Cardiac Tissue Engineering: Proposal for an Improved In Vivo Approach

December 12, 2013

Daniel Houck
Michael Warren
Andrew Vienot

Introduction
In the development of 3D tissue engineering solutions, creation of cardiac tissue of
significant volume will be required. This will require developments specifically in the areas of
angiogenesis and cell stimulation. Without angiogenesis, cardiac tissue construction is limited to
materials with cell thickness at or fewer than three cells, after which due to a lack of diffusion
cells will become necrotic [1]. Currently, methods of vascularization may be broken into two
main categories- intrinsic vascularization (development of blood vessels inside the body), and
extrinsic [3]. Secondly, due to tightly interconnected nature of cardiac cells it is necessary to
stimulate them in order for them to form into the proper shape. In this area, the current methods
are electrical stimulation and mechanical stimulation [DDD]. The longer the cell is the farther it
can contract and the alignment of the cells indicates how organized the contractions will be
[DDD]. Native heart cells are generally well aligned and elongated. While there are multiple
differing avenues being pursued in both, no studies are being performed at the juncture of these
two fields: the creation of cell constructs which are vascularized to improve host acceptance and
1

are also electromechanically optimized in order to improve the contractile response and
ultimately the ejection fraction.

Necessity
As of 2010, heart disease, in most cases myocardial infarction, is consistently the top
cause of death in the United States. Approximately 24% of reported deaths (2.46 million) were
due to diseases of the heart, and cardiovascular disease costs the United States 272.5 billion
dollars every year. Alarmingly, projections have these costs increasing over 200% to 818 billion
dollars by 2030 [4]. The advent of long term and cost effective solutions will become ever more
necessary.

Physiological effects of Cardiac Disease

The extent of damage to the cardiovascular system varies with many factors, such as
duration of cardiac incident, tissue response, and prior tissue condition. In order to develop
applicable treatments, the mechanism of cellular death and the consequences must be discussed
in detail. The most widely known type of CVD is acute myocardial infarctions, also known as
heart attacks, resulting from prolonged ischemia within the myocardial tissue(7). The infarction
occurs frequently due to plaque buildup within the coronary artery, preventing downstream blood
flow. The pathological mechanism of myocardial necrosis follows through the ischemic cascade
activated in response to the reduction of blood flow to the affected tissue. The lack of oxygen
prompts multiple side-mechanisms including anaerobic glycolysis, hydrolyzation by lysosomal
activation, and apoptosis[(7)-(8)]. Under anaerobic conditions, myocardiocytes begin to
breakdown high-energy phosphates such as adenosine triphosphate (ATP) into adenosine
diphosphates (ADP). 3
2

Figure 1: Pathogenesis of Myocardial Cell Death

The breakdown process continues until the phosphate chain is removed and adenosine is
released. The reduction of ATP and ADP effects intracellular processes specifically shutting
down the sodium and calcium pumps (9). The accumulation of calcium, sodium and water create
hazardous conditions leading to cellular hypertrophy. The cascading effect continues until
irreversible damage to the mitochondrial and lysosome structures occur, causing cellular necrosis
(10). 4 Another mechanism of cardiac tissue damage is the rate of programmed cellular death
(PCD), or apoptosis, during myocardial events. A major component to pathological development,
apoptosis aids in morphogenesis and regulates homeostasis within cell cultures (11). During
infarctions, studies report apoptosis as the leading cause of myocyte death when compared to
necrosis with approximately 1.4 million and 45,000 myocyte deaths per hour respectively (12).
Consequently, in the event of myocardial ischemia, observations of apoptosis-related

intracellular proteins binding within myocardiocytes have brought attention to the possibility of
blocking these proteins to enhance tissue survival (13).
The range of cardiac tissue destruction varies primarily on the duration of ischemic
conditions. Typically minor inflictions to the cardiac tissue occur after 15-20 minutes; in
contrast, major tissue death occurs after three to four hours (14). In other ischemic afflictions,
such as cerebral ischemia, the extent of tissue death is vastly lower than that of cardiac ischemia
(14). With current technology it is possible to retain normal function after a cardiovascular event,
however risks for additional infarctions and cardiac failure rises significantly. Therefore the
extent of tissue death is likely to determine the extent of treatment. In case of complete cardiac
failure, transplantation is the only known procedure to prevent death. With donor organ shortages
plaguing hospitals worldwide, new approaches and procedures are needed in order to combat
CVD.

Disease Treatment
Currently, treatment options are designed with the goal of a) restoring blood flow, and b)
decreasing the amount and effect of the scar tissue [6].
Two main methods which restore blood flow are thrombolysis and angioplasty. In
thrombolysis, using either mechanical or chemical methods, the blockage is broken down or
removed, restoring blood flow. In angioplasty, commonly known as balloon, a mechanical device
is used to expand a collapsed blood vessel, once again restoring blood flow [8]. Methods such as
these decrease the amount of damage done to the myocardium, consequently lowering the
amount of ventricular remodeling and scar tissue that develops [9]. These reperfusion techniques
have been shown to decrease the amount of damage by 30%.

The current treatment Captopril is the only drug aiming to inhibit the formation of scar
tissue, and does so by inhibiting angiotensin II- which aid in fibroblast and collagen formation
[10]. However, the efficacy of this drug is still contested [10].
These methods which are approved by the FDA, although valuable, do not provide
mechanisms to reverse the negative effects of the infarction- namely scar tissue development and
ventricular remodeling. Consequently, the only effective treatment option for patients with latestage heart failure is heart transplantation [11]. This has numerous issues including the need for
immune-suppressants, failure of the graft, and is further limited by the low number of donors
[12]. The shortcomings of these therapy methods provide opportunities for tissue engineers to
provide meaningful products with which to improve quality of life and in a secondary manner
provide models with which to test further products destined for cardiac uses.
Optimization in Tissue Engineering
Electrical Stimulation
Mechanical Stimulation
The inherent dynamics of the heart make mechanical stimulation a clear factor in cardiac
cell growth. The constant mechanical stress applied to natural heart tissue has been linked to
many signals for gene stimulation and protein synthesis. There is evidence that mechanical
stimulation causes several growth factors to be created and released in cardiac tissue. These
growth factors significantly affect the structure and functionality of the cardiac tissue. For
engineered cardiac tissue, two major types of mechanical stimulation have been studied: applied
pressure and stretch. Both types of mechanical stimulation have been shown to improve the
formation and behavior of engineered cardiac tissue4,2.

Applied Pressure
Applied pressure stimulation can be evenly applied to engineered cardiac tissue by a
bioreactor containing a piston device. Studies have been done comparing the cellular responses
to constant compression, intermittent compression, and no compression. Figure 4 shows images
comparing cardiomyocytes that have been stimulated with each type of compression for four
days. Continuous compression causes the cardiac cells to become round. Because the elongated
shape of cardiac cells is related to the pulsation of the tissue, the round shape is indicative of
poor cardiac tissue behavior. The cells the experienced intermittent compression became
elongated and looked similar to the control cells that received no stimulation. These cells are
much more likely to behave like natural cardiac tissue.
Applied Stretching
the effect of mechanical stretch on engineered cardiac tissue is the other widely studied
type of mechanical stimulation. It is believed that the natural beating of the heart induces
stretching which triggers the growth of cardiomyocytes2. Tissue engineers attempt to mimic this
stimulation in cultured cells to create functioning cardiac tissue. Figure 8 shows the design of a
bioreactor used to apply stretching stimulation to cardiac tissue. The reactor was used in an
experiment that was focused on determining the effect of chronic stretch on cardiac tissue. In the
experiment, cells were cultured for four days, and then exposed to unidirectional stretch in this
device for a total of six days. The amplitude of the stretch was varied from 1% of the original
area to 20% of the area to determine the optimum. The effects of the stretch on the engineered
cardiac tissue were monitored and the results are displayed in figures 9 and 10. Figure 9 shows
an image of a Hematoxylin-eosin stained section of the both the stretched and un-stretched
cardiomyocytes. The figure shows that the stretched cardiomyocytes have grown noticeably
6

longer and more organized. This result is confirmed by figure 10 which graphically shows the
width difference between stretched and un-stretched cardiac cells. The higher percentage of unstretched cells in the low width range indicate that the stretching induced the cells to elongate
like natural cardiac tissue. This improved
structure is likely to positively affect the cardiac
behavior of the cells. The chronic stretch was
also reported to have an effect on the RNA/DNA
and protein/ cell ratio2. This is generally
regarded to improve the cardiac behavior of
cells. The combined results of this experiment
indicate that the behavior of cardiomyocytes
cultured in vitro are more like native tissue when
they are applied to chronic stretch. Stretching
Figure 10: Effect of stretch on cell size2

mechanical stimulation appears to positively

contribute to the contractile function of engineered

Figure 8: Bioreactor used for strech of

cardiac tissue2

cardiac tissue.

Figure 9: Image comparing un-stretched (left) and stretched (right) cardiac tissue 2

Angiogenesis
Angiogenesis is the process by which new microcirculatory vessels grow and sprout from
pre-existing vessels [2]. This is differs from both embryonic and post-natal vasculogenesis in
which angiogenic vessels are created either de novo in the case of embryonic, or by circulating
angioblasts. Much of the research into angiogenesis has been performed by researchers in cancer
medicine, as a key issue with tumor cells is their ability to promote angiogenesis, thus allowing
them to grow past diffusion limitations [22]. Effectively, in the process of angiogenesis
endothelial cells proliferate under the control of growth factors, mechanical, and chemical
environments, and sprout new vessels from pre-existing vessels [2]. After this, the vascular
network is stabilized by smooth muscle cells. Multiple vascular endothelial growth factor
isoforms (VEGF) are necessary during the process along with differing placental, and fibroblast
growth factors [2]. Management of these varying physiological factors greatly complicate
vascularization attempts, with current attempts at in vitro vascularization producing limited,
capillary-like micro vessels [3].
In vitro vascularization is the goal of creating a vascularized tissue, in lab, which can then
be implanted or grafted to the patient. After doing so the goal is that the existing vasculature will
inosculate the construct in a timely manner so that the construct does not become hypoxic. The
method of creating vascularized sheets is accomplished by supplementing cardiocyte sheets or
scaffolds with endothelial cells and pericytes [3]. While these pre-vascularized cell sheets
perform better than simple sheets, they still face the same issue of needing timely inosculation in
order to survive. Inosculation is the process by which the artificially created construct is joined
into the existing circulatory system. Studies have this taking up to several days, a timeframe
which causes ischemic cell death [3]. Methods which have been found to decrease cell death
8

have been to include angiogenic factors such as VEGF, platelet-derived growth factor, fibroblast
growth factor, and others to decrease the time it takes to inosculate construct [3]. In combination
with the development of capillaries, methods have been found to guide the formation of micro
essels in such ways that they are uniform [2]. This is a necessary criteria in the formation of
uniform cardiac tissue. The primary methods used for guiding vascularization in vitro is the use
of porous or patterned scaffolds which can be seeded with angiogenic cytokines which create a
pro-angiogenic microenvironment [2]. This methodology makes the creation tightly controlled
constructs possible, as one can see the developing tissue create more robust scaffolds; however, it
faces a key shortcoming in the inability to properly inosculate in a timely fashion. Intrinsic
vascularization methods aim to combat this.
As the name suggests, intrinsic vascularization approaches allow use the bodys own
angiogenesis, as current methods aim first to develop a vascular system and then add the
necessary cells. Currently, a primary area of research in this methodology is the usage of
arteriovenous and flow through chambers. Conceptually, these function by constructing a

Figure 1 [16]

vascular pedicle, and isolating it from the surrounding tissue using a polycarbonate chamber [3].
In this scenario, new microcirculatory vessels and vasculature sprout from the isolated
vasculature [3]. Ideally, this chamber is supplied with stem or progenitor cells, which would then
form the cardiac tissue [23]. Researchers found however that if stem or progenitor are placed in
the chamber at the beginning of the experiment, then hypoxia occurs as would be expected. To
combat this, Tilkorn et al. created a scaffold which allow them to delay the insertion of cells until
they saw sufficient vascularization of the construct- around day 7. At this point, progenitor cells
were injected into the scaffold which had the necessary vasculature to support them. Tilkorn et al
demonstrated that implanted myoblast survival in an in vivo tissue engineering construct is
positively correlated to the vascularity of the construct.

10

Possibly the most ambitious construct methodology is the decellularized heart. Pioneered
by Ott et al, the team hypothesized that the best vascularization was provided by nature [8].
Consequently, the team developed a perfustion decellularization protocol. Rat hearts were
decellularized using a combination of three detergent solutions, PEG in deionized water, 1%
Triton X-100 deionized water, and 1% SDS in deionized water. After this process, all cellular
constituents were removed, while Collagens I and III, laminin, and fibronectin remained intact.
Also, this method circumvents entirely the issue of orientation and phenotype, as the orientation
of the myocardial extracellular matrix was preserved. After preliminary testing, freshly isolated
neonatal cardiac cells and a mixture of other cell types were seeded via intramural injection, and

11

endot
helial cells and media were perfused through the vascular conduits [8].
Figure 2 [8]

12

After eight days, the reconstructed organ showed electric and contractile responses- two
key indicators of properly functioning cardiac tissue [8]. The construct was maintained via eight
different constructs for 28 days, and after day 8 mechanical testing showed that the constructs
could generate pump function equivalent to 2% of an adult or 25% of a 16-week fetal heart.
While this method is promising, it still faces many of the shortcomings seen by other methods.
Benefits are that it appears to be the most promising method of creating the necessary vascular
system and geometry; however, the two major issues it must still face is the procurement of cells,
and also the development of mechanical function to a degree that would be viable. In the
experiment, neonatal cardiac cells and endothelial cells were obtained in amounts which would
be impossible to replicate currently in human trials (2x10^7 endothelial cells and a mixture of
75x10^6 neonatal cardiomyocytes,fibrocytes, endothelial cells, and smooth muscle cells). Also,
while the development of mechanical function was notable- after eight days it had the ejection
capabilities equating to 2% of a adult heart, this is a insufficient level for transplantation. Based
on the data that this equated to 16 weeks, based on a linear growth model this heart would
require approximately 400 days to reach the pump functionality of a working adult heart. A third
issue which is important is lowering the immunogenicity of the scaffold so that it may be
possible to reseed a cadaver heart with autologous cells to the person in need.
Aim 1: Correlate electrical and mechanical stimulation to prior results
Electromechanical stimulation has been shown to be a major influence in the behavior of
developing cardiac tissue. There have been several previous studies characterizing the effect of
electrical and mechanical stimulation individually. The first aim for this experiment is to
confirm that the procedure used to culture the cardiac tissue corresponds to the results predicted
by the literature. This step is intended to show that the method used to culture the tissue is valid
13

and account for disturbances including cell sources, scaffold type, and the method by which the
cells are stimulated. Literature predicts that the optimal electrical stimulation will be at an
amplitude of 3 V/cm and a frequency of 3 Hz and that the optimal mechanical stretch will be at
5% stretch and 1.5 Hz. The optimum electromechanical stimulation is expected to be near these
values, but may vary depending on the specific experiment setup.
The first step for this aim is to develop a bioreactor capable of simultaneously providing
electrical and mechanical stimulation to the growing cardiac tissue. The bioreactor will be
constructed by modifying a method used by Fink ET. AL. This reactor is capable of stretching
the cardiac tissue up to 20% of its original length using stretching rods attached to the tissue1.
Carbon rod electrodes will be placed lengthwise along the bioreactor to provide a constant
electrical field gradient across the scaffold. Care will be taken to ensure that the movement of
the cells is not restricted and the cells can be observed throughout the experiment.

Figure 3 Left- stretching apparatus. Right- carbon electrodes

Once a functioning apparatus to provide the necessary electromechanical stimulation has

be obtained, the optimal parameters for the stimulation can be determined. The electrical and
mechanical stimulation will be individually investigated and the optimal magnitude and
frequency will be obtained for each. For each experiment a cardiomyocyte monolayer will be
seeded into the reactor with SkGM-2 medium and held at 37oC.
14

The electrical stimulation procedure will be designed to mimic that of Tandon ET AL.2
The tissue monolayer will be subjugated to square electrical pulses 24 hours after seeding. The
stimulation will continue for three days before results are collected. The amplitude of the
electrical stimulation will be varied form 1-8 V/cm and the frequency will be varied from 1-5 Hz.
The mechanical stimulation procedure will be based off of the methods used by Fink ET AL. to
implement mechanical stretch. Unidirectional stretch will be applied to the tissue four days after
the cells were seeded in the scaffold. The stretching will continue for six days and then the
results will be collected. The stretch amplitude will be varied from 1% to 20% of the length of
the tissue and the frequency varied from 1-5 Hz.
Excitation threshold, maximum capture rate, amplitude of contraction, and histology will
be used as metrics to determine the optimum stimulation for the tissue monolayers. The
excitation threshold is a measurement of the tissues electrical excitability, and is determined by
the minimum amplitude required to cause the tissue to beat at 60 beats per minute. A low
excitation threshold value is preferred for optimal cardiac tissue. The maximum capture rate is
the frequency that the tissue will contract when exposed to electrical pulses with an amplitude
1.5 times the excitation threshold. A high capture rate value indicates strong intercellular
connectivity and is desired for cardiac tissue. The amplitude of contraction is measured in
percent change in the tissues surface area during a contraction. A large amplitude of contraction
indicates more functional cardiac tissue. The histology of the cultured tissue will be compared to
that of natural tissue. The histology will be used to give a qualitative analysis of the optimum
tissue.

15

16

Aim 2: Combine electrical and mechanical stimulation

After aim 1 has shown the validity of the stimulation procedure, the effect of combining
the electrical and mechanical stimulation will be investigated. The cells will be seeded in the
reactor, following the same procedure as in aim 1. After the cells are cultured for 24 hours the
tissue will be exposed to electrical stimulation for three days. Four days after the cells were
seeded; unidirectional mechanical stretch stimulation will be applied in addition to the electrical
stimulation. The cells will be left in the culture for a total of 10 days before they are lifted and
measurements are taken. The same metrics used in aim 1 will be used to determine the optimum
stimulation for cardiac tissues. The results of aim 2 will provide novel information regarding the
effect that multiple types of stimulation has on cardiac tissue.
Aim 3: Increasing Construct Thickness and Angiogenesis
Having created electromechanically optimized cell sheets via aims one & two, the goal of
aim three is to a create a thicker cell consruct by stacking sheets of cardiomyocytes- with

17

sparsely cultured aortic endothelial cell sheets placed in between to promote angiogenesis.
Without any angiogenesis promoters, as noted earlier the limit of construct thickness was
approximately 3 cells thick. Schimizu et al recognized the need for vascularization in these
constructs and began experimenting with the use of endothelial cell layers, with which he was
able to create a 5 layer thick construct. Building off of this, we desire to research whether a
significant increase in value is found by performing this experiment with electromechanically
stimulated cells. We will consider this aim a success granted we see higher maximum capture
rate, contraction amplitude, and excitation threshold than the control models. Our control models
will be differntiated by lacking electrochemical optimization.
The novel method designed by Schimizu relied upon two different, temperature
dependent materials: fibrinogen gel, which has a melting point at 37C and a adhering
temperature of 20C, and Poly (N-isoproplacrylamide) which has a release temp of 20C and and
adehesion temperature of 32- 37C. Due to the nature of these materials, it is possible to create
lift cell sheets and stack them in a organized manner. An overview of the method is seen in figure
XXX.

Aim 4:

Animal Model

Through the findings and

results of the previous

aims, it is necessary to

demonstrate the clinical potential using non-

human models. Previous work

has shown promise in regards to showing

sustained development of the transplanted layers. Talk about previous work

18

----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Likewise for a present model, demonstrating the ability of the optimized cell layers to regenerate
heart function would be the ultimate goal.
Materials and Methods
Animals
The study will employ using a small-animal model consisting of adult male Lewis rats around
12-14 weeks old. The Lewis rat strain is the most common strain found within the tissue
engineering field and is readily available for purchase. cite Physiologically, the rat model will
exhibit healthy conditions such as weighing around 350 grams and recording a heart rate of
around 400 per minute. # of animals needed for groupingPower analysis
All animal testing will be conducted in compliance with the Animal Welfare Act and the Guide
for the Care and Use of Laboratory Animals.
Artificial Infarction Procedure
In order to produce an infarction event within the animal model, the use of an efficient and
sustained procedure is required. Many methods of inducing an infarction contain high mortality
rates and have left some subjects unable for testing. One such method that is commonly used
with improved mortality rate is the ligation of the left coronary descending artery.
The surgery is performed under general anesthesia in which the animals will undergo intubation.
A thoracothomy is performed on the left side of the animal, by cutting the fifth and sixth ribs.
The thoracic cavity will be arranged to be able avoid injury to the heart. The surgeon will apply a
small blade an open the pericardial membrane allowing for a cardiac holder to be placed.. The
19

LAD will be localized 12 mm below the junction of the pulmonary conus and the left atrial
appendage (Fig. 2 and Fig. 3).

The left atrial appendage and the pulmonary conus can be identified by their appearance and
location above the left ventricle. The ligation will occur using a suture with a curve needle.
During the insertion of the needle, the operator should be careful to avoid puncture of the left
atrial appendage or the pulmonary conus, which can cause massive hemorrhage. After ligation
the heart will be returned back to the thoracic cavity and the chest will be closed. Postoperative
care will be continued using analgesia and hemodynamic monitoring until extubation and full
recovery from anesthesia.

Transplantation and Post OP Analysis

Transplantation of the fabricated cell layers will occur after subjects are fully recovered. The
procedure of transplantation is similar to previous works and will expect to produce the same
results in terms of mortality rate.

20

After the post-infarcted/transplant subjects recover, the extent of myocardial damage or recovery
will need to be quantified. Through the non-invasive and applicability to small animal model, the
use of ECG or Electrocardiography will be implemented in order to determine the damage.
ECGs specifically measure the strength and timing of electrical signals in the heart. From these
measurements the ejection fraction of the heart can be found. Further analysis must be

Future Work

Constant DC electric field

Mechanical pressure stimulation

Amplitude & frequency of stimuli changing over time

Stacking thicker cell layers

Conclusion

21