,
0.2 wt.-% in PBS) for 1 h and then sterilized under UV light in the
biosafety cabinent (NU-437-400E, Nuaire) for 0.5 h. JEG-3 cells
were seeded at the density of 50,000 cells cm
2
and maintained
at 37 8C with 5% CO
2
for 1–7 d before fixation, with medium
changed every day.
2.2.3. Study on Biocompatibility of PLGA Film
Cells were stained with propidium iodide (Cat. No.: P1304MP, Life
Technologies, 2 mgmL
1
in PBS) and Hochest 33342 (Cat. No.:
H1399, Life Technologies, 2 mgmL
1
in PBS) simultaneously for
15 min. After rinsing with PBS for three times, the specimens were
observed under an upright epifluorescent microscope (BX41,
Olympus). Fluorescent images were taken with a computerized
charge-coupled device (CCD) camera (12.0 Monochrome w/o IR-18,
Diagnostic Instruments). The dead cells were stained with red and
the cell nuclei blue. The viability was defined as the percentage of
live cells in each image. ImageJ (NIH) was used for counting dead
cells and cell nuclei. At least three specimens at each time point
were analyzed for both experimental and control groups.
To quantify the growth of cells with time, the specimens were
further fixedwithparaformaldehyde(Cat.No.:P6148,Sigma–Aldrich,
4 wt.-% in PBS) for 30 min, permeabilized with Triton X-1 00 (Cat.
No.: T8787, Sigma–Aldrich, 0.1% in PBS) for 30 min, and then blocked
with BSA (Cat. No.: A2153, Sigma–Aldrich, 5% in PBS) for 1h. F-actin
of cells was stained with Alexa Fluor
1
488-labeled phalloidin
(Cat. No.: A12379, Life Technologies, 1 unit mL
1
in PBS). After
staining for 1h, thesolution was replaced by PBS in succession with
double gentle washing with it. Fluorescent images were recorded
with a computerized CCD camera (7.2 Color Mosaic, Diagnostic
Instruments) coupled to an inverted fluorescent microscope (Eclipse
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TE2000-U, Nikon).ImageJ was used for measuring the area occupied
by F-actin. At least three specimens at each time point were
analyzed for both experimental and control groups.
2.2.4. Study on Cell Alignment Guidance of PLGA Film
The cell nuclei were fitted with ellipses, of which the lengths of
major and minor axes as well as their intersection angles with
respect to the orientation of microgrooves were calculated using
ImageJ. The nucleus shape index (NSI) and cell alignment index
(CAI) were calculated with a homemade program in Matlab R2013b
(MathWorks).
2.3. Engineering a Freestanding Biomimetic Cardiac
Patch
2.3.1. Differentiation of hESCs and Seeding of hESC-VCMs
on PLGA Film
hESC-VCMs were generated according to the protocol developed by
Weng et al.
[46]
Briefly, hESCs (HES2 line, NIH code ES02) were cultured
on tissue culture plate (Cat. No.: 3506, Corning) coated with Matrigel
(BD Biosciences, 1/200 dilution in PBS) and passaged with accutase
(Cat. No.: A11105-01, Gibco) when reaching 80% confluence (Day 0).
Cells were then digested into single cell suspension and maintained
on ultralow attachment six-well plate (Cat. No.: 3471, Corning) in
mTeSR1 medium (Cat. No.: 05850, Stem Cell Technologies) with
Matrigel (1/200 dilution), 1 ng mL
1
BMP4 (Cat. No.: PHC9534, Gibco)
and 10 mM Rho kinase inhibitor (ROCK) (Cat. No.: 1254, R&D) under a
hypoxic condition with 5% O
2
(Day 1). Small clusters formed after
overnight incubation. These clusters were then cultured in differen-
tiation medium StemPro34 (Cat. No.: 10640019, Gibco) with 50 mg
mL
1
ascorbic acid (Cat. No.: A4403, Sigmal–Aldrich) and 2 mM
GlutaMAX-I (Cat. No.: 35050-061, Gibco) and supplemented with
cytokines and Wnt inhibitor as follows: days 2–4.5, 10 ng mL
1
BMP4
and 10 ng mL
1
Activin-A (Cat. No.: PHC9564, Gibco); days 4.5–7,
5 mM IWR-1 (Cat. No.: BML-WN103-0005, Enzo Life Sciences). On day
8, cells were transferred to a normoxic environment and maintained
in StemPro34 SFM medium with ascorbic acid and GlutaMAX-I.
The hESC-VCMs were dissociated from the clusters by treating with
1 mg/mL collagenase IV (Cat. No.: 17104, GIBCO) and 50 mgmL
1
DNase 1 (Cat. No.: DN25, Sigma) at 37 8C for 30 min, followed by 0.05%
trypsin–EDTA (Cat. No.: 25300-054, Gibco) at 37 8Cfor7min.
PLGA film (2 cm 1.5 cm) on the agarose gel was coated with
Geltrex (Cat. No.: A15696-01, Life Technologies) at 37 8C for 1 h and
then sterilized with 75% alcohol. The isolated cells were seeded on
PLGA film at a density of 10
6
cells cm
2
and maintained in RPMI
1640 medium (Cat. No.: 72400, Gibco) supplemented with B27 (Cat.
No.: 0080085-SA, Life Technologies) and 2mM L-glutamine (Cat. No.:
21051-024, Life Technologies) to generate cardiac tissue patches.
The medium was changed every day and synchronous contraction
of the cardiac patches was observed in 7–10 d (Supporting
Information Movie 1 and 2).
2.3.2. Immunostaining
For cardiac cell identification, specimens were washed with PBS,
fixed with paraformaldehyde for 30 min, permeabilized with
Triton X-100 for 30 min and then blocked with BSA for 30 min. After
that, some of them were washed again with PBS and incubated
with mouse monoclonal anti-cardiac troponin T antibody (Cat. No.:
ab8295, Abcam, 1/100 dilution in PBS) at 4 8C overnight and then
Alexa Fluor
1
555 goat anti-mouse IgG1 antibody (Cat. No.: A21127,
Molecular Probes, 1/500 dilution) at 4 8C for 1 h. F-actin and cell
nuclei were stained simultaneously with ActinGreen 488 (Cat.
No.: R37110, Molecular Probes) and NucBlue (Cat. No.: R37606,
Molecular Probes) for 5 min.
For gap junction protein localization, other specimens were
incubated with rabbit polyclonal anti-connexin 43 antibody (Cat.
No.: ab11370, Abcam, 1/100 dilution in PBS) at 4 8C overnight,
followed by incubation with Alexa Fluor
1
555 donkey anti-rabbit
IgG antibody (A31572, Molecular Probes, 1/500 dilution) for 1 h.
After that, they were further treated with Triton X-100 (0.5% in PBS)
for 1 h and blocked with BSA for 1 h. To visualize the myofibrillar
structures, the cells were labeled with mouse monoclonal anti-
sarcomeric a-actinin antibody (Cat. No.: ab9465, Abcam, 1/100
dilution in PBS) at 4 8C overnight and then Alexa Fluor
1
488 donkey
anti-mouse IgG antibody (Cat. No.: A21202, Molecular Probes,
1/500 dilution in PBS) for 1 h. NucBlue was used again as the nuclear
counterstain.
2.4. Compliance to Regular Electrical Stimulation
and Anti-arrhythmogenicity of the Biomimetic
Cardiac Patch
Optical mapping of AP propagation was performed on the
engineered cardiac patches by using MiCam Ultima fluorescence
imaging system (SciMedia) with a 1objective and a 1
condensing lens to yield a 1 1cm
2
field of vie w. Cardiac patches
were stained with 4 mM voltage-sensitive dye di-4-ANEPPS (Cat.
No.: D8064, Sigma) for 20 min at room temperature, then replaced
with 37 8C Tyrode’s solution (140 mM NaCl, 5 mM KCl, 1 mM
MgCl
2
, 1 mM CaCl
2
,10mMglucose,and10mMHEPESatpH7.4).
The dye was excited by a halogen light source filtered by a
515 35 nm band pass filter and emission by a 590 nm high-pass
filter. For pacing of contraction, cells were stimulated by a coaxial
point stimulation electrode at 1 Hz, 8 V, and 10 ms pulse duration.
For standard (S1–S2) programmed electrical stimulation (PES), a
train of eight S1 stimuli with a basic cycle length (BCL) of 1 000 ms
was initiated, and then a premature extra-stimulus (S2) was
delivered 600 ms after S1. The S1–S2 interval was shortened by
100 ms per step until the tissue failed to capture the stimuli. Data
were collected with a sampling rate of 200 Hz and analyzed using
BV_Ana software (BrainVision, Japan) (Supporting Information
Movie3and4).Theoccurrencepercentageofspiralwaveupon
PES stimulation was measured. Power-spectral-density (PSD)
analysis was performed with a homemade program in Matlab
R2013b.
2.5. Statistical Study
Experimental data were reported as mean standard deviation.
Statistical significance was calculated by Welch’s t-test for
unpaired comparison with unequal variances. The analyses were
performed with a homemade program in Matlab R2013b.
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3. Results and Discussion
3.1. Fabrication of Microgrooved PLGA Film
As illustrated in Figure 1a,
[47]
PLGA film with microgrooves
was fabricated on FEP mold by solvent casting and the film
was readily peeled off with sticky tape from its edge. The
colored interference patterns on the various substrates
shown in Figure 1b demonstrated their micro-structures.
Under scanning electron microscope (SEM), the fine micro-
structures could be clearly seen. As shown in Figure 1c,
microgrooves were replicated from PDMS master to PLGA
film with high fidelity. The film thickness could be
controlled by the volume and concentration of PLGA
solution. In this study, PLGA film with a thickness of
30 mm was used. Based on our parallel study with PDMS
film (data not shown), the optimized geometry of groove
width ridge width groove depth ¼15 mm5mm6
mm was employed.
3.2. Biocompatibility and Cell Alignment Guidance
of PLGA Film
It has been reported that acidic products of PLGA
degradation can inhibit the spreading and growth of
cells.
[48–50]
For PLGA film with a typical thickness of dozens
of microns, the degradation rate was claimed to be much
faster compared to smaller scales (<10 mm).
[51]
Considering
these, the biocompatibility and cell alignment guidance of
PLGA film were first evaluated with an epithelial cancer cell
line JEG-3 before the fabrication of cardiac patch. JEG-3 is a
human placental choriocarcinoma cell line which has been
used to study the molecular mechanisms responsible
for the invasiveness of cancer.
[52,53]
JEG-3 cells grew fast
(doubling time <24 h) and could form a dense layer in
normal cell culture medium, making them suitable for
assessing the compatibility of a substrate for cell growth
before testing with more precious types such as pluripotent
human embryonic stem cells (hESCs) and their derivatives.
Figure 1. (a) Schematic illustration for the fabrication of a freestanding biomimetic cardiac patch using PLGA. (b) Pictures of PDMS master,
FEP mold and PLGA film. (c) Scanning electron microscope (SEM) images showing the micro-features of micropatterned PDMS master (side
view), FEP mold (top view) and PLGA film (side view).
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Y. Chen et al.
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JEG-3 cells were seeded at a density of 50 000 cells cm
2
on
microgrooved PLGA film with culture medium renewed
every day. Non-grooved PLGA film was used as a control.
The viability, growth, nucleus shape, and alignment of cells
on the substrates in 1 week duration were investigated and
evaluated. The results were summarized in Figure 2, in
which the nucleus shape index (NSI) (eccentricity of the
fitting ellipse in nature) and cell alignment index (CAI) were
defined as:
[54]
NSI ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1b
a
2
sð0NSI <1Þð1Þ
CAI ¼cosuð90u<90;0CAI 1Þð2Þ
where, aand bare the lengths of major and minor axes of
the ellipse used to fit the shape of cell nucleus, respectively. u
is the intersection angle of the major axis with respect to
the orientation of microgrooves (arbitrarily designated for
non-grooved substrates). NSI closer to 0 indicates a rounder
nuclei while closer to 1 implies a more elliptical one. CAI
closer to 1 suggests a better alignment of the cell.
Although some studies questioned the cytotoxicity of
the acidic degradation products of PLGA-based scaffolds
for tissue engineering, our result suggested high viability
(>88%) of cells on both types of PLGA film within 7 d. As the
degradation of PLGA is usually auto-catalyzed by its acidic
components,
[51,55]
the good biocompatibility of it in our
study must be attributed to the frequent renewal of cell
culture medium so that the pH was nicely maintained at
the neutral condition. This was confirmed by a preliminary
experiment in which we found the pH declined to around
6.5 after PLGA film had been incubated for 2 d without
renewal of the medium. As the presence of blood circulatory
and lymphatic systems in native tissues, the drop of pH is
believed not to be an issue in vivo. The viability at day 1 was
modestly lower than those of other days (p<0.05), which
0
20
40
60
80
100
120
140
day 1 day 3 day 5 day 7
Viability (%)
Microgrooved
Non-grooved
0
20
40
60
80
100
120
02468
Area coverage (%)
Time (day)
Microgrooved
Non-grooved
0
0.2
0.4
0.6
0.8
1
1.2
1.4
day 1 day 3 day 5 day 7
Nucleus shape index (NSI)
Microgrooved
Non-grooved
0
0.2
0.4
0.6
0.8
1
1.2
1.4
day 1 day 3 day 5 day 7
Cell alignment index (CAI)
Microgrooved
Non-grooved
a b
cd
*
***
***
*
***
***
***
Figure 2. Investigation on biocompatibility and cell alignment guidance of PLGA film. (a) Viability of JEG-3 cells versus culture time. (b)
Temporal evolution of area coverage by JEG-3 cells on the substrates. (c,d) Nucleus shape index (NSI) and cell alignment index (CAI) of JEG-3
cells as a function of culture time. (p<0.05; p<0.001).
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could be attributed to cell death from the trypsinized
suspension and the subsequent debris deposition. Howev-
er, this was followed by an increased proliferation of cells.
Although not significant, there was a trend that the viability
reduced after incubation for 5 d, implying that a proper cell
density was essential to maintain the healthy state of cells
on a substrate. NSI of cells on the microgrooved substrates
had no much difference compared to those on non-grooved
ones in the beginning, but then became significantly higher
with an average value 0.81 at day 3 and day 5, implying a
larger elongation. However, the elongation of cells on the
microgrooved PLGA film decreased by some extent from
day 5 to 7 as a result of cell crowdedness (Figure S1b). NSI of
the control group had no significant difference among all
the culture time points, with a typical average value of
0.72 as a result of random distribution. CAI presented a
similar trend as NSI, in which the experimental group was
found to be much higher, reaching a maximal mean
value 0.91 (|u|24.58) compared to the control group
(CAI 0.63, |u|50.98) at day 3 and 5, suggesting a good
alignment of cells along microgrooves.
3.3. Engineering a Freestanding
Biomimetic Cardiac Patch
Since microgrooved PLGA film had dem-
onstrated both good biocompatibility
and excellent cell alignment guidance,
our next goal was to engineer a bio-
memitic cardiac patch. Human embry-
onic stem cell-derived ventricular cardi-
omyocytes (hESC-VCMs) were seeded on
PLGA film at a density of 10
6
cells cm
2
to reach a good balance between connec-
tion and orientation of the cell layer. Non-
grooved PLGA film was used as a control
again and the cell medium was renewed
every day as well. Synchronous contrac-
tion of the cardiac patches was observed
in 7–10 d (Supporting Information Movie
1 and 2). The interaction between the
patch and the agarose hydrogel was so
weak that under immersion in cell
medium it was freestanding indeed,
allowing to be handily picked up by
tweezers. Immunostaining of ubiqui-
tously dispersed cardiac troponin T (cTnT)
(Figure 3a and b) demonstrated the
cardiomyocyte phenotype of the differ-
entiated hESC, which was consistent
with our previous studies.
[46,56–58]
The
orientations of cytoskeletal protein fila-
mentous actin (F-actin) and cell nuclei
revealed that cells were organized in
parallel with the grooves on the microgrooved PLGA film,
while those on the non-grooved ones randomly spread.
The organization of myocyte-specific protein sarcomeric
a-actinin (SAA) (Figure 3c and d) also demonstrated cell
alignment on the microgrooved substrates but the distri-
bution on the control ones was random. The wide
dispersion of gap junction protein connexin 43 (Cx43)
suggested the formation of excitation-contraction coupling
between cardiomyocytes, which was corroborated by the
synchronous contraction of the cardiac patches. Based on
these features, a biomimetic cardiac patch with anisotropic
structure mimicking native heart tissue was successfully
fabricated with microgrooved PLGA film.
3.4. Compliance to Regular Electrical Stimulation
and Anti-arrhythmogenicity of the Biomimetic
Cardiac Patch
As a potential therapy for the treatment of MI, the
contraction of the biomimetic cardiac patch should have
the compliance to be paced by regular electrical stimulation
like ventricular myocardial contraction following the
Figure 3. Confocal laser scanning microscope (CLSM) images showing the
immunostaining of the representative subcellular structures of hESC-VCMs on
microgrooved PLGA film (a, b) and on non-grooved one (c, d). (cTnT: cardiac troponin
T; F-actin: filamentous actin; Cx43: connexin 43; SAA: sarcomeric a-actinin.)
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Y. Chen et al.
Macromol. Biosci. 2015, 15, 426–436
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432 www.MaterialsViews.com
electrical impulse generated by sinoatrial (SA) node. In
addition, the patch must be refractory to sudden occurrence
of turbulence from other sources in case of arrhythmia
generation. Because of these concerns, the electrophysio-
logical properties, particularly the anti-arrhythmogenicity
of the tissue constructs, were evaluated under regular
pacing (1 Hz) and an established programmed electrical
stimulation (PES) using optical mapping.
[58]
A random
cardiac patch fabricated with non-grooved PLGA film was
used as a control and six specimens from two batches were
tested for each group.
Under steady-state pacing at 1 Hz, the patterns of
transmembrane action potential (AP) propagations on
the biomimetic (B) and random (R) cardiac patches were
elliptical and rounded, respectively, as seen in the
isochronic maps (Figure 4a, S2a and S2c; Supporting
Information Movie 3 and 4, upper panels), similar to the
results reported before and consistent with the expression
of functional juctions between cells.
[18,58]
Representative
APs as functions of time at different sites on the isochronic
maps (Figure 5a and d) were displayed and the features of
APs were further characterized. Our calculation of AP
duration at 90% repolarization (APD90) (Figure 4b) showed
no significant difference between the two types of patches
and in the propagation directions (longitudinal: L; trans-
verse: T) (APD90
B,L
¼354 75 ms, APD90
B,T
¼338 61 ms,
APD90
R,L
¼365 54 ms, APD90
R,T
¼360 47 ms). Howev-
er, the conduction velocity (CV) for the biomimetic patch
was found to be significantly higher in the longitudinal
direction than in the transverse direction (CV
B,L
¼4.60
1.91 cm s
1
,CV
B,T
¼2.31 0.95 cm s
1
,p<0.01), whereas
the conduction velocity on the random patch was
approximately the same in both directions (CV
R,L
¼2.02
0.94 cm s
1
,CV
R,T
¼2.07 0.93 cm s
1
). As reflected by
the anisotropic ratio (AR), our biomimetic cardiac patch
uniquely recapitulated the anisotropic feature of native
myocardium (AR, 2.00 0.13 of biomimetic patches vs.
0.98 0.10 of control, p<0.001),
[59]
which is in consistence
with previous studies.
[17,18,21,58,60]
Upon PES, spiral waves as a sign of arrhythmia (Figure 4a
and S2d; Supporting Information Movie 4, lower panel)
were generated on all the random patches (n¼6) but only
two of the biomimetic ones (n¼2 of 6). For insights, a
detailed study of AP signals and a frequency analysis based
on power spectral density (PSD) were performed. For a
biomimetic cardiac patch which was refractory to PES,
the representative APs at different sites showed higher
amplitude of AP after PES (Figure 5a–c, S3a, and S3b).
Biomimetic cardiac patch before PES Biomimetic cardiac patch after PES
Random cardiac patch before PES Random cardiac patch after PES
a
0
100
200
300
400
500
600
Biomimetic Random
APD90 (ms)
Longitudinal
Transverse
0
2
4
6
8
10
Biomimetic Random
Conduction velocity (cm/s)
Longitudinal
Transverse
Srb,1
Srb,2
Srb,3
Srb,4
Sra,1
Sra,2
Sra,3
Sra,4
Sbb,1
Sbb,2
Sbb,3
Sbb,4
Sba,1
Sba,2
Sba,3
Sba,4
b
**
**
Figure 4. Characterization of transmembrane action potential (AP) of cardiac patches with optical mapping. (a) Isochronic maps (20 ms) of a
biomimetic cardiac patch and a random one before and after PES. Circle indicates the location of electrode and arrow the chirality of non-
sustained spiral wave. The size of the filed of view is 11cm
2
. (b) Summaries of AP duration at 90% repolarization (APD90) and conduction
velocity (CV) of AP of the two types of patches. At least 24 AP peaks in each direction for both biomimetic and random cardiac patches were
analyzed. For biomimetic cardiac patches, the longitudinal direction is defined as the orientation of microgrooves and the transverse
direction is defined as the one perpendicular to it. For random cardiac patches, the longitudinal direction is arbitrarily defined as along the
horizon. (p<0.01).
Engineering a Freestanding Biomimetic Cardiac Patch ...
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Macromol. Biosci. 2015, 15, 426–436
ß2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 433
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Although high-frequency vibrations (5 Hz) were generat-
ed at some sites (S
b,1
and S
b,2
), the maintenance of pacing
frequency (1 Hz) as the dominant frequency and the
elliptical shape of AP propagation (Figure 4a, 5a, and S2b;
Supporting Information Movie 3, lower panel) demonstrat-
ed the resistance of the biomimetic cardiac patch against
arrhythmia. For a random cardiac patch, the influence of PES
was more prominent. While the pacing frequency was
maintained as the dominant one on three sites (S
r,1
,S
r,2
, and
S
r,4
, Figure 5d, f, S3c, and S3d), it was lost on the rest one (S
r,3
,
Figure 5d and e). In addition, the presence of high-frequency
vibrations on all the four sites upon PES suggested the
liability of the random cardiac patch to be affected by
sudden stimuli. The pattern of change in amplitude of AP
after PES was irregular. At some sites it decreased (S
r,3
and
S
r,4
), while at other places it increased (S
r,1
) or had no much
change (S
r,2
). The loss of steady-state contraction on S
r,3
and
other sites in both random and biomimetic cardiac patches
with spiral wave (data not shown) was either the cause of
arrhythmia or the result of it. Whatever the case was, they
were closely related to each other and further studies are
warranted to elucidate it. Given these results, it can be
concluded that the aligned patterns are beneficial for
maintaining the stability of the contraction of cardiomyo-
cytes upon premature stimulation, which could explain the
much smaller occurrence percentage of spiral wave on the
biomimetic cardiac patches than on the random ones. In
other words, the biomimetic cardiac patch with aligned
pattern is more stable and reliable in response to the
premature stimulation from the electrophysiolgoical
perspective.
4. Conclusion
In summary, we have successfully demonstrated a facile
method for engineering a freestanding biomimetic cardiac
patch using biodegradable PLGA and hESC-VCMs. To the
best of our knowledge, this is for the first time that
biomimetic cardiac patch was generated on thin PLGA film
with fine microgrooves. PLGA film was simply produced by
solvent casting on FEP mold and no sacrificial layer was
used. With nano-sized FEP mold which had been manufac-
tured,
[47]
we are able to fabricate PLGA film with much finer
features for the construction of various types of tissues in a
precise way. In addition, the thickness of film could be
tuned by adjusting the concentration of PLGA in the
solution or the volume of PLGA solution used. In this study,
Biomimetic cardiac patch
Random cardiac patch
0
1
2
3
4
5
6
7
8
9
0 1000 2000 3000
Fluorescence intensity (a.u.)
Time (ms)
0
0.02
0.04
0.06
0.08
0.1
012345678
Power spectral density (a.u.)
Frequency (Hz)
Before PES
After PES
0
0.02
0.04
0.06
012345678
Power spectral density (a.u.)
Frequency (Hz)
Before PES
After PES
0
1
2
3
4
5
6
0100020003000
Fluorescence intensity (a.u.)
Time
(
ms
)
0
0.01
0.02
012345678
Power spectral density (a.u.)
Frequency (Hz)
Before PES
After PES
0
0.01
0.02
012345678
Power spectral density (a.u.)
Frequency (Hz)
Before PES
After PES
Srb,1
Sra,1
Srb,2
Sra,2
Srb,3
Sra,3
Srb,4
Sra,4
Sb,3 S b,4
Sr,3 Sr,4
Sbb,1
Sba,1
Sbb,2
Sba,2
Sbb,3
Sba,3
Sbb,4
Sba,4
a)
d)
b) c)
e) f)
Figure 5. Power-spectral-density (PSD) analysis on transmembrane AP obtained by optical mapping. (a,d) Representative APs at different
sites as indicated in Figure 4 before and after PES. S is short for site, bb, ba for the biomimetic patch before and after PES, respectively and rb,
ra for the random patch before and after PES, respectively. (b,c,e,f) PSD spectra on selected sites for both cardiac patches before and after
PES.
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Y. Chen et al.
Macromol. Biosci. 2015, 15, 426–436
ß2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
434 www.MaterialsViews.com
PLGA film with a thickness 30 mm was used in case that
the patch might be pull apart by cell contraction when the it
was too thin. In fact, with this method PLGA film as thin as
a few microns could be fabricated (data not shown). The
biomimetic cardiac patch had a well-aligned organization
as the native cardiac tissue and recapitulated its anisotropic
electrophysiological feature compared to the random one
fabricated with non-grooved PLGA film. Our biomimetic
cardiac patch has demonstrated much superior resistance
against premature stimulation (33%, 2 of 6 patches with
spiral wave arrhythmia) over the random one (100%, all
6 patches with spiral wave arrhythmia), which further
proved that physical alignment could reduce the incidence
of inducible arrhythmias. Considering the good biocom-
patibility of PLGA film as demonstrated in our study and
the biodegradability of PLGA, we are convinced that our
biomimetic cardiac patch could be applied in the treatment
of MI. In the future work, implantation of the biomimetic
cardiac patch into animals such as mice with MI will be
carried out to elucidate their therapeutic effect.
Acknowledgements: We appreciate Ms. Yan Zhang for the
assistance in SEM and Mr. Qiqi Sun for the discussion on power
spectral density analysis. We also acknowledge the financial
support from Theme-based Research Scheme (TBR), General
Research Fund (GRF) and Collaborative Research Fund (CRF)
provided by the Research Grant Council (RGC) of Hong Kong, China
(Project No.: T13–706/11–2, GRF604712 and HKUST11/CRF/12).
Received: October 12, 2014; Revised: November 15, 2014;
Published online: November 25, 2014; DOI: 10.1002/mabi.201400448
Keywords: biodegradable; biomimetic cardiac patch; freestand-
ing; human embryonic stem cell; poly(lactic-co-glycolic acid)
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