Shayanti Mukherjee , Jayarama Reddy Venugopal , * Rajeswari Ravichandran ,

Seeram Ramakrishna , and Michael Raghunath

1. Introduction
Myocardial infarction (MI) is the single
leading cause of deaths due to cardiovas-
cular disease globally.
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MI is caused when
the supply of oxygen and nutrients to the
cardiac muscle is impaired, usually due to
occluded coronary arteries. A massive cell
death occurs in the affected heart region,
which forms a rather non-contractile scar
characterized by mismatch of mechan-
ical and electrical properties with native
myocardium. The substantial cell loss in
the myocardium leads to dilation of ven-
tricular wall and remodeling of the heart
and, eventually, to congestive heart failure
(CHF).
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Currently, more than 10 million
people suffer from CHF in the USA, UK
and southeastern Asia. Drugs alone might
be able slow disease progression, but they
cannot prevent it.
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Current treatments for
congestive heart failure use highly invasive
methods, such as open chest surgery and
transplantation.
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Given the patient mor-
bidity and complications involved with cur-
rent procedures, it is desirable to develop
minimally invasive technologies, such as
injectable therapeutics.
Several modes of regenerating injured
myocardium have been suggested over
time, with pioneering research being
Evaluation of the Biocompatibility of PLACL/Collagen
Nanostructured Matrices with Cardiomyocytes as a
Model for the Regeneration of Infarcted Myocardium
Pioneering research suggests various modes of cellular therapeutics and
biomaterial strategies for myocardial tissue engineering. Despite several
advantages, such as safety and improved function, the dynamic myocardial
microenvironment prevents peripherally or locally administered therapeutic
cells from homing and integrating of biomaterial constructs with the inf-
arcted heart. The myocardial microenvironment is highly sensitive due to the
nanoscale cues that it exerts to control bioactivities, such as cell migration,
proliferation, differentiation, and angiogenesis. Nanoscale control of cardiac
function has not been extensively analyzed in the eld of myocardial tissue
engineering. Inspired by microscopic analysis of the ventricular organiza-
tion in native tissue, a scalable in-vitro model of nanoscale poly( L -lactic
acid)- co -poly( -caprolactone)/collagen biocomposite scaffold is fabricated,
with nanobers in the order of 594 56 nm to mimic the native myocar-
dial environment for freshly isolated cardiomyocytes from rabbit heart, and
the specically underlying extracellular matrix architecture: this is done to
address the specicity of the underlying matrix in overcoming challenges
faced by cellular therapeutics. Guided by nanoscale mechanical cues pro-
vided by the underlying random nanobrous scaffold, the tissue constructs
display anisotropic rearrangement of cells, characteristic of the native cardiac
tissue. Surprisingly, cell morphology, growth, and expression of an interactive
healthy cardiac cell population are exquisitely sensitive to differences in the
composition of nanoscale scaffolds. It is shown that suitable cellmaterial
interactions on the nanoscale can stipulate organization on the tissue level
and yield novel insights into cell therapeutic science, while providing mate-
rials for tissue regeneration.
DOI: 10.1002/adfm.201002434
S. Mukherjee , Dr. M. Raghunath
Division of Bioengineering
9 Engineering Drive 1, Block EA #03-12
National University of Singapore, Singapore
S. Mukherjee , Dr. J. R. Venugopal , S. Ramakrishna
HEM laboratory
Nanoscience and Nanotechnology Initiative
c/o Faculty of Engineering
Block E3-05-29, 2 Engineering Drive 3,
National University of Singapore Singapore
Email: nnijrv@nus.edu.sg
R. Ravichandran , S. Ramakrishna
Department of Mechanical Engineering
9 Engineering Drive 1, Block EA, 07-08
National University of Singapore, Singapore
S. Ramakrishna
Institute of Materials Research and Engineering
A-Star, Singapore
Dr. M. Raghunath
Department of Biochemistry
Medical Drive Block MD7, #02-03, Yong Loo Lin School of Medicine
National University of Singapore, Singapore
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conducted in a variety of technologies including cell therapy
using various cell types, injection of biomaterials, bioengineered
patches, implantation of 3D construct generated in static
and bioreactor culture.
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Cellular therapy, involving the use of
live cells to repair damaged myocardium, has been extensively
studied in recent years, opening new horizons in the clinical
eld. Cellular therapeutics essentially involve the direct delivery
of suitable cell types, such as bone marrow cells (BMCs), mesen-
chymal stem cells (MSCs), endothelial progenitor cells (EPCs),
cardiac stem cells (CSCs), skeletal myoblast cells (SMCs),
embryonic stem cells (ESCs), or induced pluripotent stem cells
(iPS), to the damaged myocardial area.
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Currently, more than
25 new clinical trials are in progress in the United States, and a
similar number in Europe, using different cell types.
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How-
ever, recent experimental studies fail to answer many impor-
tant aspects of cell therapy. Despite several advantages, such
as safety and improved function, and positive results, such as
increased healing, vascular density, increased regional circula-
tion and cardiac function, the efciency of delivery and retention
is lower than expected, and the retention and survival of cells
at sites of delivery has been limited.
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Once the scarred area
has evolved, remodeling and collagen deposition changes the
histological microenvironment of infarcted areas. This changed
microenvironment may allow peripherally or locally admin-
istered stem cells to home and survive. In native tissue, cell
growth and structural development is supported by an extracel-
lular matrix (ECM) that consistently assists in coordinating the
contractility and maintenance of cardiac shape and size, as well
as the function of cardiomyocytes.
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In addition to providing
growth cues, the ECM responds actively to cellular actions.
It also undergoes remodeling by cells during development,
homeostasis and healing processes, which includes digestion
of the old matrix and deposition of a fresh one. Essentially, the
ECM is made up of 80% and 10% of collagen types I and III,
respectively.
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In the healthy heart, collagen serves to maintain
normal cardiac architecture by surrounding and bridging myo-
cytes, which consistently assist in coordinating the contractility
and maintenance of cardiac shape and size as well as the func-
tion of the cardiomyocytes. ECM proteins greatly inuence bio-
activities, such as cell migration, differentiation, proliferation,
and angiogenesis. These effects are recognizable to cells only
when the ECM is suitably modied.
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It is indeed an immense
technological challenge to mimic the entire milieu of ECM arti-
cially without evoking an immune response in such sensitive
surrounding. From a tissue engineering perspective, applying
physiological stress on immature constructs could be a way
of mimicking the natural environment.
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Many efforts by the
scientic community in the eld of myocardial tissue engi-
neering (MTE) are dedicated to identify materials possessing
specic mechanical properties that play a pivotal role.
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First
of all, it is desirable that the mechanical performance of bioen-
gineered scaffolds matches as closely as possible those of the
heart extracellular matrix in terms of stiffness, since the scaf-
fold should be exible enough to promote the contraction of
the growing cells.
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The stiffness of native heart tissue ranges
from 1020 kPa at early diastole and increases to 50 kPa at the
end of diastole, which may shoot up to 200 kPa or more in inf-
arcted hearts.
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In addition, since the myocardial tissue is
subjected to cyclical and constant deformation, materials are
requested to show elastomeric properties and possibly long-
term elasticity.
However, it is likely that the structure and function of the in
vivo cardiac tissues are regulated by much smaller, nanoscale
cues provided by the ECM, which is responsible for extensive
control over cell and tissue function.
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It is therefore important
to investigate the effects of ner control over the cellbiomaterial
interface on the nanoscale, in facilitating the creation of truly
biomimetic cardiac constructs that replicate the structural
and functional aspects of the in vivo ventricular organization.
In addition, the ability to robustly and reproducibly generate
uniformly controlled (both structurally and functionally) and
precisely dened engineered cardiac tissue will likely be neces-
sary for eventual therapeutic products. This makes nanofabri-
cation of biomaterials for MTE an attractive strategy. Ultrane
nanobers having an ECMlike topography can be achieved by
electrospinning of biomaterials.
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Such scaffolds of suitable
biomaterials with nanoscale architecture provide larger surface
areas to adsorb proteins and provide more binding sites to cell
membrane receptors, unlike microscale and at surfaces.
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At
present, there are no successful models of bioengineered car-
diac implant that can satisfactorily replicate the anatomy, physi-
ology, and biological stability of a healthy heart wall.
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To address this challenge, here we report the development
and analysis of a nanotopographically controlled in-vitro model
using nanoscale poly( L -lactic acid)- co -poly( -caprolactone)
(PLACL) and collagen-blend biopolymer scaffolds that mimic
the native myocardial environment and, specically, the under-
lying ECM architecture. The result is a nanobrous scaffold that
is scalable, biocompatible and closely imitates the myocardial
ECM, allowing healthy interaction with freshly isolated cardiac
cells over a span of 15 days in controlled environment. We inves-
tigated the mechanical properties of the nanobrous PLACL/
collagen scaffold, demonstrating that they promote higher
organizational features than unblended PLACL counterparts.
Moreover, strikingly, we found the proliferation of the cells,
their morphological patterns and expression of cardiac specic
markers such as alpha actinin, troponin T, connexin-43, and car-
diac myosin heavy chain were highly sensitive to variation in
biomaterial content as well as nanoscale topographic features,
revealing an unexpected level of regulation in the organization.
2. Results and Discussion
2.1. Fabrication and Characterization of Electrospun
Nanobrous Scaffolds
Nanobers of PLACL and PLACL/collagen were fabricated by
electrospinning, as reported by our group previously ( Scheme 1 ),
and the ber morphology examined under a scanning electron
microscope (SEM). Electrospinning enables the design and
fabrication of scalable scaffolding materials, mimicking the
structural and mechanical cues of the ECM. Moreover, the pres-
ence of puried ECM components provides sites for integrin
attachment and hence excellent binding properties to scaffolds.
Therefore, we aimed at fabrication of PLACL/collagen so that
nanoscale denitions could be extended to tissue dimensions.
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We fabricated nanobers of PLACL/collagen and PLACL with
precisely dened features, extending nanoscale control of
the magnitude of ber diameter. Figure 1 shows SEM micro-
graphs of electrospun PLACL and PLACL/collagen nanobrous
Scheme 1 . Myocardial tissue engineering aims at improving the structure and function of the in vivo cardiac tissue are regulated by much smaller,
nanoscale cues provided by the ECM, which is responsible for extensive control over cell and tissue function.
Figure 1 . Fabrication of electrospun nanobers. Nanober morphology shows porous, bead-
less, uniform nanobers of: a) PLACL with diameter measuring 332 31 nm, and, b) PLACL/
collagen measuring 594 56 nm.
scaffolds as porous, beadless, uniform nanobers with inter-
connected pores with ber diameter in the range of 332 31 nm
and 594 56 nm, respectively.
Biomaterial scaffolds are expected to provide a compliant
and highly hydrated environment, similar to
soft tissues having high water content, thus
facilitating diffusion of nutrients and cellular
waste. Thus, tailoring the surface properties
of the biomaterial in terms of hydrophilicity
inuences its biological performance.
Hydrophilicity of our fabricated nanober
scaffolds to relative of the surfaces was ana-
lyzed using water contact angle analysis.
To analyze the wettability of the nanober
scaffolds, we measured the contact angle of
water on the PLACL/collagen and PLACL
using the sessile drop method ( Table 1 ). The
PLACL/collagen nanobers had a contact
angle of 0 compared to that of 120 5 of
PLACL nanobers. This observation high-
lights the hypothesis that addition of an
ECM componentcollagenimparts superior
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hydrophilic properties to the PLACL, which is otherwise hydro-
phobic. Our results indicate that PLACL/collagen is completely
hydrophilic, unlike PLACL, and shows good tissue-engineering
potential in terms of facilitating the culture of cells.
Chemical characteristics and functional groups on the
nanobers were studied by FTIR analysis ( Figure 2 ). Figure 2
indicates uniform blending of collagen with PLACL to form
PLACL/collagen nanobers. The analysis showed bands of
NH and CH stretches at 2987.08 and 2362.08 cm
1
, respec-
tively. These form the characteristic peaks for collagen and sug-
gest the presence of amide groups on the surface of PLACL/
collagen nanobers. The amide I band of collagen for C = O
stretching, the measure of secondary structure of proteins,
was prominent at 1648.96 cm
1
in PLACL/collagen. The amide
II peak for NH bending coupled with CN stretching was
obtained at 1546.53 cm
1
and the amide III region for NH
bending was obtained between 1200 and 1250 cm
1
. The amide
region corresponds to the triple helix structure of collagen, the
integral part of ECM, and greatly favours cell adhesion and
proliferation.
The mechanical performance of electrospun PLACL/col-
lagen and PLACL mats was analyzed by tensile stressstrain
measurements. It is worth noting that stressstrain data for
electrospun materials depend not only on single ber features
but, most of all, on ber arrangement in the mat. On one hand,
the degree of ber orientation affects the mechanical properties,
since the load is mainly applied to those bers oriented parallel
to the direction of the deformation and, on the other hand, the
presence of ber-fusion at the contact sites remarkably increase
the elastic modulus.
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Moreover the ber arrangement in the
mat is expected to change during the stressstrain measure-
ment. In particular, Lu et al.
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demonstrated that bers tend to
align in the direction of applied force before getting thinner and
nally breaking. In this work we compared the tensile stress
strain analysis of PLACL/collagen nanobers with electrospun
PLACL nanobers mats of similar thickness. Figure 3 reports
representative stressstrain curves of both PLACL/collagen and
PLACL electrospun mats obtained at RT with an elastic mod-
ulus of 10 2.7 MPa and 18 2.3 MPa, respectively. Low stiff-
ness may be a suitable property with respect to integration with
native tissue, since an extremely stiff matrix inhibits the growth
properties of cardiomyocytes.
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Table 1 lists the mechanical
data of the two electrospun materials measured by carrying out
stress-strain analysis both at RT.
With the aim of observing the surface morphologies of
PLACL/collagen and PLACL nanobrous scaffolds, atomic-force
microscopy (AFM) was employed using a height mode. From
AFM images, it was observed that the surface of PLACL nanobers
was much smoother than the surface of PLACL/collagen blended
nanobers. Figure 4 shows 3D nanoscope images of PLACL and
PLACL/collagen. We observed that PLACL is extremely rough,
exhibiting a mean surface roughness (Ra) value
of 700 nm and root mean square (Rms) value
of 900 nm. However, PLACL/collagen exhibit
Ra value of 380 nm and Rms value of 463 nm.
2.2. Growth Prole and Characterization
of Cardiac Cell Cultures
Exploring the myocardial cellular structure in
detail, we performed excision of ex vivo rabbit
heart. The heart tissue displayed a complex
organization of cell sheets of varying thick-
ness. The left ventricle myocardium was the
thickest, comprising aligned myocytes, which
provide a natural direction for exertion of con-
tractile forces. We treated the tissues with col-
lagenase to breakdown the highly organized
structure and successfully obtained single,
highly elongated cells with identiable aniso-
tropic arrays. The freshly isolated cardiac cells
Figure 3 . Tensile properties of electrospun brous scaffolds: PLACL/col-
lagen (A), and PLACL (B) were determined to be 10 2.7 and 18 2.3 MPa,
respectively, using stressstrain curves.
Figure 2 . FTIR analysis of: PLACL/collagen showing characteristics amide I and II peaks at
1651.60 and 1547.50 cm
1
, respectively (A); and, PLACL (B).
Table 1. Characterization of fabricated PLACL/collagen and PLACL
nanobers in terms of hydrophilicity, ber diameter and porosity. The
data indicate differences in properties as a result of blending of an ECM
protein, collagen, with synthetic polymer PLACL.
Material Contact
Angle []
Fiber Diameter
[nm]
Porosity
[%]
Elastic Modulus
[MPa]
PLACL 120 5 332 31 85 18 2.3
PLACL/Collagen 0 7 594 56 94 10 2.7
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had a rod-shaped structure, as seen in Figure 5 , with clearly
visible striations on the rst day. Our observations suggest that
the alignment of the cells is strongly correlated to the ECM
skeleton in the tissue, which might be largely responsible in
Figure 4 . 3D AFM images of: a) PLACL/collagen (Ra = 380 nm and Rms = 463 nm) composite
nanobers, and b) PLACL nanober (Ra = 700 nm and Rms = 900 nm).
Figure 6 . Flow cytometry data of isolated cardiac cells from rabbit myo-
cardium immunostained for expression of: a) sarcomeric alpha actinin,
and b) cardiac myosin heavy chain.
Figure 5 . a, b) Freshly isolated cardiac cells from rabbit heart: clearly vis-
ible striations are seen on the rst day in the dispersed tissue. c) These
cells remain in culture and have a distinct rod-shaped morphology.
providing nanotopographic cues for guiding
myocardial alignment during cardiovascular
development. Therefore, precise control over
various aspects of the microenvironment can
be related to tissue organization and remod-
eling. In order to obtain a quantitative idea
of the functionality of the isolated cells, they
were stained with cardiac specic protein
markers and subjected to hydrodynamic ow
using a ow cytometer (Dako) to count the
per cell uorescence. The results in Figure 6
indicate that 82 12% of the isolated cells
were positive for the presence of sarcomeric
alpha actinin and 25 2% cardiac myosin
heavy chain. Figure 7 shows the expression
of sarcomeric alpha actinin and troponin T
in the isolated cells that were seeded in tissue
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culture plate and characterized at days 1, 5, and 15. The cells
maintained the rod-shape from the end of day 1, with clear stri-
ations of sarcomeric alpha actinin and troponin T. However, a
few cells were seen adhered at day 5, although the majority of
cells still appear to be rod-shaped with light striations of sar-
comeric alpha actinin and troponin T. At day 15, we observed
that the earlier rod-shaped oating cells were no longer obvious
in the ask. Rather, a monolayer was formed in the tissue cul-
ture ask out of the plated cardiac cells containing cardiomyo-
cytes and co-isolated cardiac broblasts, which were devoid of
their striated appearance hereafter. The monolayer expressed
sarcomeric alpha actinin and troponin T
uniformly. These isolated adult cardiac
cells were used as a model to understand
the cellular interaction with biomaterial,
since the mixed cell type represents a more
natural myocardium-like environment. The
myocardium is an ensemble of different cell
types embedded in the complex well-dened
structures of the ECM and arranged in nano-
scale topographical and molecular patterns.
Cardiomyocytes assemble and dissemble
myobrils prior to cell division and reas-
semble after cell division.
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Although the
structure of the cardiac tissue is highly organ-
ized in vivo, our results in Figure 7 support
the isolated cardiac cells losing their native
organization and adopting a random distri-
bution when cultured in vitro using common
cell-culture techniques.
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Immunostaining
of the isolated cardiac cells using striated
muscle specic anti-alpha actinin and anti-
troponin T revealed clear striations of alpha
actinin and troponin T until day 5. However,
the striations were no longer obvious at day
14, despite strong expression of protein in
the cytoplasm of cardiac cells. It is known
that the anisotropic architecture of the layers
of myocardial and matrix bers affects coor-
dination of mechanical contraction, as well
as electrical propagation.
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Our studies sup-
port the knowledge that isolated cardiac cell
cultures do not match the functionality of the
native tissue due to the lack of proper envi-
ronmental factors to allow cardiomyocytes
to respond morphologically, mechanically
and physiologically as they do in the native
tissue.
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Hence, we nd that development of
functional cardiac tissue from any cell thera-
peutics may require fabrication of substrates
to mimic the native environment found in
the heart in order to improve the efcacy in
terms of its mechanical and electrophysiolog-
ical properties.
In order to characterize the growth of cells
on nanobers, we tracked their metabolism
over a span of 15 days with absorbance read-
ings at regular intervals. Our results, shown
in Figure 8 , indicate that growth prolifera-
tion of cardiac cells is enhanced by blended PLACL/collagen
nanobers. The growth prole of cells on PLACL collagen was
statistically signicant ( P < 0.05) compared to that on PLACL
devoid of collagen. Furthermore, we observed a statistically sig-
nicant increment in cellular metabolism at day 15 compared to
day 10 and day 5 on PLACL collagen ( P < 0.05). These statisti-
cally signicant observations suggest that presence of an ECM
component in scaffolding material provides a suitable micro-
environment that supports cell growth and metabolism. This
property may help exercise precise control over various aspects
of tissue re-organization in the myocardium.
Figure 7 . Isolated cardiac cells stained for anti-alpha actinin, showing the cross-linked structure
of actin laments (ac) and anti-troponin T, that binds to tropomyosin (df) at Day 1 (a, d) Day
5 (d, e) and Day 14 (c, f).
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Recent studies of engineered cellbiomaterial interaction at sub-
cellular nano scale levels provide evidence for the potential impor-
tance of sub-micrometer cues for cell signaling, adhesion, growth
and differentiation. However, these initial attempts at engineering
control of cellular functions have not been inspired by ECM. Tradi-
tionally, a cardiomyocyte is considered terminally differentiated in
response to injury. However, recent evidence raises the possibility
that a natural system of myocyte repair exists. According to this
study, less than 50% of cardiomyocytes are exchanged during a
normal life span. Adult hearts have been shown to contain resident
stem cells which make the idea of cardiac regeneration through
ageing and post pathological traumas seem plausible.
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2.3. Cell and Material Interactions
The interaction of cells with substratum forms the basis of
tissue organization. Therefore in order to explore the poten-
tial of our scaffolds in promoting alignment of cells, we
observed the different scaffolds after 5, 10, and 15 days using
SEM. Figure 9 shows micrographs of isolated cardiac cells
monolayers on PLACL and PLACL/collagen. Different levels
of intercellular interaction with the scaffolding material at
Figure 9 . Cardiomyocytes culture on random nanobers of PLACL/collagen (ac) show better cell-to-cell connections, indicating cellular interactions,
compared to PLACL (df ) and TCP (gi) at days 5 (a, d, g), 10 (b, e, h), and 15 (c, f, i).
Figure 8 . Proliferation studies at days 5, 10, and 15 on TCP, PLACL
and PLACL/collagen. There is signicant growth of cardiomyocytes on
PLACL/collagen compared to PLACL ( Denotes signicance from Stu-
dents T-test, P < 0.05) indicating higher potential of blended nanober
as a suitable material for MTE.
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different time points can be observed. It is evident from Figure 9
that the cells start to stabilize and align themselves by day 10 on
PLACL/collagen. However, in the absence of collagen, the
cellular arrangement is rather random and scattered after the
same period. By day 15, PLACL/collagen shows formation of car-
diomyocyte monolayer interconnected by intercellular junctions
Figure 10 . Immunouorescence shows cardiomyocytes on random nanobers of PLACL/collagen, PLACL and TCP stained with for cardiac specic
protein at day 15: a) cardiac myosin heavy chainAlexa Fluor 594 and troponin TAlexa Fluor 488 show colocalized expression, with PLACL/collagen
showing the strongest expression; b) sarcomeric alpha actininAlexa Fluor 488 and connexin 43Alexa Fluor 594, a few cells on PLACL collagen show
re-appearance of a fribrillar pattern of alpha actinin expression (arrows).
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that are well aligned and guided in an evidently parallel fashion,
while the cells remain scattered and poorly aligned on PLACL.
The intercellular network on PLACL/collagen was also observed
to be superior to that on TCP on day 15. This may be attributed
to an increased number of integrin binding sites provided by
collagen at a nanoscale, since the nanobrous architecture of the
scaffolds provide more surface area for binding of cell receptors.
From a clinical perspective, it is of utmost importance that an
implantable material integrates with the host organ cells and initi-
ates cell spreading. The latter is also indicative of acceptance from
the host. The inability of a biomaterial to promote cell spreading
and integration creates a rift for itself from being accepted at a
clinical level. Our study indicates that, although the rough surface
to an extent would be benecial for cell adhesion and proliferation,
extreme roughness may inhibit the same properties. Lack of a suit-
able microenvironment in myocardium post infarction is the main
cause of cell loss in cellular therapeutics. Our results conrm that
PLACL/collagen provides a suitable microenvironment where cells
can interact well with other cells as well as the nanober scaffold.
2.4. Inuences on Protein Expression of Cardiac Cells
The biomaterial scaffold inuences cells not only with respect
to their morphology but also their cellular activities. To analyze
the cellular responses of the cardiac cell isolated grown onto the
nanober scaffold, we studied the expression of the markers
specic to cardiac lineage, such as sarcomeric alpha actinin,
troponin T, cardiac myosin heavy chain and connexin 43. Each
of these proteins plays an indispensable role in the functioning
of cardiomyocytes. Alpha actinin is an actin cross-linking pro-
tein that cross-links two laments of actin.
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Tropomyosin is
yet another actin binding protein that is associated with muscle
contraction along with troponins.
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Myosin heavy chain is the
main motor proteins of the thick muscle laments.
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Connexin
43 is the main gap junction protein of the cardiac cells that
indicates cell-cell communication.
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We studied the expression
of these cardiac specic markers, in the isolated cardiac cells
grown on the nanobers in comparison with cells grown on
TCP, representative of standard in vitro culture.
Our observations in Figure 10 indicate strong expression of
alpha actinin and connexin 43 in cells grown on PLACL, PLACL/
collagen and TCP. Our results indicate that strongest expression
was obtained in cells grown on PLACL/collagen. Interestingly,
we noticed the beginning of a striated appearance of actinin in
few cells on PLACL/collagen. Such appearance was not seen in
cells grown on PLACL or TCP. The expression of troponin T
and myosin heavy chain was studied as a dual staining. PLACL/
collagen seeded cells showed strongest expression of both
indicating a co- localization of the proteins. Troponin T helps
binding of tropomyosin to actin and facilitates contraction of stri-
ated muscle. PLACL/collagen favors alignment of cells and their
normal activity of cardiomyocytes. The unaffected expression of
major cardiac specic proteins results in the ability of PLACL/
collagen to work not only as a biocompatible biomaterial, but
also as scaffolding platform promoting functional activity of cells.
On the other hand, the absence of collagen in PLACL meshes
hampers the integration between cells and biomaterial. There-
fore, our results strongly support that presence of nanoscale
cues from ECM component biomaterial collagen provides robust
scaffolding for growth and functioning of cells.
3. Conclusion
The application of cellular therapies to an infarcted heart
requires retention of the applied cells in a scarred area and suc-
cessful integration with the native tissue. We demonstrate fabri-
cation of a nanobrous substrate for cellular support on the basis
of our observation of arrangement of myocytes in adult native
myocardium in the form of parallel alignment. The hydrophilic,
biocompatible nanobrous scaffolds made of PLACL/collagen
blend provide superior attachment and growth of adult cardiac
cells favoring native myocardiumlike alignment of newly seeded
cardiac cells compared to purely synthetic PLACL counterparts.
Moreover, PLACL/collagen allows cellcell interaction without
attenuating the functional activity of cells and cardiac specic
protein expression. These nanobrous scaffolds have an elastic
modulus of a magnitude nearing to that of native heart tissue.
Thus, our results indicate that PLACL/collagen is an attractive
biomaterial for myocardial regeneration that can be potentially
applied in cell based therapies for myocardial regeneration.
4. Experimental Section
Fabrication of Nanober Scaffolds : A solution of PLACL was prepared by
dissolving the polymer in 1,1,1,3,3,3-hexauoro-2-propanol (HFP) to form a
10% (w/v) clear solution. PLACL and collagen were dissolved and mixed in
HFP in a ratio of 1:1 (w/w) to form a 10% (w/v) solution. Both the solutions
were subsequently used for electrospinning to form random nanobers.
For electrospinning, the polymer solutions of PLACL and PLACL/collagen
were separately fed into a 3 mL standard syringe attached to a 27 G blunted
stainless steel needle using a syringe pump (KDS 100, KD Scientic,
Holliston, MA) at a ow rate of 1 mL h
1
with an applied voltage of 18 kV
(Gamma High Voltage Research, USA). On application of high voltage the
polymer solution was drawn into bers and collected on 15 mm cover slips
spread on collector plate wrapped with aluminum foil, kept at a distance of
1213 cm from the needle tip. These nanobers were dried overnight under
vacuum and used for characterization and cell culture studies.
Characterization of Nanober Scaffolds : The surface morphology of
electrospun nanobrous scaffolds was studied under eld-emission
SEM (FESEM; FEI-QUANTA 200F, Netherland) at an accelerating voltage
of 10 kV, after sputter-coating with gold (JEOL JFC-1200 ne coater,
Japan). Diameters of the electrospun bers were analyzed from the SEM
images using image analysis software (Image J, National Institutes of
Health, USA). Fourier-transform infrared (FTIR) spectroscopic analysis
of electrospun nanobrous scaffolds was performed on an Avatar 380,
(Thermo Nicolet, Waltham, MA, USA) over a range of 5003800 cm
1
at
a resolution of 2 cm
1
. Hydrophilic nature of the electrospun nanobrous
scaffolds was measured by sessile drop water contact angle measurement
using a VCA Optima Surface Analysis system (AST products, Billerica,
MA). Distilled water was used for a drop formation. The tensile properties
of electrospun brous scaffolds were determined using a tabletop tensile
tester (Instron 3345, USA) at 10 N load capacities. Rectangular specimens
of dimensions 10 mm 20 mm were used for testing at a rate of 5 mm
min
1
. The data were recorded at ambient conditions (25 C and 34%
humidity) and a tensile stressstrain curve was obtained. AFM analyses
were performed with a Nanoscope IIIa from Digital Instruments operating
in tapping mode at room temperature. A commercial silicon-tip cantilever
was used, with a stiffness of about 40 N. All the images were recorded as
is without any lter or image treatment. Height images were recorded at
90 scanning direction at a given set point and a xed frequency.
Isolation of Cardiomyocytes : Cardiomyocytes were freshly isolated from
adult rabbit hearts. The left ventricle of the heart was cleaned thoroughly
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with phosphate buffered saline (PBS) (1
st
Base) containing antibiotics
(Sigma) for removal of fat tissue and clots and minced into ne pieces.
The processed tissue was treated with 5 mg of collagenase (Sigma) for
20 min at 37 C. Freshly isolated cardiac cells were cultured in DMEM
media supplemented with 10% FBS and 1% antibiotic and antimycotic
solutions (Invitrogen Corp, USA) in a 75 cm
2
cell-culture ask. Cells were
incubated at 37 C in a humidied atmosphere containing 5% CO
2
for
6 days and the culture medium was changed once in every 2 days.
Cardiomyocyte Culture on Nanobers : Each of the nanobrous scaffolds
on 15 mm coverslip was placed in a 24-well plate and secured with a
stainless steel ring to ensure complete contact of the scaffolds with
wells. The specimens were sterilized under UV light, washed thrice with
PBS and subsequently immersed in DMEM overnight before cell
seeding. Cardiomyocytes grown in 75 cm
2
cell culture asks were
detached by adding 1 mL of 0.25% trypsin (Sigma) containing 0.1%
EDTA. Detached cells were centrifuged, counted via the trypan blue
assay using a hemocytometer and seeded on the scaffolds at a density
of 2.0 10
4
cells well
1
and left in incubator for cell growth.
Cell Morphology Studies : Morphological studies of in vitro cultured
cardiomyocytes on nanobers were performed after 5, 10, and 15 days of cell
culture, by processing them for SEM studies. The scaffolds were rinsed twice
with PBS and xed in 3% glutaraldehyde (Sigma) for 3 h. Thereafter, scaffolds
were rinsed in DI water and dehydrated with increasing concentrations
of ethanol (30%, 50%, 70% 90%, 100%) twice for 10 min each. Final
washing with 100% ethanol was followed by treating the specimens with
hexamethyldisilazane (HMDS). The HMDS (Sigma) was air-dried by keeping
the samples in a fume hood. Finally, the scaffolds were sputter-coated with
gold and observed under SEM to observe the morphology of cardiomyocytes.
Proliferation Studies : The cell adhesion and proliferation on the
scaffolds were determined using the colorimetric MTS assay (CellTiter 96
AQueous One solution, Promega, Madison, WI). The reduction of yellow
tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2(4-sulfophenyl)-2H-tetrazolium] in MTS to form purple formazan crystals
by the dehydrogenase enzymes secreted by mitochondria of metabolically
active cells forms the basis of this assay. The formazan dye shows
absorbance at 492 nm and the amount of formazan crystals formed is
directly proportional to the number of cells. For the MTS assay, samples were
rinsed with PBS to remove unattached cells and incubated with 20% MTS
reagent in a serum-free medium for a period of 3 h at 37 C. Absorbance
of the obtained dye was measured at 490 nm using a spectrophotometric
plate reader (FLUOstar OPTIMA, BMG lab Technologies).
Immunouorescence : Cardiomyocytes grown on nanobers were
washed with PBS, and xed with 100% chilled methanol for 10 min. The
cardiomyocytes were permeabilized with 0.1% Triton X-100, blocked for
nonspecic staining using 3% BSA and then immunostained with anti-alpha
actinin, anti-troponin T, anti-cardiac myosin heavy chain (Abcam) and anti-
connexin 43(Sigma) for 1 h at room temperature. Then, they were revealed
with anti-species-specic Alexa Fluor 488 or 594 secondary antibodies for
30 min and DAPI (Invitrogen). The samples were mounted using anti-
quenching mounting medium (Vectashiled, Vector Laboratories) and
observed under a confocal microscope (Olympus Fluoview FV1000). The
images taken were analyzed using Olympus FV10-ASW imaging software.
Statistical Analysis : All the data presented are expressed as mean
standard deviation (SD) and were analyzed using Students t -test for the
calculation of signicance level of the data. Differences were considered
statistically signicant at P < 0.05.
Acknowledgements
The authors gratefully acknowledge the NRF-Technion (Grant No.:
R-398-001-063-592), Division of Bioengineering, Nanoscience and
Nanotechnology Initiative, National University of Singapore for nancial
support.
Received: November 18, 2010
Published online: Published online: April 26, 2011
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2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 2300

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www.MaterialsViews.com
wileyonlinelibrary.com Adv. Funct. Mater. 2011, 21, 22912300