Fisher John P. e.a. (ed.) Tissue Engineering
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(4) architectural differences and relative position between implant and host tissue. Understanding how
normal functioning of the cardiac cell network changes with the presence of different types of cellular,
structural, and functional heterogeneities is a necessary step in the design of safer and more efficient cell
and tissue transplantation therapies.
For example, it is already known from computational and experimental studies that contact between
two cells with different resting potentials (e.g., injured and healthy cell [130]) or action potential durations
(e.g., purkinje and ventricular cell [131], epicardial and M cell [132]), may trigger afterdepolarizations or
create conduction block depending on the degree of their electrical coupling. It is also known that partial
decoupling between the cells may actually increase the safety of propagation [133,134] as is the case in
transverse vs. longitudinal propagation in healthy cardiac muscle [47]. On the other hand, excessive gap
junction decoupling [134], presence of fibrotic regions [135], geometrical expansions [136], and repetitive
tissue branching [137] (e.g., sparse cardiac network in ischemic or infarcted area [138]) can yield extreme
slowing of impulse propagation and occurence of conduction block and introduce the susceptibility of
cardiac tissue to formation of micro- or macroreentrant circuits. In the ideal world, the safest and most
efficient therapy would be to engineer a cardiac patch that exactly mimics the geometry, architecture, and
function of the “missing region” in the heart, and to perfectly couple it to the host tissue, as “a piece of
the jigsaw puzzle.” In the “semi-real” world, even if one is to produce a patch with perfect anisotropic
architecture, its optimal orientation relative to host tissue when implanted may change depending on
the electrical differences between the donor and host cells, or capability of patch to couple with the host
tissue. In the real world, things are very complex due to interplay of many factors. For example, the use of
skeletal myoblasts or stem cell-derived cardiomyocytes may demand different patch design and different
implantation strategy as determined by the functional differences between these cell types (e.g., shorter vs.
longer action potential, different resting potentials, none vs. significant expression of gap junctions, etc.).
The simulation may be further complicated by the presence of a fibrous capsule at the implantation site.
Due to the complexity of the problem, carefully designed studies on possible implantation scenarios will
be crucial as a prescreening tool before the actual implantation. The author’s view is that there are at least
two simplified settings that could be used to systematically and reproducibly study the factors affecting the
likelihood of arrhythmia arising from cell and tissue cardiomyoplasty. One is the use of micropatterned
cocultures [139,140] of host cardiomyocytes and different types of donor cells or mixtures of donor
cells, with the possibility for well-controlled studies based on (1) simplified cellular composition and
tissue architecture compared to the 3D heart, (2) precise control of the cell microenvironment, cellular
geometry and distribution, and geometry of cellular interactions between donor, host, and donor and
host cells, and (3) the possibility to optically assess and exactly correlate electrical/mechanical activity
and underlying tissue architecture at microscopic and macroscopic spatial scales. Optical mapping of
transmembrane potentials and intracellular calcium [141] are well-suited not only for these studies, but
also for electrophysiological evaluation of engineered cardiac patch pre and postimplantation ex vivo.
The other setting is the use of computer models that incorporate cell-specific membrane properties, cell
geometry, distribution of intercellular connections, and discrete tissue microarchitecture. With increase
in computing efficiency, these detailed models could be used as counterparts to different in vitro or in vivo
implantation scenarios to help interpretation and design of experiments, and eventually yield safe and
efficient therapies.
27.4.6 Spontaneous Activity
Another electrophysiological parameter for consideration is a somewhat misinterpreted presence of
spontaneous contractile activity in cardiac cell cultures. It is important to understand that although
spontaneous contractions in cardiac cultures represent convenient way to visually identify cardiac cells,
they are by no means a physiologically normal state for adult or neonatal ventricular muscle (which is most
often the source tissue for cell dissociation). While early embryonic ventricular cells still spontaneously
depolarize and contract [142], their resting potential hyperpolarizes with maturation such that neonatal
ventricular cells already exhibit steady resting potentials (less than −70 mV), fast action potential upstroke
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Cardiac Tissue Engineering 27-15
(more than 100 V/sec), and no spontaneous activity [76,142,143]. Rather, spontaneous activity of cardiac
cells observed in vitro is an artifact of the cell culture possibly caused by (1) membrane depolarization
due to cell injury during or after dissociation, (2) dedifferentiation of cultured cells to more embryonic
state due to presence of high serum and inadequate media formulation, (3) large presence of nodal or
other pacemaking cells due to unselective cell dissociation, and/or (4) decreased intercellular coupling
compared to native tissue due to 2D or sparse 3D arrangement of ventricular cells. The first three causes
can be alleviated with time in culture, proper cultivation conditions, and careful cell dissociation. How-
ever, the sparse cardiac network, if present, will always result in less electrotonic load “seen” by a cell and
facilitate propagation from single or small group of pacemaking cells into the quiescent tissue, favoring
spontaneous activity [144,145]. Our observations that spontaneous contractions in cardiac constructs
can be more readily observed when cells are less coupled (e.g., at lower seeding density, in the beginning
of cultivation, or on the perturbing polymer fibers at the edges of the tissue construct) are in agreement
with this reasoning. After 5 to 7 days in culture, tissue constructs in our studies usually become quiescent,
presumably due to increased packing density and connectivity of cardiac cells. The low percentage of
nonmyocytes in tissue constructs as assessed by immunostaining, and increase in conduction velocity
between culture days 4 and 7, exclude fibroblast overgrowth as a possible cause of cardiac cells quiescence.
Although reported spontaneous beating rates in cardiac constructs are usually lower than regular heart
rate, presence of paracrine chronotropic factors (e.g., epinephrine, adrenaline) after implantation in
the heart may result in faster activity and possible arrhythmogenic hazard. For electrical safety reasons,
a nonstimulated ventricular cardiac patch should be electrically quiescent, but readily excitable and fast
conducting, similar to native ventricular muscle. Moreover, a quiescent avascular patch may have lower
metabolic demand and increased chance for survival after implantation compared to a spontaneously
contractile patch. However, persistent contractions and presence of mechanical load are essential for
maintenance of ventricular mass in vivo [146], and for cell hypertrophy [147], spreading to confluence
[148], and establishment of cell contacts in vitro [149,150]. (Our experience is that if cardiac cell culture
is noncontracting for more than 3 days, cells start to atrophy and loose cell contacts.) Therefore, spontan-
eously quiescent engineered cardiac constructs still need to be maintained mechanically active in culture
either by use of electrical, mechanical, or chemical (e.g., small concentrations of epinephrine) stimulation
[150], or by coculture with spatially distinct population of pacemaking cells, which can be easily dissected
before the implantation in the ventricle.
In any case, thorough evaluation of electrical properties and susceptibility to conduction block and
arrhythmic behavior should be routinely done in cultured cardiac constructs and used as one of the
tissue design criteria. From our experience, it is important to assess the properties of engineered tissue in
challenging regimes such as fast or premature stimulation, similar to standard clinical pacing protocols for
testing the susceptibility to arrhythmias [151]. Forexample, a tissue construct can support macroscopically
continuous and relatively fast electrical propagation at low pacing rates, but yield conduction blocks and
high incidence of reentrant arrhythmias [122] when paced rapidly (Figure 27.4) due to large spatial
dispersion of refractoriness, or abundance of microscopic anatomic heterogeneities.
27.5 Future Work
The crucial technical aspect of this and all other cell-based therapies will be a choice of appropriate cell
source, which is to be primarily determined by developmental biologists, geneticists, and immunologists.
Engineering of a functional cardiac tissue patch using embryonic or adult stem cells and subsequent
implantation studies are still to be done. Simultaneous efforts on design of appropriate scaffolds and
control over the cellular connectivity and tissue architecture in three dimensions will be essential to the
development of functional cardiac patch for use in laboratory and clinics. Systematic in vitro and in vivo
studies on the role of heterogeneities on structure and function of cardiac tissue will help in designing more
efficient and safer therapies. Possible engineering of the patent capillary- and microcapillary-like networks
inside the tissue constructs may enable culture of thick tissue slices, and facilitate immediate perfusion
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27-16 Tissue Engineering
0 ms 40 140
240 340 420
(a)
(b)
200 ms
FIGURE 27.4 Functional reentry in a cardiac tissue construct induced by rapid point pacing, and assessed by optical
mapping of transmembrane potential. (a) Single rotation of a counterclockwise reentrant wave shown through a series
of voltage snapshots in time. Frames progress left to right, top to bottom. Time in milliseconds is marked at the top
left corner of each frame. Arrows denote the direction of wave front. + denotes 2-mm spaced recording sites. Gray
scale bar next to the first frame corresponds to a normalized transmembrane voltage with bottom and top denoting
rest and peak of an action potential, respectively. (b) Recording of transmembrane voltage from the site marked by a
white square in the frames of panel A. Reentrant activity appears stationary and periodic.
during implantation by attachment to one of the host arteries. The success will only be accomplished by a
profound understanding of a number of complex topics, and achieved through a joint effort among basic
scientists, engineers, and clinicians.
27.6 Conclusions
Cardiac tissue engineering is a new and exciting field with many obstacles to be surmounted before start of
clinical trials. With technical aspects resolved, the main advantage of implanted cardiac tissue patch over
the injected cells will be a structural and functional repair of large tissue defects. Given that implantation
of cardiac patch will repair a centimeter-size tissue region, inference about the quality of tissue construct
sprior to implantation should be based not only upon different assessments at cellular or subcellular
level, but also upon detailed evaluation of both electrical and mechanical function at centimeter-size scale
(i.e., measurements of impulse propagation and contractile force in constructs relative to those in native
cardiac muscle). Similarly, only systematic micro- and macroscopic assessments of heart structure and
electromechanical function postimplantation will provide necessary selectivity towards the design of safe
and efficient clinical therapies.
Acknowledgment
The author would like to thank P. Bursac and K. Cahill for reviewing the manuscript. This manuscript
was submitted for press on May 1, 2004. Due to rapid developments in the field, some of the most
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contemporary work will not be reviewed, and certain statements may be outdated at the time of press.
Author apologizes in advance if this is the case.
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