Download Engineering Myocardial Tissue

Reviews
This Review is part of a thematic series on Cardiovascular Tissue Engineering, which includes the following
articles:
Custom Design of the Cardiac Microenvironment with Biomaterials
Heart Valve Tissue Engineering
Engineering Myocardial Tissue
Small-Diameter Artificial Arteries Engineered In Vitro
Regenerative Cardiomyocytes for Cardiovascular Tissue Engineering
Richard T. Lee, Guest Editor
Engineering Myocardial Tissue
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Thomas Eschenhagen, Wolfram H. Zimmermann
Abstract—To create an artificial heart is one of the most ambitious dreams of the young field of tissue engineering, a dream
that, when publicly announced in 1999 (LIFE initiative around M. Sefton), provoked as much compassion as scepticism
in the scientific and lay press. Today, it is fair to state that the field is still far away from having built the “bioartificial
heart.” Nevertheless, substantial progress has been made over the past 10 years, and a realistic perspective exists to
create 3-dimensional heart muscle equivalents that may not only serve as experimental models but could also be useful
for cardiac regeneration. (Circ Res. 2005;97:1220-1231.)
Key Words: tissue engineering 䡲 heart 䡲 stem cells 䡲 regeneration
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preliminary (for review of the “bioartificial heart,” see also
ref 10). This review will give an overview on the evolution of
cardiac tissue engineering and today’s state-of-the art concepts. Tissue engineering heart valves and blood vessels will
not be covered here because these are topics of other reviews
in this Series.
he term “Tissue Engineering” was introduced in 1987 by
members of the US National Science Foundation (NSF)
in Washington, D.C. and defined a year later at an NSForganized conference on tissue engineering in Lake Tahoe,
California as “Application of principles and methods of
engineering and life sciences toward fundamental understanding of structure–function relationship in normal and
pathological mammalian tissues and the development of
biological substitutes to restore, maintain, or improve
functions.”
The “repair” part of tissue engineering overlaps, but is not
synonymous with, “cell therapy” which intends to promote
the formation of new tissue or to improve the function of an
existing tissue by injecting or infusing suspensions of isolated
cells. This concept has gained much attraction over the past
years and is currently tested in controlled clinical trials (for
review see references 1– 6). Tissue engineering aims at generating functional 3D tissues outside of the body that can by
tailored in size, shape, and function according to the respective needs before implanting them into the body. First clinical
experiences have been published using bioengineered skin,
cartilage, and vascular grafts,7–9 but the present data are still
History of Cardiac Tissue Engineering
The spontaneous beating of heart tissue explants and their
cellular outgrowth when placed in culture dishes under
suitable conditions have fascinated researchers for generations.11–15 Cardiac tissue engineering has several roots: developmental biology, cardiac muscle cell biology, and material science. The first man-made 3D heart tissues have been
generated long before the term tissue engineering was introduced.11–15 In the late 1950s, Moscona generated spheroid
aggregates from embryonic chick heart cells by cultivating
freshly isolated cells in Erlenmeyer flasks under continuous
gyration (Figure 1A).16 After 18 hours in this simple bioreactor aggregates formed containing up to 200 cells. Many
researchers adapted Moscona’s model and found that the
aggregates were functionally more similar to intact heart
Original received July 5, 2005; revision received September 20, 2005; accepted November 3, 2005.
From the Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Germany.
Correspondence to Thomas Eschenhagen, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail t.eschenhagen@uke.uni-hamburg.de
© 2005 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000196562.73231.7d
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Engineering Myocardial Tissue
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Figure 1. Strategies to generate 3D striated muscle tissues in vitro. A, Phase contrast microscopic view of aggregates generated by
gyratory shaking of embryonic chick heart cells in an Erlenmeyer flask.16 B, Macroscopic view of a device to stretch skeletal myocyte
monolayers to generate thin strips of 3D muscle (top) and immunostaining of myosin heavy chain (bottom).23 C, Scheme of a method to
generate fibroblast-populated collagen matrices from chick fibroblasts.30 D, Macroscopic (top) and microscopic view (bottom) of synchronously beating and force developing cardiac myocyte-populated collagen matrices anchored between two Velcro-coated glass
tubes.31 E, Scheme to prepare and culture polyglycolic acid-based cardiac constructs in a bioreactor (top) and microphotograph (bottom) of construct.37 F, Electron microscopic photograph of alginate matrix (top) and microphotograph (bottom) of an H&E stained
alginate-based cardiac construct.39 G, Macroscopic view of the setup to cast, culture and phasically stretch engineered heart tissue
(EHT) from neonatal rat heart cells and to determine contractile force (top and middle) and microphotograph of H&E stained EHT (bottom).35 H, Macroscopic (top) and microscopic view (H&E stain bottom) of stacked monolayers forming a beating 3D tissue.18 I, Tissue
formation (H&E stain, top) and sarcomere structure (bottom) in electrically stimulated collagen⫹matrigel-based constructs from neonatal rat heart cells.42
tissue than standard 2D-monolayer cultures (Figure 1A).17
This early work proved that isolated cells from immature
hearts retain the capacity to reform heart-like tissues under
cell culture conditions.
Another fascinating finding supports the notion that cardiac myocytes have an inherent preference to aggregate.
When cultured at high density for extended time periods in
serum-containing culture medium researchers often see that
entire rhythmically contracting monolayers start to detach
from the culture surface forming spontaneously beating
cardiac sheets floating in the dish. Because these sheets,
devoid of mechanical load, quickly retract and stop to beat,
the detachment has been considered a shortcoming of cell
culture experiments rather than an exploitable feature until
Shimizu and colleagues have used free floating monolayer
sheets to generate essentially exogenous matrix free cardiac
tissue constructs.18 In fact, in the late 1980s, Vandenburgh
and colleagues searched for a solution to overcome the
detachment problem of differentiated skeletal myotubes
(showing a similar contractile behavior as cardiac myocytes)
and came up with the idea to cover the cultured myotubes
with a layer of freshly neutralized collagen type I to chronically impose load on the cells.19 This led to an improved
differentiation state of the myotubes. Similar observations
had actually been made in various other cell types before,
either by placing collagen on top of or below cellular
monolayers or by embedding cells in collagen gels. For
example, epithelial thyroid cells cultured in collagen organized into follicles,20 and kidney and mammary gland epithelial cell lines formed lumina resembling those of the tissues
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from which they were derived.21 Collectively, these data
nicely demonstrate that the 3D environment in a collagen gel
promotes tissue formation and cellular differentiation of
various cell types in vitro.
Besides cell composition and differentiation, tissues are
also defined by cell orientation. In this respect, two factors
have been shown to be important: Mechanical strain and
orientation of the growth substratum. The groups of Vandenburgh and Terracio independently designed motorized
computer-controlled devices to impose stretch on cultured
skeletal and cardiac myocytes and showed that mechanical
stimulation had a favorable effect on muscle cell orientation
and differentiation.19,22 When repetitive cycles of stretch and
relaxation were imposed on skeletal myotubes cultured at
very high density, thin, 3D strips of longitudinally aligned
and well developed skeletal myofibers developed.23The strips
even initiated tendon development and performed work in
response to depolarization, and therefore may be considered
to be the first engineered 3D skeletal muscle (Figure 1B). An
important conclusion from this work was that mechanical
load is crucial for the orientation and differentiation of
(skeletal) muscle cells and muscle tissue development.
Others showed that cell growth can be influenced by the
underlying matrix substrate. For example, when a scraper was
drawn across the surface of culture dishes coated with freshly
neutralized collagen type I before gelation, Simpson and
colleagues observed that neonatal rat cardiac myocytes
aligned themselves in parallel with the collagen and adopted
a tissue-like organization.24 High magnetic fields have been
used as an alternative means to orient collagen,25 and
Tranquillo and colleagues showed that this method can be
used to improve longitudinal orientation of fibroblasts26 and
smooth muscle cells.27 Others used photolithographic patterning of culture surfaces to govern the growth of cardiac
myocytes in specific patterns28 or biodegradable elastomeric
polyurethane films patterned by microcontact printing with
laminin lanes.29
Another approach to cardiac tissue engineering originated
in the search for an improved in vitro heart model for target
validation that would allow both the measurement of contractile force and genetic, pharmacological, and mechanical
manipulations under controlled conditions in vitro. The solution was an adaptation of a method previously developed for
embryonic fibroblasts by Kolodney and Elson in St. Louis
(Figure 1C).30 Essentially, cardiac myocytes were cultured in
collagen gels as described above,19 –21 but between two
Velcro covered glass tubes that were positioned in a rectangular well and held at a fixed distance with a metal spacer.
Under these culture conditions the cells formed spontaneously contracting biconcaval lattices that were anchored on
the Velcro covered glass tubes (Figure 1D) and could be
attached to mechanical force transducers to record contractile
force.31 Neonatal rat cardiac myocytes initially failed to
exhibit differentiation, growth, and tissue formation inside
collagen I gels as described previously.32 Only after addition
of extracellular matrix from Engelbrecht Swarm tumors
(Matrigel) to the initial reconstitution mix, engineered heart
tissue (EHT) could be generated also from rat heart cells.33
Interestingly, chronic cyclic stretch of the EHTs improved
contractile force by a factor of 3 and induced better cardiac
tissue development.34 A further improvement was the introduction of circular casting molds that (1) allow large scale
production with minimal handling and are reusable, (2) can
be easily miniaturized for high through-put screening, and (3)
lead to better tissue formation than the original lattice design
because the circular geometry causes a homogeneous force
distribution throughout the tissue (Figure 1G).35 A main
conclusion from these observations is that, similar to what has
been found before by Vandenburgh and Terracio and colleagues, mechanical load is crucial for a proper heart tissue
development.
Two other, principally different tissue engineering approaches were developed in parallel. One originated from
the material sciences field and can be viewed as an
adaptation of “classical” tissue engineering principles36 to
cardiac cells. One of the pioneering groups (VunjakNovakovic and colleagues at the MIT in Cambridge, MA)
initially used polyglycolic acid as a scaffold in combination with bioreactor cultures (Figure 1E).37 Alternatively,
the group of Li seeded fetal rat ventricular cells onto
gelatin scaffolds, cultured them for 7 days in vitro and
implanted them onto cryo-infarcted rat hearts.38 Spontaneous contractile activity, both in vitro and in vivo, was
reported, but the histological microphotographs showed
mainly cells of unknown identity embedded in the scaffold
material and poor sarcomere development in the few
cardiac myocytes present in the construct. Leor and colleagues generated alginate scaffolds seeded with fetal
cardiac cells and implanted these graft onto rat hearts
(Figure 1F).39 The constructs were highly vascularized, but
did not exhibit true integration into the host myocardium.
The conceptionally important advantage of the classical
tissue engineering approach using preformed matrices as
compared with the liquid matrix cell entrapment approach
is that technically fabricated, solid materials can be shaped
in any 3D geometric form, on a macroscopic, but also, at
least in theory, at a microscopic level. Thus, one could
fabricate the ideal patch by computer design and then seed
it with cardiac myocytes and potentially other cell types,
hoping that they would use the matrix as a scaffold to
generate a tissue similar to the native heart. In essence, this
strategy either uses the natural organ as a blueprint
(exemplified in the famous “ear-mouse”40) or decellularized grafts that are already successfully utilized in clinical
use such as porcine heart valves. One could term this latter
approach “biologization” of tissue grafts. The great promise of these strategies with regard to cardiac muscle tissue
engineering remains unproven, so far. Limitations include
unfavourable matrix properties such as limited diffusion
capacity, low mechanical compliance, liberation of potentially toxic substances during degradation, and incompatibility with physiological cell growth. Essentially, cardiac
myocytes appear to survive for some time and start to beat
inside such artificial scaffolds, but remain largely isolated
and do not form coherently beating cardiac tissue. New
materials, the molecular refinement of surfaces and creation of microstructures that promote spreading of cells
may offer solutions to this problem (review in reference
Eschenhagen and Zimmermann
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41). Alternatively, Vunjak-Novakovic and colleagues successfully developed a somewhat mixed approach by seeding preformed collagen sponges/foam with neonatal rat
heart cells suspended in Matrigel together with electrical
stimulation for extended times.42 This led to the generation
of cardiac muscle constructs with improved cardiac tissue
morphology, contractile function, and molecular marker
expression when compared with nonstimulated cultures
(Figure 1I). Hence, electric stimulation seems to induce a
similar degree of cardiac myocyte differentiation as mechanically stimulation.34,35 Whether electrical activity per
se is a stimulus for advanced differentiation and tissue
formation or the resulting contractile activity is not clear
and may be difficult to experimentally separate. Yet, the
work by Radisic et al also supports the notion that
biological extracellular matrices mainly composed of collagen and laminin (present in high concentration in matrigel) are presently the optimal scaffold material in cardiac
tissue engineering.
The third approach can be termed tissue engineering
without matrix and is a refinement and systematization of
the phenomenon of detachment of cellular monolayers
after prolonged culture time (see above). Shimizu and
colleagues used a thermo-sensitive surface coating for
culture dishes which allows cardiac myocytes (or any other
cell type) to attach at 37°C as in other normal dishes, but
to detach under controlled conditions at room temperature
as an intact sheet.18 These beating cardiac myocyte monolayers can be stacked on one another and grown together to
form interconnected 3D tissue sheets of up to 50 to 75 ␮m
thickness (Figure 1H). The advantages of this technique
are its relative ease and the independence from potentially
immunogenic or pathogenic scaffold materials. Disadvantages are its fragility (leading to handling problems),
restriction in terms of geometric form and difficulties to
impose mechanical load on the contractile sheets.
Other studies have described variations of the above
techniques. One report described generation of coherently
beating aggregates from neonatal rat heart cells by exposing
cell suspensions with fibronectin coated polystyrene microcarrier beads or oriented collagen fibers to microgravity in
bioreactor cultures.43 Other studies seeded commercially
available collagen sponges with neonatal rat heart cells alone
and observed cardiac tissue formation.44 Van Luyn and
colleagues experimented with liquid collagen type I matrices
and used a bioreactor to improve tissue formation.45 The
group of Dennis developed a technique that allows spontaneously detaching heart cell monolayers to form cylindrical
tissues spanning between two laminin-coated silk sutures.45a
Finally, another strategy may be to create new cardiac tissue
by injecting liquid, “cardiogenic” cell-matrix mixtures into
the heart, either with or without cells.46 This “tissue engineering in situ” is based on the premise that suitable matrices
provide appropriate signals for cardiac progenitors such as
c-kit,47 sca-1,48 and isl-149 positive cells to proliferate and
form new cardiac tissue. The idea is interesting, but has to be
proven more rigorously.
Taken together, various techniques have been developed
over the past 10 years that allow engineering of beating 3D
Engineering Myocardial Tissue
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cardiac tissues. Such tissues develop force and can be formed
at different shapes and sizes in a directed manner.
How Far Has Cardiac Tissue Engineering
Gone and What Are the Present Limitations?
Tissue Development
It has been questioned whether the term “tissue” may actually
be used for engineered myocardial constructs. This argument
is based on the classical definition of a “tissue” as being “a
part of an organism consisting of an aggregate of cells having
a similar structure and function.” Yet other widely used
definitions state that a tissue is “a group of similar cells united
to perform a specific function” or “a grouping of cells that are
similarly characterized by their structure and function,” a
definition that applies to engineered tissue constructs. In fact,
use of stretch34 or electrical stimulation42 and mixed populations of heart cells including cardiac myocytes, fibroblasts,
smooth muscle cells, endothelial cells, macrophages among
others35 in cardiac tissue engineering leads to the formation of
heart muscle constructs with a high degree of organotypic
differentiation on the single cell level and also a high native
tissue-like cellular complexity. The cellular complexity can
be further enhanced by using unpurified heart cell populations instead of purified cardiac myocytes.50 Cardiac myocytes themselves showed many structural features of terminal
differentiation including well-developed sarcomeres, desmosomes, gap junctions and fasciae adherentes. Interestingly,
T-tubule-sarcoplasmic reticulum junctions were found regularly that are not or only rarely observed in neonatal rat
hearts.51 Interestingly, EHTs developed approximately twofold higher contractile force if generated from an unpurified
cell preparation than from a relatively pure cardiac myocyte
fraction.50 Similarly, endothelial cells can promote cardiac
myocyte survival and spatial organization when cocultured
on hydrogels.52 These in vitro data are well in line with the
established role of the endocardial endothelium for normal
cardiac development,53,54 where disruption of normal endothelial cell function led to severe cardiac malformation.
Collectively, these data suggest that endothelial cells, smooth
muscle cells, fibroblasts, macrophages and other noncardiomyocytes play an important or even essential role in the
formation of engineered cardiac tissue grafts.
Important structural characteristics of cardiac myocyte
terminal differentiation are sarcomere organization in registry
with a cross-striation pattern including M-bands,55 formation
of cell-cell contacts through desmosomes, gap junctions, and
fasciae adherents, as well as development of T-tubulesarcoplasmic reticulum junctions (dyads) at the Z-band level.
These features have been observed in engineered cardiac
constructs that have been chronically stretched35 or electrically stimulated42 indicating that these conditions promote
terminal cardiac differentiation. Interestingly, M-band formation was found to be enhanced after engraftment of EHTs
indicating that additional differentiation factors are provided
in the intact adult heart that are lacking in vitro.56
Thus, critical factors identified today for cardiac tissue
development in vitro are (1) a suitable biological matrix such
as collagen I, IV and laminin (which, in case of the sandwich
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technique of Shimizu, may be entirely cell-derived), (2)
mechanical load, (3) a total heart cell mix, and (4) either
electrically stimulated or spontaneous contractile activity. In
summary, the various techniques presently used generate
constructs with a very high degree of cardiac tissue development (Figure 1E, 1H, and 1I).
Contractile Function
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Also functionally, artificial heart constructs resemble intact
heart tissues in terms of force-frequency behavior, forcelength relationship (Frank-Starling mechanism), response to
extracellular calcium and the ␤-adrenergic agonist isoprenaline as well as its antagonism by acetylcholinederivatives.31,33 These data support the above conclusion of
true heart tissue-development in vitro from a functional point
of view. However, differences exist as well. For example,
EHTs exhibit a higher calcium sensitivity, and absolute forces
remain lower than in the intact heart. Initially, the maximal
twitch tension was 0.5 to 1 mN, optimized EHTs today reach
up to 4 mN. Others reported active forces between 0.05 mN44
and 1 to 2 mN18 (overview in reference 50). Values of 4 mN
are similar to the force of intact muscle preparations such as
rat papillary muscles, but experiments in the latter underestimate maximal forces developed by adult, mature cardiac
myocytes due to core ischemia. Very thin adult rat heart
muscle strips develop forces of 56 mN/mm2.57 The diameter
of mature rat EHTs is 0.8 mm, therefore the diameter of the
two sides of the rings together is 1 mm2. Thus, the normalized
optimal force of EHT is 4 mN/mm2. The difference of EHT
force generation as compared with mature heart muscles most
likely reflects both a quantitative and a qualitative aspect, as
for example a lower fractional occupancy of the EHT tissue
by cardiac myocytes and the lower degree of sarcomere
development. Both are likely to be important for tissue
formation in the absence of perfusion in vitro, but are, at least
partly, overcome after implanting onto the heart.56
Critical Size
One of the central unresolved problems of all tissue engineering approaches is the limitation of maximum size which is
defined by maximum diffusion distances for nutrients and
oxygen. Indeed, none of the various tissue engineering
approaches developed today generate cardiac tissue-like,
contracting constructs in which compact muscle strands
exceed 50 to 100 ␮m. Early tumor angiogenesis studies have
shown that, in the absence of capillarization and perfusion,
tumors implanted into nude mice did not reach a size of more
than 2 to 3 mm.58 These values, however, depend both on the
metabolic demand and the cellular density of the respective
tissue. Beating cardiac myocytes obviously have a very high
metabolic activity. Accordingly, the density of capillaries in
the adult heart is very high, amounting to approximately 2400
to 3300/mm2 in rat and human at different postnatal stages
(intercapillary distance ⬇19 to 20 ␮m).59,60 On the other
hand, the early embryonic rat heart (until ED 16) as well as
the adult frog heart are avascular and nourished exclusively
from the lumen by blood circulating within the trabecular
system.61,62 Thus, large heart muscles either develop physio-
logically through vascularization or through intense trabecularization with single strands smaller than 50 to 75 ␮m. The
latter pattern is recapitulated in EHTs, in which the network
of cardiac myocytes strands is loose in most parts and
consists of muscle bundles with a diameter of 30 to 50 ␮m
(see Figure 2 in reference 35). Only in some parts compact
muscle strands form that reach up to 100 ␮m. Similarly, 3 to
4 cardiac myocyte monolayers can be successfully stacked,
more did not improve the thickness of tissue constructs.18
Some groups increase oxygen and nutrient delivery by
cultivating constructs in bioreactors, increasing ambient oxygen concentrations and/or by adding oxygen carriers such as
perfluorocarbon.63,64 Indeed, such optimization of culture
conditions has had beneficial effects on cell density and
metabolic activity.63 and has been modeled to allow construction of cardiac tissues with a clinically meaningful thickness
of up to 500 ␮m.64 Another strategy is to incorporate a
perfused natural vessel into engineered heart muscle constructs65 or to stimulate angiogenesis in cardiac tissue constructs. In EHTs, primitive capillaries and larger vessel-like
structures lined with CD31 positive endothelial cells develop
spontaneously.35 It seems unlikely that these vessel structures
play a significant role in oxygen and nutrient exchange under
in vitro conditions, but they may facilitate perfusion after
implanation. Levenberg and colleagues have recently mixed
endothelial cells and fibroblasts to a culture of skeletal
myoblasts on a synthetic polymer and observed generation of
skeletal muscle constructs with increased density of vascular
structures.66 A third strategy to overcome size limitations in
vitro is to weave several EHTs or comparable constructs
together, thus forming a network in which each individual
construct remains accessible for unlimited diffusion and
exchange of nutrients. Large networks could be useful as an
improvement of the “ACORN” technology (CorCap Cardiac
Support Device), a therapeutic device currently tested in
patients with terminal heart failure with predominant ventricular dilatation.67 Such a “chain mail” biological network
(Figure 2) could have the advantage over the synthetic
CorCap Cardiac Support Device in that it could support both
diastolic and systolic function of a failing heart. Taken
together, several potential strategies have been developed to
overcome size limitations, both in vitro and after implantation
in vivo. Yet, critical size remains an unresolved problem and
its solution is one of the most important tasks in the field.
Implantation Studies
The early studies by Li and Leor indicated that cardiac
myocyte-populated gelatin and alginate-grafts, respectively,
were visible for extended time after implantation as a patch
onto the heart and contained cells, despite the absence of
immunosuppression.38,39 No definite proof for the development of new cardiac tissue was provided. Shimizu et al
demonstrated survival and vascularization of their sandwich
constructs and recorded beating activity after implantation
into nude rats.18 Survival, vascularization and terminal cardiac differentiation of cardiac myocytes was observed in
EHTs after implantation onto rat hearts in the presence of
immunosuppression.56 Even though these data collectively
Eschenhagen and Zimmermann
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Figure 2. Perspectives of cardiac tissue engineering exemplified with the EHT technology. Constructs will be made from ES cells that
are ideally autologous, derived from a nuclear transfer strategy, or from adult cardiac progenitors that may be obtained from a heart
muscle biopsy. By miniaturizing the setup and combining casting, culturing, and force-measurement in one 96-well plate, engineered
cardiac tissues can be used for drug screening and target validation. For cardiac regeneration, several circular constructs can be
woven together to form a multi-loop construct. Several multi-loop constructs together can form large “chain-mail”–like networks for the
treatment of dilated cardiomyopathy or a patch for the treatment of myocardial infarction.
suggest that constructs are rapidly vascularized after implantation and that hypoxia during and after implantation is not a
major problem, several important questions remain unanswered: Is vascularization sufficient? How many of the
cardiac myocytes initially implanted survive over time? Do
the constructs couple electrically and mechanically to the host
myocardium? Does the implantation of cardiac constructs
beneficially affect cardiac performance? The early studies
reported improved function as evidenced by enhanced developed pressure in isolated Langendorff-perfused hearts38 and
improved left ventricular dimensions as well as fractional
shortening.39 However, the effects were generally small and
similar effects have been reported in numerous cell injection
studies, apparently independent of the cell type injected.
Thus, the specific functional contribution of grafting 3D
constructs as compared with cell injection remains unclear at
present. Thorough evaluation of this issue is indispensable for
the further development of the field.
Future Developments
Matrix Materials
As outlined above, the optimal scaffold for engineering
cardiac muscle tissue has not yet been found. Alginate,
gelatin, polyglycolic or polylactic acid, as presently used for
engineering cartilage, bone, ear, skin, blood vessels, or heart
valves, will not likely be the materials of choice. The reason
lies in the physiological properties of cardiac muscle tissue,
particularly the need for high mechanical stability coupled
with great compliance. Moreover, muscle fibers and bundles
in the heart form layers with different orientation to provide
optimal pump function. At present it is difficult to imagine
that any technically fabricated material will ever mimic such
sophisticated structure and even if it would do so, the cardiac
cells, stem cells or stem cell-derived cardiac myocytes still
have to grow into the free spaces, interconnect and thereby
form an electrical and mechanical syncytium. On the other
hand, material sciences and particularly nanotechnology are
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progressing rapidly and it may be that in a collaboration
between engineers, physicists, biotechnologists, biologists
and morphologists the ideal scaffold material for creating
truly “artificial” heart tissues will be designed.68,69 Recent
examples are tubular scaffolds for the engineering of blood
vessels that were generated by electrospinning of collagen
and elastin70 and collagen tubes for cardiac tissue engineering.71 Another innovative approach is to use MRI-derived 3D
images of an organ as a blueprint for the fully automated
fabrication of a synthetic copy.72 It will be interesting to see
whether these sophisticated methods in nanotechnology and
imaging will prove useful in creating optimized cardiac
constructs.
Cell Sources for Cardiac Tissue Engineering
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The greatest conceptional problem of cardiac tissue engineering (as for all cell-based therapies) relates to a suitable cell
source. It has been estimated that the adult human left
ventricle contains 5⫻109 cardiac myocytes,73 ie, 40 million
cardiac myocytes per gram of native myocardium. Thus, even
the creation of a small tissue patch would require tens of
millions of cardiac myocytes, a number impossible to obtain
from a primary human cardiac cell source, not to speak of the
ethical aspect. Thus, the primary cells used today for cardiac
tissue engineering can only serve as a proof-of-principle.
Great hope has been created by recent findings indicating that
stem cells exist in the adult organism that could give rise to
the formation of autologous cardiac myocytes as well as
endothelial and smooth muscle cells. Such cells have been
found in various tissues, including the bone marrow,74,75
peripheral blood,76,77 umbilical cord,78 and adipose tissue.79
Initial studies in mice showed that lin-, c-kit⫹ cells can be
isolated from bone marrow and regenerate new myocardium
when injected into the heart of recipient mice after myocardial infarction.75 These data prompted the initiation of clinical
studies in patients with acute myocardial infarction indicating
that such treatment is safe and may provide functional benefit
(review in reference 5). The first results of larger, randomized
and placebo-controlled studies are eagerly awaited in late
2005. Yet, recent studies in mice using similar cells as the
original study75 reported no cardiac regeneration at all.80 – 82
Thus, the capacity of bone marrow stem cells to regenerate
the heart is currently under intensive debate and a detailed
discussion is beyond the scope of this article (for review see
references 5 and 7).
New hope was raised by the demonstration of progenitor cells
resident in the heart that have the capacity to differentiate into
functional cardiac myocytes and repair the heart.47– 49,75,83 The
first report by Beltrami and colleagues described small, round
c-kit⫹ cells organized in niches in the adult rat heart that could be
clonally expanded and, when injected into infarcted hearts,
generated new viable myocardium.47 This study has been subsequently supported by data showing that a c-kit⫹ cell population
can be propagated from murine and human heart specimens and,
in case of murine cells, form spontaneously contracting cardiospheres even in the absence of coculture with primary heart
cells.84 Moreover, c-kit⫹ cells could be mobilized to regenerate
new myocardium in infarcted dog hearts by injecting IGF-1 and
HGF-1.83 Another study isolated sca-1⫹ cells from a total adult
murine heart cell population and found that these cells can
acquire a cardiac phenotype after treatment with 5⬘-azacytidine,
a compound known to induce muscle cell differentiation.48 By
using an extensive genetic labeling strategy it was also shown
that sca-1⫹ cells home to the injured myocardium when injected
intravenously and have the capacity to differentiate into cardiac
myocytes as well as to fuse with host cells. Subsequent studies
extended these findings showing that sca-1⫹ expressing cells can
be slowly expanded in vitro and, in the absence of primary heart
cells, differentiate to beating cardiac myocytes when treated with
oxytocine.85 Moreover, data were presented showing that cardiac differentiation of sca-1⫹ cardiac stem cells depends on
FGF-286 and that only a CD31--, isl-1--subset of sca-1⫹ cells may
actually represent cardiac precursors.87 Finally, the LIM domain
transcription factor isl-1 has been identified by genetic means to
mark a cardiac progenitor population of which few cells appear
to remain in the postnatal heart.49 Isl-1⫹ cells have also been
expanded and differentiated into contracting cardiac myocytes
under coculture with neonatal rat cardiac myocytes. Fusion was
made unlikely by pretreatment of myocytes with formaldehyde.
It is not clear at present whether c-kit, sca-1 and isl-1 indeed
label three different cardiac progenitor populations as indicated
by some of the above studies49,87 or whether expression of these
markers reflects the dynamic phenotype of the same one. In any
case, these exciting findings open the door not only to an
autologous cell therapy approach but also to the engineering of
autologous cardiac tissues. Such tissues would be ideal for
cardiac repair as being likely devoid of tumorigenic and immunological problems. However, tissue engineering from autologous stem cells has yet to be shown.
The alternative to adult stem cells or cardiac progenitors
are embryonic stem cells which are able to form virtually any
cell type of the body in vitro under appropriate conditions.88 –90 Genetically selected ES cell-derived cardiac myocyte form electrical connections with the host myocardium
when injected into the heart.91 The pluripotency of ES cells
may be of particular importance for cardiac tissue engineering when considering the role of noncardiomyocytes in EHT
formation (see above). Indeed, recent experiments showed
that spontaneously contracting EHTs can be generated from
mouse ES cells and produce forces similar to EHTs from
primary cardiac cells (own unpublished data). Human ES
cells possess principally the same features as mouse ES cells
including the propensity to spontaneously differentiate into
cardiac myocytes92–94 and a recent study showed that transplanted human ES cell-derived cardiomyocytes survived in
the swine hearts under immunosuppression and acted as a
rate-responsive biological pacemaker.95 The potential of cardiac differentiation apparently strongly depends on the cell
line, but also on the quantification algorithm. Some lines
seem to have a very limited or basically no potential for
cardiac differentiation at all. This raises questions with regard
to the present federal restrictions to a limited number of
already existing human ES cell lines (listed by the NIH at
http://stemcells.nih.gov/research/registry/). Moreover, it calls
for studies deciphering the mechanisms of cardiac differentiation from ES cells. On this basis pharmacological strategies
could be developed to direct cardiomyogenic differentiation.
Because ES cells can be easily and quickly propagated in
Eschenhagen and Zimmermann
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unlimited quantities, even the immense cell number theoretically needed to produce cardiac muscle patches to cure
human heart diseases appears a realistic option.
Major problems of ES cells are their immunogenicity96 and
their potential to form tumors.7,97 Data exist that ES cells at
least partly escape the normal immune responses98 and some
groups have even injected mouse ES cells into rat hearts and
found large numbers of surviving cardiac myocytes up to 32
weeks thereafter without evidence for tumor formation.99,100
In contrast, significant immune responses were seen after
injecting GFP-labeled mouse ES cells into allogenic mouse
hearts.96,101 The reasons for these discrepancies are not clear
at present, but it is evident that immune responses have to be
anticipated. Options to deal with this problem could be to
create ES cell banks containing immunologically diverse
phenotypes from which the best fit for the respective patient
could be chosen. And finally the option exists to derive
potentially autologous ES cells by nuclear transfer.102–104
The most significant problem of ES cells in regenerative
approaches is the problem of tumor formation as noted in
careful studies.7,101,105,106 Even if the risk can be minimized
by various means, it is likely that even 1 tumorigenic cell in
1 million can create problems in a cardiac repair approach in
which 100 million cells are needed. Thus, injection of
undifferentiated ES cells for cardiac regeneration must be
viewed with scepticism. Two principal strategies are currently used to reduce this risk. One is to positively select cells
that acquire a cardiac phenoytpe, for example by introducing
into the genome of an ES cell line a neomycin resistance gene
under control of a cardiac specific promotor.91 With this
strategy noncardiac myocytes can be eliminated before implantation of the cells/tissue by adding neomycin-derivatives
into the culture medium. Alternatively or in addition, it may
be necessary to introduce a suicide gene into the ES cell clone
such as herpes simplex virus thymidine kinase which makes
all undifferentiated potentially tumorigenic ES cell-derived
cells sensitive to drugs such as ganciclovir. This strategy
could be used as an emergency treatment if tumor formation
occurs. However, none of these genetic approaches is 100%
efficient or safe. In fact, any approach to alter a stem cells’
genome may by affected by genetic and epigenetic instability.107 Moreover, the genetic manipulation itself could create
new tumorigenic problems by deleterious integration of the
vector into the genome.108 It will therefore be essential to
apply rigorous tests to exclude such problems before any
application, because safety will be crucial for the success of
the entire ES cell concept. Other unresolved issues relate to
insufficient vascularization, one of the likely reasons for graft
size reduction after ES cell injection.101
Immune Response
All allogenic cell-based therapies face the problem of immunorejection and it is questionable whether patients and doctors would accept lifelong immunosuppression with potentially fatal consequences such as tumor development,
cyclosporine-induced renal failure, Cushing syndrome, and
osteoporosis. This issue is in fact a strong argument for
autologous stem cell therapies, most likely with resident
cardiac stem cells. Alternatively, ES cell-based approaches,
Engineering Myocardial Tissue
1227
will require either a way to circumvent immunorejection or
the opportunity to do therapeutic cloning, either with oocytes
derived from volunteers104,109 or from ES cells.110
Another somewhat neglected problem is that most of the
commonly used media supplements are xenogenic (eg, fetal
calf serum, horse serum, chick embryo extract, matrigel,
collagen). This results in several problems: Infectious risks,
nonadherence with standards of good laboratory and manufacturers practice (GLP, GMP), and the generation of immune responses even with autologous or syngenic cells. For
example, complete immune rejection was seen when EHTs
made from neonatal Fischer rats and Fischer rat tail collagen
I were implanted into adult syngenic Fischer rats.56 The most
likely explanation is that, despite extensive washing before
implantation, remnants of the chick embryo extract and horse
serum, or Matrigel had impregnated the genetically autologous cells or the extracellular matrix and provoked an
immune response. Another explanation could be induction of
self-antigens during prolonged culture. It will be necessary
therefore to identify suitable culture conditions for engineered heart tissues that are devoid of xenogenic growth
supplements. Some critical factors for cardiac myocyte cultures including epidermal growth factor, hydrocortisone,
L-thyroxine, albumin, selenium, and transferrin have already
been identified.111
Realistic Perspectives of Cardiac Tissue
Engineering in the Next 10 Years
Statements on the perspectives of science share the limitations of stock market predictions and remain essentially
speculations, founded on facts and spiced with personal
convictions. With this in mind, some predictions can be
made. The total artificial heart will likely remain fiction
for a while, but some important milestones toward this
ambitious goal have already been achieved as outlined
above. The following summarizes realistic accomplishable
achievements:
Drug Screening Assay Based on Engineered
Cardiac Constructs
Engineered cardiac tissues will be useful for drug development and target validation in a format that allows for
medium-to-high throughput assays (Figure 2). Experiments
are on the way to construct a 96-well plate ES-cell EHT
format to eventually generate a humanized experimental
model for drug research.
Serum-Free Culture Conditions for Cardiac
Tissue Grafts
Experiments to define such culture conditions are currently
being pursued in several laboratories. High throughput
screening systems as mentioned above will be helpful to
identify cardiogenic or cardiac growth supportive factors in
the right concentration and combination as well as the right
timing. Given the availability of serum-free media for many
other cell types there is little reason to assume that a similar
condition cannot be established for cardiac cells and tissues.
1228
Circulation Research
December 9/23, 2005
Prevention/Slowing of Remodeling After
Myocardial Infarction by Tissue Grafts in Large
Animal Models
A realistic perspective is that large networks composed of
several engineered cardiac grafts can be generated and
sutured around the heart. Such “biological chain-mail” as
exemplified with the EHT-technology in Figure 2 could be
designed both from primary cardiac and ES cells and tested in
rats, mice and pigs after myocardial infarction.
Correction of Heart Defects by a Contracting
Patch in a Large Animal Model
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Congenital heart defects such as complex Tetralogy of Fallot,
and aplastic right and left ventricles would profit from the
implantation of actively contracting engineered tissue
patches. This approach will likely be tested first in the low
pressure system of the right heart with patches made from ES
cell-derived myocytes.
Autologous Cardiac Graft From an Autologous ES
Cell Line Generated by Nuclear Transfer
Techniques to generate autologous blastocysts by nuclear
transfer and to produce autologous ES cell lines (ntES cell)
from these early embryos have been successfully established
over the past 5 years, in various animal species and recently
also in human.104 Thus, despite the present formal hurdles and
the ongoing ethical debate, it seems realistic to create autologous artificial heart muscle from ntES cell lines and test
their applicability in an animal model in the next years.
Autologous Cardiac Graft From Resident
Cardiac Precursors
Given the discovery of cardiac progenitors it should also be
possible to isolate such cells from a cardiac biopsy, amplify
and differentiate them into functional cardiac myocytes and
create a 3D tissue construct.
Conclusions
Cardiac tissue engineering is a relatively young, actively
developing field. Its great attraction, both for researchers and
the public, lies, first of all, in the fascinating natural capacity
of heart cells to form spontaneously beating, well organized
heart-like tissues in the culture dish—a finding that was made
more than 50 years ago. Apart from the naive enthusiasm this
phenomenon excites, there are good reasons to assume that
engineered cardiac tissues will be of practical use in the near
future, as an improved experimental model, as a model
system for cardiac development and, much later, as a therapeutic option for cardiac repair. The latter optimism is mainly
based on the enormous progress in stem cell research. What
has been pure fantasy 10 years ago now becomes a realistic
prospect—to create contracting heart muscles from the patients own stem cells and to use them to alleviate heart
disease. Many critical caveats remain, however, and intense
and stringent scientific work is mandatory, before such
strategies should head into the clinic. And finally, future
developments in mechanic devices, xenotransplantation, cell
therapy and not at last pharmacology may decide whether or
not cardiac tissue engineering will indeed find its place in the
practical therapy of heart disease.
Acknowledgments
The work of the authors has been supported by Bundesministerium
für Forschung und Technologie (BMBF 01GNO124 to T.E.), Deutsche Forschungsgemeinschaft (DFG Es 88/8-2 to T.E.), Deutsche
Stiftung für Herzforschung (W.H.Z.), and Novartis Foundation
(W.H.Z. and T.E.). We thank L.J. Field, Indianapolis, and L. Carrier,
Hamburg, for critical reading of the manuscript.
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Engineering Myocardial Tissue
Thomas Eschenhagen and Wolfram H. Zimmermann
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Circ Res. 2005;97:1220-1231
doi: 10.1161/01.RES.0000196562.73231.7d
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Correction
In an article by Eschenhagen et al (Circ Res. 2005;97:1220 –1231), the authors incorrectly cited
the source of Figure 1E. The top of Figure 1E is reproduced with permission from Bursac et al
(Am J Physiol Heart Circ Physiol. 1999;277:433– 444). The bottom of Figure 1E is reproduced
with permission from Papadaki et al (Am J Physiol Heart Circ Physiol. 2001;280:168 –178). The
authors apologize for this error.
e19