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Cardiac Muscle Engineering: Strategies to
Deliver Stem Cells to the Damaged Site
Giancarlo Forte1, Stefania Pagliari2, Francesca Pagliari2,
Paolo Di Nardo2 and Takao Aoyagi1
1Biomaterials Center, International Center for Materials Nanoarchitectonics (MANA),
National Institute for Materials Science (NIMS), Tsukuba,
2Laboratorio di Cardiologia Molecolare e Cellulare, Dipartimento di Medicina Interna,
Università di “Tor Vergata”, Roma,
1Japan
2Italy
1. Introduction
In healthy human hearts, only 10-20% of the total cells are contractile cardiomyocytes and, at
the age of 25 years, no more than 1% of them are annually substituted by progenitor cells,
this percentage reducing to less than 0.5% at the age of 75. In total, less than 50% of
cardiomyocytes are renewed during a normal human life span [1]. For this reason, the topic
of cardiac repair is among the major challenges for the tissue engineers worldwide. In fact,
cardiac diseases are a predominant cause of mortality and morbidity in industrialized
countries, despite the recent advancements achieved in pharmacological treatment and
interventional cardiology procedures. Nonetheless, end-stage heart failure management still
relies on organ transplantation as unique approach, and, notwithstanding the use of massive
immunosuppressive drugs, still a percentage falling within 20%-40% of patients encounters
immune rejection during the first year post-transplant [2]. Among the patients not facing
severe immune rejection, almost 70% is forced to retire or reduce their working activity,
their survival rate falling below 70% during the first five years post organ transplantation
[3]. Last, but not least, the economic impact of cardiovascular diseases and stroke has been
estimated in 2010 at $503.2 billion [4].
Currently, post-infarction myocardial revascularization protocols include the administration
of raw bone marrow stem cells, while a number of clinical trials have been performed or are
currently in progress in which different cell subsets are implanted in the damaged tissue by
means of surgical techniques. The results of such trials are still controversial. In fact, when
autologous skeletal myoblasts were injected into the heart of patients suffering from
ischemic cardiomyopathy, the modest functional improvement obtained was impaired by
the arising of arrhythmia events, thus requiring the adoption of a pacemaker [5]. On the
other side, intracoronary administration of bone marrow mesenchymal stem cells resulted
in minimal improvements in cardiac contractile function in patients with dilated
cardiomyopathy [6]. These mild results were mostly ascribed to a paracrine effect exerted on
host tissue, rather than to a direct contribution of stem cells to the contractile activity.](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-35-2048.jpg)
![Tissue Engineering for Tissue and Organ Regeneration
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Thus, among the criticisms to be challenged before efficient cell therapy protocols for
cardiac diseases can be setup, the choice of the appropriate cell subset to generate new
vessels and contractile cardiomyocytes, as well as the route of cell delivery remain key steps.
The solution of such problems requires additional efforts in basic research to clarify the
processes leading to stem cell differentiation as well as technological advancements to setup
efficient protocols to implant the cells.
In principle, adult stem cells could be extracted from patient’s own tissues and expanded in
culture by means of well-known techniques (Figure 1).
Fig. 1. Cardiac Tissue Engineering paradigm. Adult stem cells can be harvested, purified
from the patient and expanded in culture. Such cells can be delivered to the injured heart by
injection (intramural or through bloodstream with or without injectable carriers), or in the
form of solid bio-constructs. Stem cell-derived bio-constructs can be obtained by culturing
the cells on scaffolds or by scaffold-free technology
Nonetheless, a number of issues should be challenged before safe procedures to
manipulate stem cells in vitro for cardiac transplant can be setup. In fact, stem cells should
be amplified in vitro to reach a critical number (Figure 1). During this passage, malignant
transformation is likely to occur in ex vivo cells when standard culture conditions are
adopted to expand stem cells [7, 8]. On the other side, stem cells could encounter
senescence after a short number of passages in vitro [9]. Moreover, the use of animal-
derived supplements during the phase of cell expansion would hinder the use of stem
cells for cardiac cell therapy.](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-36-2048.jpg)
![Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 21
The employment of autologous stem cells would avoid the problem of immune rejection
and the need for immune-suppressive drugs, while, in the treatment of pathologies for
which a genetic basis is suspected the use of autologous cells is hampered. As far as the use
of autologous cells is concerned, the possibility that a significant patient-to-patient
variability in stem cell quality exists should be taken into account [10]. Finally, the use of
cellular and tissue-based products in human disease therapy is subjected to regulations
issued by the European Union and Food and Drug Administration (FDA) aimed at
establishing classification criteria for advanced therapy medicinal products (ATMP). In
particular, the European Regulation states that human cells to be used in cell therapy have
to comply with the principles of Good Manufacturing Practice (GMP) protocols [11, 12].
2. Adult stem cells for cardiac repair
A number of stem cells and progenitors have been so far proposed for cardiac repair, due to
the inability of cardiomyocytes to proliferate after birth [1]. Among the cell sources
challenged for the possibility to produce new cardiomyocytes, skeletal myoblasts have
proven to be able to acquire a contractile phenotype in vitro [13]. Moreover, when implanted
in vivo in a canine model of dilated cardiomyopathy (DCM), they attenuated cardiac
remodeling [14]. This result is likely to be due to the fusion of skeletal myoblasts with the
surrounding myocardium rather than to direct cell differentiation, as suggested by in vitro
experiments [15]. As discussed in the following section, clinical trials demonstrated that
skeletal myoblasts are not able to couple electrically with host tissue, leading to arrhythmia
events [5].
The role of hematopoietic stem cells (HSC) in cardiac repair has been investigated by several
research groups and their contribution to cardiac regeneration in vivo has been heavily
debated, being the ability of HSC to transdifferentiate to other lineages still questionable.
Indeed, evidence of the ability of bone marrow-derived c-kit+ HSC to help cardiac tissue
healing has been given using two different approaches: c-kit+ cells were (i) either delivered
to the infarcted site by intramural injection [16] or (ii) mobilized from bone marrow through
growth factor administration [17]. More recently, elegant experiments compellingly clarified
that HSC are not able to acquire contractile phenotype in vivo [18-20]. Nonetheless, a subset
of bone marrow hematopoietic precursors expressing CD34 and CD133 has been proven to
contain endothelial progenitors. Thus, they have been tested for revascularization protocols
in hind limb ischemic animals and could be proposed for cardiac infarction therapy [21]. On
the other hand, the results obtained in preliminary investigations in which another bone
marrow-derived stem cell subset, mesenchymal stem cells (BM-MSC or MSC) were
challenged as a candidate for cellular cardiomyoplasty, raised great enthusiasm for such a
cell subpopulation. Recent studies clarified that the direct contribution of MSC to cardiac
repair in terms of production of new contractile cells is minimal if any, while a paracrine
effect on the diseased tissue of such cells is universally recognized [22]. Such cells are also
appealing for their ability to induce a certain degree of immune tolerance [23].
The presence of a small reservoir of cardiac resident progenitor cells (CPC or CSC) has been
recently demonstrated in human as well as in other mammals’ heart [24]. Such tissue-
resident cells participate in myocardial homeostasis and retain a limited regenerative
capacity throughout organism lifespan [1]. All the subsets so far identified through the
expression of stemness markers (c-kit+, Sca-1+, Islet-1+) demonstrated the ability to give
birth to new contractile cells in vitro, while only c-kit+, Sca-1+ progenitors were shown to be](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-37-2048.jpg)
![Tissue Engineering for Tissue and Organ Regeneration
22
involved in post-natal cardiac tissue homeostasis in vivo [25]. In fact, the presence of Islet-1+
cells appears to be limited to fetal life and their contribution to the endogenous program of
cardiovascular repair is still unknown; on the other hand, the very low number of c-kit+ and
Sca-1+ cells in the myocardium is considered the limiting factor of cardiac regeneration [26].
Furthermore, among the adult stem cells, a novel “artificial” subset can be recognized:
induced pluripotent stem cells (iPSC, Figure 2). This cell type can be produced in vitro by
transducing somatic cells with a combination of transcription factors able to induce the
nuclear reprogramming of differentiated cells. These cells, which display the functional
features of pluripotent embryonic stem cells, have been credited of the ability to produce
new cardiomyocytes. They could thus be the source of autologous, although genetically
modified, patient-specific contractile cells [27]. Moreover, the possibility to directly obtain
functional cardiomyocytes by the genetic reprogramming of postnatal cardiac or dermal
fibroblasts has been demonstrated [28]. Such a result was firstly obtained in vitro but also
when the cells were transplanted into mouse hearts one day after transduction of
transcription factors (GATA-4, MEF-2c, Tbx-5) known to be involved in cardiac muscle
development. Nonetheless, the reprogramming and differentiation efficiency of these cells
appears to be really low, thus requiring an efficient purification step before they can be
implanted in vivo. Additionally, safety concerns due to the use of genetically modified cells
and / or viral vectors remain.
Fig. 2. Induced Pluripotent Stem Cell Generation. Induced pluripotent Stem Cells (iPSC) can
be generated by reprogramming somatic cells through their transduction with four
transcription factors. iPSC share functional similarities with Embryonic Stem Cells (ESC)
and can be differentiated towards cardiomyocytes, thus representing an autologous source
of contractile cells](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-38-2048.jpg)
![Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 23
3. Stem cell delivery to the injured heart
As previously said, cell route of delivery to damaged heart represents the major topic in
the setup of efficient, minimally invasive techniques to treat cardiac pathologies.
Recently, a number of techniques to deliver stem cells to the injured site have been
proposed but questions remain regarding the optimal approach able to favor high cell
retention, differentiation rate and clinically relevant improvement in cardiac
performance.
a) Direct injection
Stem cell direct intramural injection, including trans-epicardial and trans-endocardial cell
injection, is the elective strategy for patients with severe occlusion of coronary vessels. In
particular, trans-epicardial approach consists in the direct injection of a high number of cells
into the infarcted area or around the border zone. Endocardial stem cell injection is
performed using catheters such as MyoStarTM injection catheter (Biosense Webster)
integrated with imaging systems like NOGA® system (Cordis Corp., Warren, NJ, USA),
which allows real-time three-dimensional reconstruction of left ventricle as well as the
targeting and functional assessment of specific myocardial area [29]. Such procedures are
highly invasive since they require open-heart surgery and gave contrasting results so far.
For example, pre-clinical studies performed on experimental animals demonstrated that,
although a certain extent of cardiac repair was achieved when bone marrow Stro-3+
perivascular cells are implanted in vivo, the cells vanished from the application site within
few days [30]. In other reports, when Sca-1+ cardiac resident stem cells were injected in
infarction border zone, a modest but significant improvement in cardiac function was
reported, with evidence of cell engraftment and differentiation [31]. Finally, in another
pre-clinical study, bone marrow-derived c-kit+ cells were shown to repair entire
ventricular areas while massively engrafting and differentiating in contractile and
vascular figures in vivo [32]. Of interest, independent groups already demonstrated that c-
kit+ bone marrow-derived hematopoietic stem cells fail to acquire contractile phenotype
when implanted in diseased myocardium [19, 20]. Such discrepancies are not surprising
since different stem cell subsets or preparation protocols were probably used in these
studies.
Stem cells can be delivered intravenously to the heart, through coronary arteries or even
through retrograde coronary sinus. The major drawback of stem cells being infused through
peripheral venous system seems to be the low retention of cells into infarcted area. Results
obtained in pre-clinical animal models showed that this minimally invasive approach results
in a significant percentage of injected cells being sequestered in lungs, liver or spleen, due to
blood flow [33]. On the other hand, intracoronary or retrograde coronary sinus infusion of
the cells are mainly performed after acute myocardial infarction using an angioplasty
balloon and high pressure to deliver cells to heart muscle [34]. The coronary route was
proven to be free of stem cell systemic delivery, while a limited number of cells could be
found in the infarcted area [35].
Finally, an interesting attempt with stem cells being injected into the pericardial cavity has
been proposed. By this means, a higher number of cells could be deposited and retained
in the pericardial cavity, while migration across the visceral pericardium is required
(Table 1).](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-39-2048.jpg)
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Table 1. Advantages and disadvantages of injecting stem cells by intravenous,
intracoronary, intramyocardial, retrograde coronary sinus or intra-pericardial route
b) Injectable scaffolds
Injectable scaffolds are defined as materials offering the unique solution of replacing
damaged myocardial ECM and/or delivering cells directly to the infarcted region while
holding the potential for minimally invasive delivery [36]. Such scaffolds can be composed
of biocompatible microspheres or in situ gelling materials having reasonable dimensions as
to surpass capillary barrier. They are considered a promising tool for stem cell delivery to
damaged myocardium. In situ gelling materials are generally made of components of
extracellular matrix (ECM), which are induced to a transition after being implanted in situ.
Complex injectable gelling materials have been prepared by decellularization technique out
of ventricular or epicardial ECM, thus possibly avoiding animal-derived components and
paving the way to the definition of patient-specific treatments.
The use of injectable, synthetic microspheres has already been proven promising in the
treatment of neurological diseases in vivo [37]. Recently the possibility of using injectable
scaffolds in cardiac cell therapy has been explored by interfacing murine mesenchymal
(mMSC) and cardiac stem cell (mCSC) lines with poly-lactic acid (PLA) microspheres
having a diameter of 30 and 100 μm. Preliminary in vitro experiments demonstrated that
such cells can be grown onto PLA microspheres while preserving their phenotype, but the
formation of cell clumps can hamper the application of this technique [38]. The use of
dynamic seeding techniques (i.e. bioreactors) would favor a more homogeneous distribution
of the cells. An interesting approach has been recently proposed to deliver human
mesenchymal stem cells to the injured myocardium: RGD-modified alginate microsphere
(diameter: 200-700 μm) encapsulation of hMSC was setup. In vitro experiments showed that
hMSC could survive, proliferate and migrate through the porous material. When
intramyocardially injected in a rat model of myocardial infarction by left anterior
descendant coronary (LAD) ligation, cell-loaded alginate microspheres promoted
angiogenesis and prevented LV negative remodeling [39]. Nonetheless, few human cells
were found in the injection area after few days, while microbead remains were still present](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-40-2048.jpg)
![Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 25
within host myocardium 10 weeks after the injection. The aspect of microbead resorption
should thus be addressed before clinical perspectives could be foreseen.
c) Scaffold-based technology
The possibility of using biocompatible scaffolds to deliver stem cells to the injured heart has
been explored by a number of independent research groups so far. The scaffolds proposed
are natural of synthetic but when designing cardiac-specific constructs, a number of
requirements should be fulfilled. For example, it cannot be neglected that myocardial
contractile function relies on the transmission of electrical and mechanical forces throughout
a functional syncytium. So, the integrity of the tissue has to be preserved. For this reason, a
cardiac-specific scaffold should comply with tissue architecture and thus be deformable
enough to indulge and, if possible sustain cardiac contraction. Moreover, as far as stem cell
engraftment is concerned, scaffolds should be able to start at least cell alignment and
commitment to favor stem cell electromechanical coupling with host tissue. In this respect,
the work of Mandoli and collaborators using Cerium Oxyde nanoparticles to affect poly-
lactic acid film surface and obtain a controlled nanorugosity appears intriguing [40]. In fact,
far from being a noxious compound for stem cells, ceria was able to induce cardiac stem cell
alignment and growth. Nonetheless, cardiac tissue is extremely complex and highly
demanding in terms of blood supply and catabolite removal, so that porous scaffolds that
could allow microvascular branches formation and oxygen perfusion are to be preferred. To
fulfill such requirements, the first attempts were performed by the group of Thomas
Eschenhagen. Neonatal cardiomyocytes were seeded in Collagen I + Matrigel to produce
Engineered Heart Tissue (EHT). Continuous contractile activity up to 1 week in vitro as well
as cell survival and integration in vivo in singenic rat hearts were reported [41]. In another
attempt, anisotropic accordion-like honeycomb scaffolds were prepared by excimer laser
microablation using poly(glycerol sebacate) as an elastomeric tool to mimic anisotropic
cardiac muscle stiffness distribution [42]. Although the authors demonstrated that such
scaffolds promote neonatal rat cardiomyocyte alignment and contraction, in vivo testing has
not been performed so far. The same material has been utilized to produce elastomeric
patches on which human embryonic stem cell-derived cardiomyocytes were grown,
showing that it is indeed possible to observe spontaneous beating activity in vitro up to 3
months [43]. Such patches were shown to be suitable as delivery systems and, when sutured
in the absence of cells onto healthy rat left ventricle, they did not affect cardiac contractile
activity. More basic studies were also conducted to study the ability of stem cells to interface
with different synthetic and natural materials. In this respect, few research groups focused
on the possibility to drive a certain extent of stem cell commitment through tailoring
scaffold physical and chemical properties, independently of biological cues. In this respect, a
common agreement on the ability of stem cells to sense substrate rugosity and elasticity has
been reached [44]. Thus, in order to rule out the occurrence of spontaneous events of
differentiation in implanted cells, the possibility to induce in vitro stem cell commitment on
scaffolds towards a desired phenotype is being investigated. Indeed, Engler and
collaborators compellingly demonstrated that the possibility to affect stem cell fate
determination by simply tuning substrate elasticity as to match tissue-specific stiffness,
exists. Recently, this concept has been corroborated by other research groups, showing that
cardiac resident progenitors (Sca-1+ CPC) can be committed to cardiac phenotype by the
physico-chemical signals arising from matrix, but biological factors are needed to complete
the differentiation process [45, 46].](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-41-2048.jpg)
![Tissue Engineering for Tissue and Organ Regeneration
26
d) Preparation of thick cardiac substitutes by Scaffold-free technology
To overcome the problem of poor cell retention reported in cell injection experiments in the
heart [30] and avoid the release of possibly harmful scaffold byproducts, scaffold-free
technology has been developed, in which cells are grown in a monolayer onto thermo-
responsive surfaces and easily detached in the form of cell sheet by lowering the
temperature [47]. Such technology takes advantage of the ability of polymers like poly-N-
isopropylacrylamide (PNIPAAm) to shift between hydrophobic and hydrophilic status
when the temperature ranges from 37ºC to 32ºC. Cell sheets can be serially stacked to obtain
multilayered scaffoldless constructs (Figure 3). Such an approach has already been applied
to obtain cell sheets composed of rodent [48, 49] and human [50] cells. Given the need for
thick cardiac substitutes suited to comply with cardiac muscle continuous contractility,
thermo-responsive technology has been envisaged as a possible answer to the lack of heart
donors. Pre-clinical trials performed onto experimentally infarcted animals demonstrated
that when a murine adipose-derived monolayer sheet is leant onto injured myocardium, it
can be retained and help tissue repair [48]. Similarly, striking results are obtained when a
Sca-1+ cardiac progenitor cell-derived sheet is used [49]. Finally, an interesting approach has
been recently proposed to deliver cardiac stem cells cultured in the form of cardiospheres to
the injured heart: cardiospheres were embedded into a cardiac stromal cell-derived sheet
obtained by using poly-lysine/ collagen IV-coated dishes [51]. The formation of mature
vessels as well as new cardiomyocytes in vivo was reported after 3 weeks.
Fig. 3. Generation of scaffoldless multilayered bio-constructs by means of thermo-
responsive technology: cells grown in a monolayer onto thermo-responsive poly-N-
isopropilacrylamide (PNIPAAm)-coated dishes can be detached by lowering the
temperature below 32°C. At 37°C the surface is highly hydrophobic and allows cell
adhesion. When the temperature is lowered, PNIPAAm becomes hydrophilic, the cell sheet
is detached and extracellular matrix (ECM) preserved. Multilayered cell sheets can be
obtained by serially stacking monolayered sheets](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-42-2048.jpg)
![Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 27
4. Clinical trials
In the attempt to transfer bench experience to bedside, a number of clinical trials in which
different stem cell or progenitor subsets are used have been approved (see
http://www.clinicaltrials.gov). Most of them are still in the recruitment phase while some
already gave indications and preliminary results. Since most of the ongoing trials are based
on the injection of raw stem cell preparations (mostly bone marrow-derived cells), the time
and route of cell application remain the key problems to be addressed before proceeding to
routine clinical practice. In this respect, recent animal experiments demonstrated that the
acute phase of myocardial infarction is probably not suitable for stem cell engraftment and
differentiation [52]. Therefore, the right moment in which stem cells should be delivered is
to be studied. An overview on some of the ongoing clinical trials is given below.
1. MAGIC (Myoblast Autologous Grafting in Ischemic Cardiomyopathy). In one of the
first phase II clinical trials setup to study the possibility to use stem cells to treat cardiac
pathologies, ninety-seven (97) patients undergoing coronary artery bypass grafting
(CABG) were enrolled. 400-800 X 106 autologous myoblasts harvested from patient
muscle biopsy were implanted in the akinetic area of ventricular wall 21 days after in
vitro culture. The follow-up after 30 days and 6 months demonstrated the arising of
arrhythmia events, thus requiring the implantation of pacemaker. Moreover, no cardiac
function improvement was reported. Such negative results were ascribed to the
inability of skeletal myoblasts to balance cell death and achieve complete
electromechanical integration with the recipient myocardium. Finally, skeletal myoblast
administration was reported to determine no enhancement in major cardiac adverse
events and mild effects on left ventricular remodeling process [53, 54]. More recently,
final results from SEISMIC [Safety and Effects of Implanted (Autologous) Skeletal
Myoblasts (MyoCell) Using an Injection Catheter] Trial, a phase II-a study
encompassing 40 patients experiencing congestive heart failure and receiving
percutaneous intramyocardial injection of autologous skeletal myoblasts, reported the
feasibility and safety of this procedure without significant arrhythmogenic events
recorded at 6-month follow-up with respect to control groups, although left ventricular
ejection fraction did not result significantly improve. These encouraging results suggest
that myoblast cell therapy could be considered as a potential effective treatment when
associated with standard medical therapy in patients with previously implanted cardiac
defibrillators [55].
2. TOPCARE-CHD, -AMI, -DCM (Transplantation of Progenitor Cells and
Regeneration Enhancement in Acute Myocardial Infarction, Chronic Stable Ischemic
Heart disease or Dilated Cardiomyopathy). In this complex clinical trial, a total of 346
patients were classified to CHD, AMI or DCM pathologies and infused either with bone
marrow cells (BMCs), blood-derived stem cells, or no infusion. In TOPCARE-CHD, 121
patients (mean age: 59) with chronic stable ischemic heart disease (CHD) were treated.
Although complications occurred in 21% of the patients during 3 months follow-up,
BMC intracoronary administration was related with a reduction of both brain and atrial
natriuretic peptide (NTP) serum levels (indicators of LV remodelling process) in the
remaining population (79%), especially in patients with higher NTP levels at baseline
and receiving a greater BMC number with a high functional capacity. Moreover, these
results were also correlated with a left ventricular ejection fraction (LVEF) increase and
better survival during the further follow-up, suggesting that cell therapy could be](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-43-2048.jpg)
![Tissue Engineering for Tissue and Organ Regeneration
28
associated with cardiac function enhancements in patients with advanced chronic post-
infarction heart failure [56]. Similarly, two hundred and four (204) patients were treated
using bone-marrow-derived progenitor cells directly into the infarct artery three to
seven days after an acute myocardial infarction (AMI). A statistically significant 2.5%
improvement in left ventricular ejection fraction at four months was reported for
patients randomized to the bone marrow injection [57]. Finally, intracoronary infusion
of bone marrow cells was performed in 33 patients with dilated cardiomyopathy
(DCM) by using an over-the-wire balloon catheter. Three month follow-up
demonstrated an improvement in left ventricular pump function while a modest
improvement in Brain Natriuretic Peptide (BNP) levels was reported after 1 year [6].
Importantly, the conditions chosen in the present clinical trial were representative of
different conditions (acute, chronic phase) encountered in the clinic. Unfortunately, no
clear indication on stem cell characterization or on their actual ability to regenerate
contractile cells is available.
3. TRACIA STUDY (Intracoronary Autologous Stem Cell Transplantation in ST
Elevation Myocardial Infarction). The phase II/ III clinical trial aimed at evaluating the
effects of intracoronary administration of adult stem cells on LV ejection fraction and
major adverse cardiovascular events (MACE) after 6 months follow-up. For this reason,
1-2 million CD34+ cells were injected through the infarct-related artery few days after
post-infarct angioplasty using an "over-the-wire" catheter in 80 patients aging from 20
to 75 years. The results of this study are still to be published.
4. Combined CABG and Stem-Cell Transplantation for Heart Failure. Intramyocardial
delivery of autologous bone marrow cells extracted from iliac crest and purified by
Ficoll centrifugation, during cardiac surgery for CABG intervention in 30 patients, as
compared to 30 patients undergoing CABG without cell infusion. Although information
on the number and characteristics of cells to be injected has not been given, the trial is
currently ongoing and the follow-up is scheduled in 6-12 months
(http://clinicaltrials.gov).
5. POSEIDON-Pilot Study (The Percutaneous Stem Cell Injection Delivery Effects on
Neomyogenesis Pilot Study) Poseidon-pilot Study is a phase I/ II multi-center trial in
which the trans-endocardial injection of autologous Mesenchymal Stem Cells (20-, 100-,
200 X 106) is compared to autologous non-purified bone marrow cells and to allogeneic
human Mesenchymal Stem Cells. The implant is performed during cardiac
catheterization using the Biocardia Helical Infusion Catheter in fifty (50) patients
suffering from chronic ischemic left ventricular dysfunction secondary to myocardial
infarction. The data collection is currently ongoing.
6. SCIPIO (Cardiac Stem Cell Infusion in Patients With Ischemic CardiOmyopathy).
This phase I clinical trial is aimed at assessing the safety and effectiveness of
intracoronary autologous cardiac stem cell therapy. As such, forty (40) patients
suffering from ischemic cardiomyopathy are exposed to intracoronary injection of
cardiac resident stem cells (CSC). Cardiac stem cells are harvested from right atrial
appendages and selected for c-kit expression, cultured and expanded in vitro prior to
injecting them via intracoronary route, three to five months after CABG surgery. The
hypothesis is that CSC infused into nonviable myocardial segments will regenerate
infarcted myocardium by differentiating into cardiomyocytes and vascular cells. The
preliminary results are encouraging: in the nine patients treated at four months after](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-44-2048.jpg)
![Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 29
CSC infusion, LVEF increased from 31.3 + 2.5 percent before CSC infusion to 38.8 + 3.2
percent four months after CSC infusion. Moreover, in the five patients in whom data
are available at 12 months after stem cell infusion, the improvement in LVEF observed
at four months was even greater, averaging 15% at 12 months. The follow-up is
scheduled in 1,5 years.
7. ALCADIA (AutoLogous Human CArdiac-Derived Stem Cell to Treat Ischemic
cArdiomyopathy). In this phase I, multicenter clinical trial, a rather different approach
is followed. In fact, patients’ own cardiac stem cells obtained by endo-myocardial
biopsies are delivered by a single intramyocardial injection. The cells injected are 0.5
million cells/kg (patient body weight) and their engraftment should be favored by the
concomitant implantation of gelatin hydrogel sheet releasing human recombinant beta
Fibroblast Growth Factor (bFGF), during CABG surgery. The study has been designed
to treat refractory heart failure, ischemic cardiomyopathy or ventricular dysfunction
cases. Importantly, this is the first clinical trial, to our knowledge, in which a human
recombinant growth factor is used. Unfortunately, the number of enrolled patients is
limited to six (6).
8. REGEN-IHD (Bone Marrow Derived Adult Stem Cells for Chronic Heart Failure). In
this phase II/ III study, granulocyte-colony stimulating factor (G-CSF) is
subcutaneously administered for 5 days to patients with heart failure secondary to
ischemic heart disease to mobilize CD34+ bone marrow stem cells. A concomitant
intracoronary or intramyocardial administration of bone marrow derived stem cells is
performed. The number of enrolled patients is high (165) and the aim of the study is to
compare the effects of G-CSF and autologous bone marrow progenitor cell infusion on
the quality of life and left ventricular function in the patients. The follow-up timepoint
is scheduled in 6-12 months.
A number of papers reporting statistical analyses and comparisons among the clinical trials
in which stem and progenitor cells have been adopted are currently available. [For further
information, please refer to www.clinicaltrials.gov].
5. Conclusions
The possibility to treat cardiac diseases by cell therapy techniques is an extraordinary
promise. While a number of different approaches has been so far proposed to setup
minimally invasive techniques for cardiac repair, few of them being already in the clinical
experimental phase, basic questions still need to be addressed. In fact, the molecular
processes leading to cardiac differentiation still need to be fully clarified, while the impact of
novel, genetically modified cell types obtained from adult differentiated cells on cardiac
microenvironment deserve further investigations. More importantly, the seek to identify
suitable delivery systems (i.e. scaffolds) able to foster stem cell survival, growth and
differentiation, while degrading without negative effects as the formation of new tissue
occurs is still open. A look at the literature reveals that an impressive effort to translate the
information obtained by in vitro and pre-clinical studies to the bedside is being produced. In
particular, a number of stem cell subsets, which have been previously tested in vitro and in
animal models, are currently being tested in phase I, II clinical trials. As expected, the
predominant delivery system used in the ongoing clinical trials is intracoronary or
intramural injection of stem cells. The possibility to adopt tissue engineering techniques to](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-45-2048.jpg)
![Tissue Engineering for Tissue and Organ Regeneration
30
design patient-specific cardiac substitutes containing synthetic or natural scaffolds is still far
from being taken into consideration for clinical application, since any single formulation will
have to be approved before clinical testing.
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34
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![Tissue Engineering for Tissue and Organ Regeneration
38
patients, as the volume of sampled blood needs to be kept to an absolute minimum.
Therefore, the use of alternative precipitation methods with different chemicals has been
evaluated: (1) ethanol (Kjaergard et al., 1992; Weis-Fogh, 1988), (2) ammonium sulphate
alone, and (3) in combination with the cryoprecipitation method (Wolf, 1983), (4) albumin
plus cryoprecipitation, and (5) polyethylene glycol (PEG) plus cryoprecipitation (Epstein et
al., 1986). Heselhaus investigated each of these different precipitation methods with regard
to their efficiency and their use in the development of fibrin scaffold materials for
cardiovascular applications (Heselhaus, 2011):
Fig. 2. [A] Fibrin gel after polymerisation in a 6-well plate. [B-D] Scanning electron
microscopy (SEM) images demonstrating the nano-fibre network structure of the fibrin,
which enables the gentle embedding of cells, with a vascular smooth muscle cell (SMC)
shown in [D] immediately after the gelation of the fibrin within a web-like network
surrounded by cell culture medium
Figure 3 demonstrates that all of the reported alternative methods are more efficient than
the standard cryoprecipitation method. Here, the technique using ethanol as the
precipitation reagent is observed as the most efficient method, with an isolation efficiency of
~80%, ~4-times higher than the efficiency of the standard method. The technique applying
both albumin and cryoprecipitation indicates a false positive high result due to
contamination of the precipitate with albumin (shown by a significant band in SDS gel
electrophoreses) (Heselhaus, 2011).](https://image.slidesharecdn.com/2057541-250604171406-a72741d5/75/Tissue-Engineering-For-Tissue-And-Organ-Regeneration-D-Eberli-54-2048.jpg)
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- 6. Tissue Engineering forTissue and Organ Regeneration Edited by Daniel Eberli Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2011 InTech All chapters are Open Access articles distributed under the Creative Commons Non Commercial Share Alike Attribution 3.0 license, which permits to copy, distribute, transmit, and adapt the work in any medium, so long as the original work is properly cited. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published articles. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Romina Krebel Technical Editor Teodora Smiljanic Cover Designer Jan Hyrat Image Copyright Arun K. Sharma, 2011. First published August, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected] Tissue Engineering for Tissue and Organ Regeneration, Edited by Daniel Eberli p. cm. ISBN 978-953-307-688-1
- 9. Contents Preface IX Part 1Cardiac Muscle 1 Chapter 1 Myocardial Tissue Engineering 3 Tatsuya Shimizu Chapter 2 Cardiac Muscle Engineering: Strategies to Deliver Stem Cells to the Damaged Site 19 Giancarlo Forte, Stefania Pagliari, Francesca Pagliari, Paolo Di Nardo and Takao Aoyagi Chapter 3 Cardiovascular Tissue Engineering Based on Fibrin-Gel-Scaffolds 35 Stefan Jockenhoevel and Thomas C. Flanagan Chapter 4 Rapid Prototyping of Engineered Heart Tissues through Miniaturization and Phenotype-Automation 49 Tetsuro Wakatsuki Part 2 Skeletal Muscle 59 Chapter 5 Tissue Engineering of Skeletal Muscle 61 Klumpp Dorothee, Horch Raymund E. and Beier Justus P. Chapter 6 Skeletal Muscle Tissue Engineering Using Biological Scaffolds for Repair of Abdominal Wall Defects in a Rabbit Model 81 Zuki Abu Bakar, Ayele Taddese Tsedeke, Noorjahan Banu Mohamed Alitheen and Noordin Mohamed Mustapha Chapter 7 Skeletal and Adipose Tissue Engineering with Adipose-Derived Stromal Cells 107 Jeong S Hyun, Emily R Nelson, Daniel Montoro, Benjamin Levi and Michael T. Longaker
- 10. VI Contents Part 3Ligaments 129 Chapter 8 Tissue Engineering of Ligaments 131 Sarah Rathbone and Sarah Cartmell Chapter 9 Potential of Tissue-Engineered Ligament Substitutes for Ruptured ACL Replacement 163 Goulet F., Chabaud S., Simon F., Napa I.D., Moulin V. and Hart D.A. Part 4 Cartilage 179 Chapter 10 Joint Cartilage Tissue Engineering and Pre-Clinical Safety and Efficacy Testing 181 Thomas G. Koch, Lorenzo Moroni, Younes Leysi-Derilou and Lise C. Berg Chapter 11 Cartilage Regeneration from Bone Marrow Cells Using RWV Bioreactor and Its Automation System for Clinical Application 217 Toshimasa Uemura, Masanori Nishi, Kunitomo Aoki and Takashi Tsumura Chapter 12 Cartilage Tissue Engineering: the Application of Nanomaterials and Stem Cell Technology 233 Adelola O. Oseni, Claire Crowley, Maria Z. Boland, Peter E. Butler and Alexander M. Seifalian Part 5 Hollow Organs 267 Chapter 13 Bioengineering of Colo-Rectal Tissue 269 Roman Inglin, Lukas Brügger, Daniel Candinas and Daniel Eberli Chapter 14 Aspects of Urological Tissue Engineering 285 Arun K. Sharma and Dorota I. Rozkiewicz Part 6 Craniofacial Tissues 315 Chapter 15 Tooth Organ Engineering: Biological Constraints Specifying Experimental Approaches 317 Sabine Kuchler-Bopp, Laetitia Keller, Anne Poliard and Herve Lesot Chapter 16 Transplantation of Corneal Stroma Reconstructed with Gelatin and Multipotent Precursor Cells from Corneal Stroma 347 Tatsuya Mimura, Yasuhiko Tabata and Shiro Amano
- 11. Contents VII Chapter 17Human Ear Cartilage 363 Lu Zhang, Qiong Li, Yu Liu, Guangdong Zhou, Wei Liu and Yilin Cao Part 7 Central Nervous System 377 Chapter 18 Advances in the Combined Use of Adult Cell Therapy and Scaffolds for Brain Tissue Engineering 379 Elisa Garbayo, Gaëtan J.-R. Delcroix, Paul C. Schiller and Claudia N. Montero-Menei Part 8 Endocrine Organs 415 Chapter 19 Regenerative Medicine and Tissue Engineering for the Treatment of Diabetes 417 Matsumoto S, SoRelle JA and Shimoda M Chapter 20 Perspectives of Islet Cell Transplantation as a Therapeutic Approach for Diabetes Mellitus 435 Prabha D. Nair and Neena Aloysious
- 13. Preface Over the lastdecade Tissue Engineering progressed rapidly and first biological substitutes were developed for several tissues in the body. Today, Tissue Engineering is one of the major approaches of Regenerative Medicine and represents a growing and exciting field of research. With the understanding and application of new knowledge of structure, biology, physiology and cell culture techniques, Tissue Engineering may offer new treatment alternatives for organ replacement or repair deteriorated organs. Among the clinical applications of Tissue Engineering are the production of artificial skin for burn patients, tissue engineered trachea, cartilage for knee-replacement procedures, urinary bladder replacement, urethra substitutes and cellular therapies for the treatment of urinary incontinence. The classical principle of Tissue Engineering is to dissociate cells from a tissue biopsy, to expand them in culture, and to seed them onto a scaffold material in vitro in order to generate a viable tissue construct prior to re-implantation into the recipient's organism. In the appropriate biochemical and biomechanical environment these tissues will unfold their full functional potential and serve as native tissue equivalents. Tissue Engineering products may be fully functional at the time of treatment, or have potential to integrate and evolve into the expected functional tissue after implantation. While these steps may seem logical and easy to understand, the underlying biology is far more complicated and more profound questions have to be answered before the engineering of tissue and organs becomes a routine practice. Even so, the Tissue Engineering approach has major advantages over traditional organ transplantation and circumvents the problem of organ shortage. Tissues reconstructed from readily available biopsy material implicate only minimal or no immunogenicity when reimplanted in the patient. This eventually conquers several limitations encountered in tissue transplantation approaches. This book is aimed at anyone interested in the application of Tissue Engineering in different organ systems. With a colorful mix of topics which explain the obstacles and possible solutions, it offers insights into a wide variety of strategies applying the principles of Tissue Engineering to tissue and organ regeneration. As more and more applications move toward clinical application, a reliable preclinical model system to
- 14. X Preface evaluate thedeveloped techniques becomes crucial. Several animal models and Tissue Engineering approaches for a variety of organ systems are presented in this book. Finally, I would like to thank all the authors who have supported this book with their contributions. Daniel Eberli University Zürich Switzerland
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- 19. 1 Myocardial Tissue Engineering TatsuyaShimizu Institute of Advanced Biomedical Engineering & Science Tokyo Women’s Medical University Japan 1. Introduction Many lives are lost due to heart diseases including myocardial infarction and cardiomyopathy. Recent reports have demonstrated that regenerative medicine has promising potential for recovering severe heart failure. Regenerative therapies for heart failure include cytokine, gene and cell therapy. Because many types of cardiovascular stem cells have been identified and their clinical potentials have been demonstrated for the past decade, cell injection therapy has most attracted both researchers and clinicians (Wollert 2008). On the other hand, significant cell loss due to washing out and cell death has become problematic in cell injection technique. So, as next generation of regenerative therapy for impaired heart, transplantation of myocardial patches fabricated by tissue engineering technology are emerging and are clinically applied. Furthermore, several challenges for fabricating functional myocardial tissues/organs, which are electrically communicated, pulsate synchronously and evoke contraction power, have also started (Zimmermann, Didie et al. 2006). These ambitious challenges may lead to reconstruction of malformed hearts and become alternative therapy for heart transplantation. Heart tissues are composed of high-dense cylindrical cardiomyocytes and fibroblasts with abundant vascular network and collagen-based extracellular matrix (ECM). Cardiomyocytes pulsate via sodium and calcium ion transient through cell membrane. They are also electrically coupled by gap junctions composed of connexion 43 and rapid electrical propagation realizes simultaneous beating as a whole. Continuous blood flow supplies oxygen and nutrition, and withdraw the waste for high metabolic demand of heart tissues. These structure and function produce mechanical contractions as a blood pump. Therefore the researchers should take into account high density culture of cardiomyocyte and surrounding cells, sufficient micro blood vessel fabrication, cell/ECM orientation and proper cell-to-cell coupling for engineering heart tissues/organs. Here, previous and current status of cell injection therapy, myocardial patch transplantation and pulsatile myocardial tissue fabrication is described with some future views. 2. Cell injection therapy Cell injection therapy for damaged heart has been researched since the early 1990’s. Many researchers have demonstrated the therapeutic potential of isolated cell transplantation into myocardium using various types of cell sources both in animal models and in some clinical
- 20. Tissue Engineering forTissue and Organ Regeneration 4 trials (Puceat 2008). The mechanism of myocardial tissue regeneration has not been completely cleared, but most researchers have agreed that transplanted cells secrete several cytokines which promote neovascularization, prohibit fibrosis, decrease cell death and recruit stem cells, leading to heart function improvement. It has been also asserted that some of injected cells differentiate into functional cardiomyocytes and may directly contribute to heart contraction improvement. Although some differences may exist in according to cell types, multifactorial mechanisms seem to relate with myocardial tissue regeneration. In addition to cell sourcing, different routes are used for cell administration. Systematic intravenous infusion is performed through a central or peripheral vein. This method is simple and less invasive, however widespread distribution cause low ratio of cell engraftment. Most popular approach is intracoronary cell infusion via a balloon-catheter. Injected cells are reached directly in the target myocardial region, however, cells have to transmigrate across endothelium wall. Intracardiac injection is performed via pericardium during open heart surgery and via endocardium by a catheter with a 3-D electromechanical mapping system (NOGA mapping system). These methods realize relatively targeted delivery, but myocardial damage and arrhythmia induction are problematic. Future clarification will be needed to decide which is the best approach for cell injection. 2.1 Skeletal myoblasts Skeletal myoblasts were the first cell source to enter the clinical application for heart tissue repair. They lie in a quiescent state on the basal membrane of myofibers and have the potential to start to proliferate and differentiate into functional skeletal muscle in response to muscle damage. They can be isolated autologously and be expanded from a single biopsy. In addition, skeketal myoblasts are relatively resistant to ischemia. Menasche and colleagues first applied skeletal myoblast injection via epicardium for patients undergoing coronary artery bypass grafting (CABG) (Menasche, Hagege et al. 2001). The phase I clinical study (MAGIC I) have shown the feasibility of skeletal myoblast implantation, however, increased risk of ventricular arrhythmias after the operation. Then, MAGIC II trial was performed to clarify the safety and efficacy, in which all patients received preventive medication and an implantable cardioverter-defibrillator for rescuing critical ventricular arrhythmias. In result, skeletal myoblast injection failed to significantly improve heart function, leading to sample size reduction (Menasche, Alfieri et al. 2008). On the other hand, the trial indicated the possibility that high dose cell injection might recover left ventricular dilatation. In addition, the other clinical trials of catheter-based myoblast implantation via endocardium have revealed functional efficacy (Opie and Dib 2006). According to these results, not the regenerative potential of myoblasts themselves but the amount of injected cells and delivery system may affect the efficacy. Therefore, it seems that skeletal myoblasts should not be excluded as a cell source for heart tissue repair. More optimization of cell delivery and comparison of cell sources can address these critical issues. 2.2 Bone marrow-derived cells Bone marrow-derived cells are the most used cells in clinical trials for myocardial tissue repair (Wollert 2008). The discovery of circulating progenitor cells originated from human bone marrow has stimulated research and clinical use of bone marrow-derived cells
- 21. Myocardial Tissue Engineering5 (Asahara, Murohara et al. 1997). Bone marrow cells contain different stem and progenitor cells which will differentiate into various types of cells including endothelial cells, smooth muscle cells and cardiomyocytes. Bone marrow mononuclear cells (BMNCs), which can be isolated simply by gradient sedimentation after bone marrow aspiration without culture expansion, have been clinically injected via coronary artery from the first. BMNCs include heterogeneous cell population of monocytes, hematopoietic stem cells and endothelial progenitor cells (EPCs). Therefore, some groups have used BMNCs selected by surface markers (CD34+, CD133+) and demonstrated more efficacy of their injection. As another cell population, mesenchymal stem cells (MSCs) have been researched and clinically used. Although, MSCs represent between 0.01 and 0.001% of all nucleated cells in bone marrow, they can be readily expanded in culture. MSCs have the potential to differentiate into various types of cells and injected MSCs in heart seem to differentiate into myocardial composing cells. Recent studies have revealed rare happening of cardiomyocyte differentiation, therefore MSCs seem to recover heart function via their cytokine secretion and partial differentiation into vascular cells. As a unique feature, MSCs have the potential to escape from immune detection due to the direct inflammatory inhibition and the lack of cell-surface molecules. This property has realized allogenic mesenchymal stem cell transplantation in clinic and has given high impact on cell therapy research field. Recent randomized controlled trials of bone marrow-derived cell injection revealed overall feasibility and safety. However the data has revealed only marginal increases of ejection fraction (EF) even in positive studies (0-5%) (Martin-Rendon, Brunskill et al. 2008). For establishing more effective bone marrow-derived cell therapy, optimization of cell source, cell dose, delivery method and deliver timing will be needed. 2.3 Adipose-derived stem cells In addition to bone marrow-derived MSCs, stem cells isolated from the stroma of adipose tissues have represented regenerative potential for heart tissues (Psaltis, Zannettino et al. 2008). Adipose tissue-derived stem cells (ASCs) display features similar to that of bone marrow-derived MSCs and their angiogenic potential have been reported. Some studies have also revealed cardiomyocyte differentiation from ASCs. It has not been clarified which mesenchymal stem cells are superior to other cell types, however, reatively easy isolation of adipose tissue may push the clinical application of ASCs. 2.4 Cardiac stem cells Cardiac stem cells (CSCs) are also possible cell source for myocardial tissue regeneration. Two groups first reported CSC existence in 2003 (Beltrami, Barlucchi et al. 2003; Oh, Bradfute et al. 2003). Until then, it was common knowledge that heart was a post mitotic organ, but those reports accelerated the researches for identifying surface marker of CSCs and culturing them. Islet-1, Sca-1 and c-kit have been known as CSC markers. Recently, it has been also confirmed that heart has renewal ability at normal state and the annual rate of turning over is 1% at the age of 25 (Bergmann, Bhardwaj et al. 2009). Although the ability of CSCs may increase after heart injury, newly formed cardyomyocytes are not sufficient for replacing damaged muscle tissues. Therefore isolation and expansion of CSCs have been extensively examined. Some groups have used a different approach to make cardiospheres from biopsied myocardium, which lead to efficient CSC expansion (Lee, White et al. 2011).
- 22. Tissue Engineering forTissue and Organ Regeneration 6 Clinical trials for injection therapy of autologous CSCs isolated from biopsy sample are now on going. 2.5 Embryonic stem cells Although abundant studies demonstrated that MSCs, ASCs and CSCs have the potential of cardiomyocyte differentiation regarding gene and protein expression, there are no studies clearly showing beating cardiomyocytes differentiated from those stem cells. On the other hand, many researchers have confirmed that embryonic stem cells (ESCs) can differentiate into beating cardiomyocytes in vitro and implantation of ESC-derived cardiomyocytes improves damaged heart function. Several signal pathways for cardiac differentiation have been already clarified and various molecules have been reported as its promoters. For example, noggin increased cardiac differentiation efficacy via regulation of Bone morphogenetic protein (BMP) signalling pathway (Yuasa, Itabashi et al. 2005) and insulin- like growth-factor-binding protein 4 (IGFBP4) promotes cardiogenesis by inhibitor of canonical Wnt signalling (Zhu, Shiojima et al. 2008). In addition, fibloblast growth factor (FGF), retinoic acid, ascorbic acid and cyclosporine A have been reported to have the potential to enhance cardiac differentiation from ESCs. The important issue as well as cardiac differentiation is purification of cardiomyocytes from heterogeneous cell mixture, because contamination of immature cells leads to teratoma formation. Although gene- modified ESCs harboring neomycin resistance gene or green fluorescent protein (GFP) gene in the cardiac-specific gene locus are very useful in non-clinical experiments, safe and efficient isolation technologies will be needed for clinical application. Culture media control focusing on the differences of cell metabolism may be useful for safe cell selection. Moreover immune response of the host is another critical issue. Nucler transfer or cell banking is possible approach avoiding immunoreaction. Electrical communication and simultaneous beating of implanted ESC-derived cardiomyocytes should be also requested for improving damaged heart function without arrhythmia. In vivo electrophisiological analyses and the transplantation technology for synchronization will be essential for clinical application of these cells. 2.6 Induced pluripotent stem cells Induced pluripotent stem cells (iPSCs) also hold great promise for myocardial tissue engineering (Vunjak-Novakovic, Tandon et al. 2010). Terminally differentiated cells can be reprogrammed to have the same potential as ESCs by introducing 3 or 4 transcriptional factor genes. Furthemore none-gene transfer technologies have been developed in the world. The superiority of iPSCs to ESCs is autologous cells, which do not cause immune response. Cardiac differentiation of human iPSCs has been reported in the same manner with ESCs. Several critical issues must be clarified for clinical use, but ESCs/iPSCs-derived cardiomyocytes should contribute to myocardial tissue engineering in the view point of their pulsatile function and scaling-up. 2.7 Problems of cell injection therapy Cell injection therapies for heart failure are now world-widely performed. While moderate success of direct cell injection has been observed, the efficacies seem not to reach the level that general clinicians think cell therapy a reliable treatment for heart failure. More
- 23. Myocardial Tissue Engineering7 optimization of cell source, cell preparation process, injection route, injection timing and patient population may increase the effectiveness; however one of the essential issues is cell delivery methodology. Cell injection therapy has significant difficulties about cell retention in the target tissue. The shape, size, and position of the grafted cells are often uncontrollable and large amount of the cells are washed-out. Moreover, once retaining cells die due to necrosis and apoptosis. Time course quantification with TUNEL assay demonstrated that a large number of the grafted cells die within a few days after injection in rat models (Zhang, Methot et al. 2001). In the clinical trial using bone marrow-derived cells, it has been also demonstrated that only 1-3% of the cells infused via coronary arteries could be detected by 3D positron emission tomography (PET) imaging of the patient heart. In this study, a large percentage of cells were found in the liver and spleen immediately after the procedures (Hofmann, Wollert et al. 2005). To clear the problem of cell loss, hydrogel-cell mixture injection has been pursued. Fibrin, collagen and alginate hydrogels are now used. Hydrogels with cells are injected as a liquid phase through syringe or catheter, then, they are polymerized and fixed in the target tissues (Kofidis, de Bruin et al. 2004). In hydrogel-cell mixture injection therapy, local tissue damage due to space occupation of hydrogel itself and inflammatory reaction due to hydrogel biodegradation are problematic. Therefore, more advanced cell delivery systems have been requested to spread the regenerative therapy as one of the reliable treatments for heart failure. 3. Tissue engineering Recent advance of tissue engineering technologies have realized the transplantation of tissue-engineered construct “myocardial patch” covering over damaged heart surface instead of simple cell injection into myocardium. Grafted cells within myocardial patches can survive more and secrete more cytokines, resulting in more heart function improvement. Furthermore pulsatile myocardial tissues have been successfully engineered by using cardiomyocytes as a seeding cell source. These tissues may directly help heart contraction and total heart wall replacement may be possible in future. There are several contexts of tissue engineering. 3.1 Scaffold-based tissue engineering Most popular technology of tissue engineering is to seed cells into 3-D pre-fabricated biodegradable scaffolds which are made from synthetic polymer and biological material. Hydrogel formation after mixing cells and scaffold solution is another approach. Decellularized tissues have been also used as scaffolds. These scaffolds play as alternatives for extra cellular matrix (ECM), therefore, their cell-adhesiveness and porosity affect survived cell amount and engineered tissue quality. Scaffold modification can control its biodegradation and tissue formation. Growth factor linkage leads to accelerating tissue formation. Now these scaffold-based tissue engineering has been widely applied to cardiovascular tissue regeneration as well as other tissue repair (Vunjak-Novakovic, Tandon et al. 2010). 3.2 Cell sheet-based tissue engineering In contrast to scaffold-based tissue engineering, our group have developed unique technique involving cell sheet stacking to fabricate 3-D tissues (Shimizu, Yamato et al. 2003).
- 24. Tissue Engineering forTissue and Organ Regeneration 8 Cell sheets are 2-D connecting pure cells without any scaffolds, therefore cell-dense 3-D tissues can be fabricated by stacking cell sheets. Cell sheets are harvested from intelligent culture surface “temperature-responsive culture surface”, which are covalently grafted with temperature-responsive polymer, poly (N-isopropylacrylamide) (PIPAAm) (Okano, Yamada et al. 1993). The surfaces are slightly hydrophobic and cell-adhesive at 37˚C, on the other hand, the surface changes to hydrophilic and not cell adhesive below 32˚C. Confluently cultured cells on the surface can detach as a contiguous cell sheet simply by reducing temperature. Furthermore, biological molecules underneath cell sheets are also preserved and play a critical role as an adhesive agent during cell sheet stacking. Cell sheet-based tissue engineering has been applied for a wide range of regenerative medicine including corneal epithelial replacement, heart tissue repair, pneumothorax repair, liver tissue repair and so on. According to the spread of the concept fabricating 3-D tissues from 2-D confluent cells, several other technologies using this concept have emerged. Cell sheet fabrication techniques using fibrin coated dishes or nanofibrous polycaprolactone meshes have been reported (Shin, Ishii et al. 2004; Itabashi, Miyoshi et al. 2005). Cell sheet-like constructs have been also engineered using magnetite nanoparticles (Ito, Hibino et al. 2005). Magnetically labelled cells are attached on culture materials by magnetic force and confluent cells are harvested as a cell sheet by magnetic force release. Thus, cell sheet-based tissue engineering has now spread in the world as scaffold-free tissue engineering. 4. Myocardial patch transplantation Both scaffold-based and cell sheet-based tissue engineering have been used for myocardial patch fabrication. Not only cardiomyocytes but also other types of cells have been used for creating myocardial patches and some myocardial patches using non-cardiomyocytes have been already clinically transplanted over damaged hearts. (Fig. 1.) 4.1 Scaffold-based mayocardial patch In myocardial patch fabrication, synthetic polymer, biological material and decellularized tissue have been used as prefabricated scaffolds. Li and colleagues, who were one of the pioneer groups of myocardial tissue engineering, first demonstrated that gelatine sponges seeded with cardiac cells have therapeutic potentials for cryoinjured rat hearts (Li, Jia et al. 1999). Leor and colleagues reported that bioengineered heart grafts using porous alginate scaffolds attenuated left ventricular dilatation and heart function deterioration in infarction model (Leor, Aboulafia-Etzion et al. 2000). Eschenhagen and Zimmermann’s group have developed innovative myocardial tissue engineering approach (Zimmermann, Schneiderbanger et al. 2002). They have fabricated 3-D tissues by gelling mixture of cardiac cells and collagen solution. The constructs induced systolic wall thickening of the left ventricle infracted area and improved fractional shortening of damaged hearts in rat myocardial infarction model (Zimmermann, Melnychenko et al. 2006). Small intestinal submucosa (SIS) has also been used as a scaffold for myocardial patch. MSC-seeded SIS improved heart contraction in rabbit infarction model (Tan, Zhi et al. 2009). There have been various types of myocardial patches using different scaffolds and different cell sources. Although implantable human myocardial patches using beating cardiomyocytes have not been established now, clinical trials of collagen-based myocardial patch with bone marrow
- 25. Myocardial Tissue Engineering9 cells (MAGNUM trial) (Chachques, Trainini et al. 2007) and vicryl mesh-based myocardial patches with fibroblasts (Anginera) ((Mirsadraee, Wilcox et al. 2006)) have revealed feasibility and safety of myocardial patch transplantation. Diseased heart Cells Stacking 3. Cell sheet-based TE Temperature-responsive culture surface Cells & ECM mixture Gelling Scaffold 1. Scaffold-based TE 2. Hydrogel-based TE Cell sheet Seeding Fig. 1. Tissue engineering (TE) strategies for myocardial patch fabrication 4.2 Cell sheet-based myocardial patch Many types of cell sheets have been reported to improve impaired heart function (Shimizu, Sekine et al. 2009). Cell sheets are transplanted onto heart surface directly via open heart surgery and cells can be more effectively delivered as thin, but large-area cell-dense grafts than isolated cell injection. Scaffold-based myocardial patches are usually transplanted on myocardium with suture, on the other hand, cell sheets are transplanted with no suture because biological adhesive proteins underneath cell sheets promote the attachment. When neonatal rat cardiac cell sheets were transplanted onto infracted rat hearts, grafted cardiomyocytes communicated with host myocardium via gap junctions and blood vessels formed within the graft, resulting in significant improvement of heart function (Miyagawa, Sawa et al. 2005; Sekine, Shimizu et al. 2006). Sawa and colleagues have started to use skeletal myoblasts for cell sheet fabrication, because myoblasts can be isolated autologously and are relatively resistant to ischemic condition.
- 26. Tissue Engineering forTissue and Organ Regeneration 10 The recovery of heart function by skeletal myoblast transplantation has been confirmed in rat ischemic model, in dilated cardiomyopathy hamster model, in pacing-induced canine heart failure model and in pig infarction model (Memon, Sawa et al. 2005; Hata, Matsumiya et al. 2006; Kondoh, Sawa et al. 2006; Miyagawa, Saito et al. 2010). Regarding stacking cell sheet number, 3-5 layers are optimal and more layering cause primary necrosis of the constructs (Sekiya, Matsumiya et al. 2009). They have demonstrated more hematopoietic stem cells and less fibrosis in cell sheet transplantation than in isolated cell injection in accordance with more expression of stromal-derived factor 1 (SDF-1), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). Based on these results, clinical trial of autologous myoblast sheet transplantation for severe heart failure has started and the detailed results will appear soon. In the same manner with cell injection therapy, MSCs are used as a candidate cell source for human implantable cell sheet. Adipose tissue-derived MSCs and menstrual blood-derived MSCs have improved damaged heart function in rat infarction model (Miyahara, Nagaya et al. 2006; Hida, Nishiyama et al. 2008). MSCs can gradually grow to form a thick stratum containing newly formed blood vessels and some cells seem to differentiate into cardiomyocytes at least by histological analyses. Further studies will be needed to confirm the differentiation into functional beating cardiomyocytes and possibilities to differentiate into unexpected cell types. As emerging cell source, cell sheets of stem cell antigen 1-positive (Sca-1-positive) CSCs ameliorates cardiac dysfunction in mouse infarction model through cardiomyocyte differentiation and paracrine mechanisms mediated via soluble vascular cell adhesion molecule 1 (VCAM-1)/very late antigen-4 (VLA-4) signaling pathway (Matsuura, Honda et al. 2009). In addition, cardiac cell sheets originated from ESCs/iPSCs have been successfully fabricated and their transplantation into animal models is now ongoing. For enhancing the efficacy of cell sheet transplantation, gene-modified cell sheets have been examined. Bcl-2 expressed myoblast sheets prolonged survival, increased production of proangiogenic paracrine mediators, and enhanced the therapeutic efficacy (Kitabayashi, Siltanen et al. 2010). HGF overexpression in myoblast sheets enhances their angiogenic potential in rat chronic heart failure model (Siltanen, Kitabayashi et al. 2011). As another concept, cell sheets co-cultured with endothelial cell sources have been transplanted in rat infarction models. Transplantation of EPC co-cultured fibroblast sheet improved heart function more than only fibroblast sheet implantation or EPC injection (Kobayashi, Shimizu et al. 2008). Furthermore, endothelial cell co-culture within cardiomyocyte sheets induced more neovascularization and more improvement of cardiac function than only cardiomyocyte sheets (Sekine, Shimizu et al. 2008). These studies indicate advanced strategies of cell sheet transplantation. As mentioned previously, it is considered that the main mechanism of heart function improvement is neovascularization, fibrosis inhibition, apoptosis inhibition and stem cell recruitment due to various cytokines secreted from grafted cells. In comparison with cell injection approach, increase of cell survival within myocardial patches leads to more cytokine secretion, then, resulting in more function improvement. In addition to cytokine secretion, myocardial patches may have girdling effect and prohibit heart dilatation. Therefore, myocardial patch transplantation is quite different cell delivery method from cell injection and has more potential to rescue diseased hearts. In the case of myocardial patches using beating cardiomyocytes, direct enhancement of contraction power is additionally
- 27. Myocardial Tissue Engineering11 expected, however, electrical synchronization between host hearts and transplanted patches is a critical issue to be clarified. 5. Engineering plusatile myocardial tisssue Beyond myocardial patch fabrication, several research groups have challenged to fabricate pulsatile myocardial tissues by their original tissue engineering strategies. Bioengineered contractile myocardial tissues may realize new therapeutics for severe heart diseases and be useful as alternatives for animal models. 5.1 Pre-fabricated scaffold-based myocardial tissue fabrication The first approach for engineering functional myocardial tissue is seeding cardiomyocytes into synthetic or biological 3-D scaffolds. Vunjak-Novakovic and colleagues first reported that seeding primary cultured cardiomyocytes onto disc-shaped polyglycolic acid (PGA) scaffolds in rotating bioreactor system resulted in spontaneously pulsatile myocardial tissues (Papadaki, Bursac et al. 2001). Optimization of cell population, serum concentration and scaffold coating improved electrical conduction velocity of engineered constructs. Radisc and colleagues seeded rat cardiomyocytes in Matrigel onto collagen sponges and stimulated the constructs electrically. The stimulation improved the conductive and contractile properties in accordance with increased expression of myosin heavy chain and connexion 43. Furthermore, cardiomyocytes in the electrically stimulated constructs were more aligned and elongated as same as those in native heart tissue (Radisic, Park et al. 2007). Following these studies, many research groups have started to engineer myocardial tissue in vitro by using various types of scaffolds. Scaffold porosity is one of the critical factors for pre-fabricated scaffold-based tissue engineering. High porosity increases seeded cell number and facilitates mass transport. Surface modyfication is also important for cell attachment and survival. Laminin coating improved cardiomyocyte adhesiveness. In addition, scaffold elasticity and degradability affect contraction property of engineered myocardium. Further studies are ongoing to development appropriate scaffold materials for myocardial tissue engineering. 5.2 Hydrogel-based myocardial tissue fabrication The second approach is to form 3-D tissues by gelling of cardiac cell and matrix solution mixture. Eschenhagen and Zimmermann have continuously developed this strategy using collagen gel and successfully engineered macroscopically beating cardiac tissues (Zimmermann and Cesnjevar 2009). First, neonatal rat cardiomyocytes were suspended in collagen I solution and the mixture was poured into the mold. After gelling, the constructs were unidirectionally stretched with the mechanical devise. They have also realized contraction force measurement. Cyclic stretch introduced cell alignment along the stretching direction and increased mitochondrial density, leading to native heart-like tissue. The contraction force of engineered myocardium was comparative with native heart tissue and responded to pharmacological agents properly. Ring-shaped myocardial tissues were also fabricated and combined 5 constructs were transplanted onto infarcted rat hearts. Interestingly, the constructs synchronized to each other and improved damaged heart function. They have also confirmed that co-culture constructs including cardiomyocytes,
- 28. Tissue Engineering forTissue and Organ Regeneration 12 fibroblasts and endothelial cells were superior to cardiomyocyte rich constructs in morphology and function. Recently, they have also started to utilize human cardiomyocytes differentiated from ESC/iPSC as cell source and challenged to create human myocardial tissues (Zimmermann 2011). In contrast to pre-fabricated scaffold usage, relatively homogeneous myocardial tissues are engineered by hydrogel-based approach. Therefore collagen gel-based myocardial tissue engineering has now become popular in the world. 5.3 Cell sheet-based myocardial tissue fabrication The third approach is to engineer 3-D pulsatile myocardial tissues by stacking cardiac cell sheets. As mentioned previously, 2-D cell sheets can be harvested from temperature- responsive culture dishes only by lowering temperature and do not include any materials. 3- D tissues are constructed by layering cell sheets. Because 2-D confluent cells are directly stacked without any scaffolds, resulting constructs are cell-dense 3-D tissues. It is well- known that 2-D confluent cardiomyocytes connect to each other electrically via gap junctions resulting in synchronized beating. Cardiac cell sheets harvested from temperature- responsive culture dishes maintain this synchronized pulsation (Shimizu, Yamato et al. 2002). For creating 3-D functional heart tissues by layering cardiac cell sheets, morphological and electrical communications between cell sheets are critical. Multiple-electrode extracellular recording system revealed that double-layer rat cardiac cell sheets coupled electrically about one hour after layering and histological analysis showed the existence of connexin 43 between two cardiac cell sheets. Adhesive proteins deposited on cell sheet surface are considered to promote these rapid electrical communications (Haraguchi, Shimizu et al. 2006). Stacked cardiac cell sheets beat synchronously in macroscopic view and the constructs transplanted into rat subcutaneous tissues also pulsated continuously at least up to one year and eight months after implantation. Morphological analyses showed elongated cardiomyocytes, well-differentiated sarcomeres, gap junctions and multiple blood vessels, which were characteristic structure of native heart tissue (Shimizu, Yamato et al. 2002). Long-term observation revealed that their size, conduction velocity, and contractile force increased in proportion to the host growth (Shimizu, Sekine et al. 2006). Recently, fabrication of cardiac cell sheets using ESC-originated cardiomyocytes have just started and human cardiac cell sheets will appear in near future. 5.4 Fabrication of vascularized myocardial tissue One of the major obstacles in myocardial tissue engineering is scaling-up of the constructs. Insufficient supply of oxygen and nutrient, and waste accumulation limit their thickness. Actually, cells are sparse in the central area, on the other hand, cells are dense in the outer surface (100-200μm) area in scaffold-based myocardial tissue engineering. In the case of cell sheet-based myocardial tissue engineering, thickness limit is approximately 80μm (3 layers) (Shimizu, Sekine et al. 2006). Several approaches have been examined in the point of view overcoming diffusion limit. Perfusion of culture media through the constructs using porous scaffolds is one possible approach. Media penetration increased cell migration depth and improved cell metabolism. However shear stress due to media flow may prohibit tight cell attachment on the scaffold material. Media perfusion with oxygen carrier, perfluorocarbon (PFC) has been also examined for improving oxygen transport. PFC usage increased cell proliferation and improved pulsatile function. Media penetration is useful to some extent, however, it becomes more difficult as cell density increases.
- 29. Myocardial Tissue Engineering13 To overcome this problem, it has been requested to develop new technologies for introducing vasculature or vascular-like structure into engineered tissues. Several researchers have tried to generate microchannel network within porous 3-D scaffolds by microfabrication techniques including CO2 laser ablation. The technology has not reached to mimicking native micro capillary network. On the other hand, recent studies have revealed that co-cultured endothelial cells within cardiac constructs can spontaneously form vascular-like network in vitro and tubular formation has been found in some parts. It has been also confirmed that this pre-vascular structure connected to host blood vessels immediately after transplantation and the newly developed vessels within the constructs were blood-supplied within a few days (Sekiya, Shimizu et al. 2006). We have already demonstrated that the tissue thickness of cardiac cell sheets co-cultured with endothelial cells were just twice as the thickness of cardiac cell sheets without endothelial cells (Sekine, Shimizu et al. 2008). Although endothelial cell co-culture is helpful for accelerating blood vessel formation, more scaling-up is still limited due to primary ischemia until sufficient vascularization. One possible idea for scaling-up is utilizing in vivo vascularization power. Our group has reported that triple-layer cardiac cell sheets were repeatedly implanted after waiting enough vascular formation within previously implanted tissues. In result, synchronously beating thick myocardial tissues with sufficient micro capillaries were successfully fabricated and 10-times transplantation of triple-layer constructs (totally 30 sheets) formed 1-mm thick, pulsatile myocardial tissues. Furthermore, when triple-layer grafts were transplanted repeatedly over a surgically connectable artery and vein in leg, the multilayer constructs were blood-supplied from the thick artery and vein. The constructs were successfully resected with the connectable blood vessels and were ectopically transplanted in neck with direct vessel anastomoses (Shimizu, Sekine et al. 2006). Recently several groups have also utilized in vivo power for myocardial tissue engineering. Cardiomyocytes, ECM alternatives and native blood vessels were packed in the special chamber and incubated in vivo. Vascularized heart-like tissues were created in the body (Morritt, Bortolotto et al. 2007; Birla, Dhawan et al. 2009). Furthemore, next challenge is now in vitro fabrication of vascularized myocardial tissues. Kofidis and colleagues have constructed fibrin gel-based myocardial tissues containing rat aortas (1-2mm), through which culture media was perfused (Kofidis, Lenz et al. 2003). Cell survival and metabolism were improved, however formation of functional blood vessels connecting with central aortas were not clear. We are now trying to promote endothelial cell tubular formation within in vitro engineered cardiac tissues and to perfuse culture media through the newly formed vessels using perfusion bioreactors. Further studies will be needed to break through the obstacles for in vitro scaling up. 5.5 From tissue engineering to organ engineering For future organ engineering, some groups have challenged to engineer myocardial constructs with pumping function. Ott and colleagues have used decellularized organ as a scaffold. They decellularized rat whole hearts and re-seeded cardiac cells into decellularized hearts. Heart contraction was recovered and pump function was generated (Ott, Matthiesen et al. 2008). Zimmermann’s group developed pouch-like myocardial tissue by their technology as previously described and covered heart with pouch-like constructs (Yildirim, Naito et al. 2007).
- 30. Tissue Engineering forTissue and Organ Regeneration 14 Regarding cell sheet technology, myocardial tubes have been fabricated by wrapping rat cardiac cell sheets around fibrin tubes and rat resected aortas. The engineered myocardial tubes revealed spontaneous, synchronized pulsation and small but significant inner pressure changes (about 0.1 mmHg) in vitro (Kubo, Shimizu et al. 2007). On the other hand, resected rat aortas wrapped with cardiac cell sheets were micro surgically transplanted in place of the abdominal aorta. After 1 month, in vivo myocardial tubes demonstrated spontaneous beating and evoked independent blood pressures (about 6 mm Hg). The value of in vivo myocardial tubes was much bigger than in vitro myocardial tubes (Sekine, Shimizu et al. 2006). Comparing in vitro and in vivo, it was considered that pulsation due to host blood flow has induced cardiomyocyte hypertrophy, leading to improvement of pumping function. Therefore pulsatile perfusion bioreactors may improve pumping function of in vitro engineered myocardial tubes. Thus, small size myocardial constructs evoking pumping function have been realized. Expansion and selection of cardiomyocytes, and sufficient blood vessel formation for scaling-up are now critical issues for organ engineering. 6. Conclusions As the first generation of cardiac regenerative therapy, many clinical trials of cell injection therapy have been already performed. The controversial arguments about its effectiveness will be settled in next several years. Tissue engineered myocardial patches have now emerged as the second generation and previous studies indicate promising potential for rescuing damaged heart. As the third generation, tissue-engineered pulsatile myocardial tissues should support heart contraction physically. Furthermore, future development of cell sourcing and scaling-up technologies may realize “bioengineered hearts”. 7. Acknowledgment This work is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). 8. References Asahara, T., T. Murohara, et al. (1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science 275(5302): 964-967. Beltrami, A. P., L. Barlucchi, et al. (2003). Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 114(6): 763-776. Bergmann, O., R. D. Bhardwaj, et al. (2009). Evidence for cardiomyocyte renewal in humans. Science 324(5923): 98-102. Birla, R. K., V. Dhawan, et al. (2009). Cardiac cells implanted into a cylindrical, vascularized chamber in vivo: pressure generation and morphology. Biotechnol Lett 31(2): 191- 201. Chachques, J. C., J. C. Trainini, et al. (2007). Myocardial assistance by grafting a new bioartificial upgraded myocardium (MAGNUM clinical trial): one year follow-up. Cell Transplant 16(9): 927-934.
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- 35. 2 Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site Giancarlo Forte1, Stefania Pagliari2, Francesca Pagliari2, Paolo Di Nardo2 and Takao Aoyagi1 1Biomaterials Center, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Tsukuba, 2Laboratorio di Cardiologia Molecolare e Cellulare, Dipartimento di Medicina Interna, Università di “Tor Vergata”, Roma, 1Japan 2Italy 1. Introduction In healthy human hearts, only 10-20% of the total cells are contractile cardiomyocytes and, at the age of 25 years, no more than 1% of them are annually substituted by progenitor cells, this percentage reducing to less than 0.5% at the age of 75. In total, less than 50% of cardiomyocytes are renewed during a normal human life span [1]. For this reason, the topic of cardiac repair is among the major challenges for the tissue engineers worldwide. In fact, cardiac diseases are a predominant cause of mortality and morbidity in industrialized countries, despite the recent advancements achieved in pharmacological treatment and interventional cardiology procedures. Nonetheless, end-stage heart failure management still relies on organ transplantation as unique approach, and, notwithstanding the use of massive immunosuppressive drugs, still a percentage falling within 20%-40% of patients encounters immune rejection during the first year post-transplant [2]. Among the patients not facing severe immune rejection, almost 70% is forced to retire or reduce their working activity, their survival rate falling below 70% during the first five years post organ transplantation [3]. Last, but not least, the economic impact of cardiovascular diseases and stroke has been estimated in 2010 at $503.2 billion [4]. Currently, post-infarction myocardial revascularization protocols include the administration of raw bone marrow stem cells, while a number of clinical trials have been performed or are currently in progress in which different cell subsets are implanted in the damaged tissue by means of surgical techniques. The results of such trials are still controversial. In fact, when autologous skeletal myoblasts were injected into the heart of patients suffering from ischemic cardiomyopathy, the modest functional improvement obtained was impaired by the arising of arrhythmia events, thus requiring the adoption of a pacemaker [5]. On the other side, intracoronary administration of bone marrow mesenchymal stem cells resulted in minimal improvements in cardiac contractile function in patients with dilated cardiomyopathy [6]. These mild results were mostly ascribed to a paracrine effect exerted on host tissue, rather than to a direct contribution of stem cells to the contractile activity.
- 36. Tissue Engineering forTissue and Organ Regeneration 20 Thus, among the criticisms to be challenged before efficient cell therapy protocols for cardiac diseases can be setup, the choice of the appropriate cell subset to generate new vessels and contractile cardiomyocytes, as well as the route of cell delivery remain key steps. The solution of such problems requires additional efforts in basic research to clarify the processes leading to stem cell differentiation as well as technological advancements to setup efficient protocols to implant the cells. In principle, adult stem cells could be extracted from patient’s own tissues and expanded in culture by means of well-known techniques (Figure 1). Fig. 1. Cardiac Tissue Engineering paradigm. Adult stem cells can be harvested, purified from the patient and expanded in culture. Such cells can be delivered to the injured heart by injection (intramural or through bloodstream with or without injectable carriers), or in the form of solid bio-constructs. Stem cell-derived bio-constructs can be obtained by culturing the cells on scaffolds or by scaffold-free technology Nonetheless, a number of issues should be challenged before safe procedures to manipulate stem cells in vitro for cardiac transplant can be setup. In fact, stem cells should be amplified in vitro to reach a critical number (Figure 1). During this passage, malignant transformation is likely to occur in ex vivo cells when standard culture conditions are adopted to expand stem cells [7, 8]. On the other side, stem cells could encounter senescence after a short number of passages in vitro [9]. Moreover, the use of animal- derived supplements during the phase of cell expansion would hinder the use of stem cells for cardiac cell therapy.
- 37. Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site 21 The employment of autologous stem cells would avoid the problem of immune rejection and the need for immune-suppressive drugs, while, in the treatment of pathologies for which a genetic basis is suspected the use of autologous cells is hampered. As far as the use of autologous cells is concerned, the possibility that a significant patient-to-patient variability in stem cell quality exists should be taken into account [10]. Finally, the use of cellular and tissue-based products in human disease therapy is subjected to regulations issued by the European Union and Food and Drug Administration (FDA) aimed at establishing classification criteria for advanced therapy medicinal products (ATMP). In particular, the European Regulation states that human cells to be used in cell therapy have to comply with the principles of Good Manufacturing Practice (GMP) protocols [11, 12]. 2. Adult stem cells for cardiac repair A number of stem cells and progenitors have been so far proposed for cardiac repair, due to the inability of cardiomyocytes to proliferate after birth [1]. Among the cell sources challenged for the possibility to produce new cardiomyocytes, skeletal myoblasts have proven to be able to acquire a contractile phenotype in vitro [13]. Moreover, when implanted in vivo in a canine model of dilated cardiomyopathy (DCM), they attenuated cardiac remodeling [14]. This result is likely to be due to the fusion of skeletal myoblasts with the surrounding myocardium rather than to direct cell differentiation, as suggested by in vitro experiments [15]. As discussed in the following section, clinical trials demonstrated that skeletal myoblasts are not able to couple electrically with host tissue, leading to arrhythmia events [5]. The role of hematopoietic stem cells (HSC) in cardiac repair has been investigated by several research groups and their contribution to cardiac regeneration in vivo has been heavily debated, being the ability of HSC to transdifferentiate to other lineages still questionable. Indeed, evidence of the ability of bone marrow-derived c-kit+ HSC to help cardiac tissue healing has been given using two different approaches: c-kit+ cells were (i) either delivered to the infarcted site by intramural injection [16] or (ii) mobilized from bone marrow through growth factor administration [17]. More recently, elegant experiments compellingly clarified that HSC are not able to acquire contractile phenotype in vivo [18-20]. Nonetheless, a subset of bone marrow hematopoietic precursors expressing CD34 and CD133 has been proven to contain endothelial progenitors. Thus, they have been tested for revascularization protocols in hind limb ischemic animals and could be proposed for cardiac infarction therapy [21]. On the other hand, the results obtained in preliminary investigations in which another bone marrow-derived stem cell subset, mesenchymal stem cells (BM-MSC or MSC) were challenged as a candidate for cellular cardiomyoplasty, raised great enthusiasm for such a cell subpopulation. Recent studies clarified that the direct contribution of MSC to cardiac repair in terms of production of new contractile cells is minimal if any, while a paracrine effect on the diseased tissue of such cells is universally recognized [22]. Such cells are also appealing for their ability to induce a certain degree of immune tolerance [23]. The presence of a small reservoir of cardiac resident progenitor cells (CPC or CSC) has been recently demonstrated in human as well as in other mammals’ heart [24]. Such tissue- resident cells participate in myocardial homeostasis and retain a limited regenerative capacity throughout organism lifespan [1]. All the subsets so far identified through the expression of stemness markers (c-kit+, Sca-1+, Islet-1+) demonstrated the ability to give birth to new contractile cells in vitro, while only c-kit+, Sca-1+ progenitors were shown to be
- 38. Tissue Engineering forTissue and Organ Regeneration 22 involved in post-natal cardiac tissue homeostasis in vivo [25]. In fact, the presence of Islet-1+ cells appears to be limited to fetal life and their contribution to the endogenous program of cardiovascular repair is still unknown; on the other hand, the very low number of c-kit+ and Sca-1+ cells in the myocardium is considered the limiting factor of cardiac regeneration [26]. Furthermore, among the adult stem cells, a novel “artificial” subset can be recognized: induced pluripotent stem cells (iPSC, Figure 2). This cell type can be produced in vitro by transducing somatic cells with a combination of transcription factors able to induce the nuclear reprogramming of differentiated cells. These cells, which display the functional features of pluripotent embryonic stem cells, have been credited of the ability to produce new cardiomyocytes. They could thus be the source of autologous, although genetically modified, patient-specific contractile cells [27]. Moreover, the possibility to directly obtain functional cardiomyocytes by the genetic reprogramming of postnatal cardiac or dermal fibroblasts has been demonstrated [28]. Such a result was firstly obtained in vitro but also when the cells were transplanted into mouse hearts one day after transduction of transcription factors (GATA-4, MEF-2c, Tbx-5) known to be involved in cardiac muscle development. Nonetheless, the reprogramming and differentiation efficiency of these cells appears to be really low, thus requiring an efficient purification step before they can be implanted in vivo. Additionally, safety concerns due to the use of genetically modified cells and / or viral vectors remain. Fig. 2. Induced Pluripotent Stem Cell Generation. Induced pluripotent Stem Cells (iPSC) can be generated by reprogramming somatic cells through their transduction with four transcription factors. iPSC share functional similarities with Embryonic Stem Cells (ESC) and can be differentiated towards cardiomyocytes, thus representing an autologous source of contractile cells
- 39. Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site 23 3. Stem cell delivery to the injured heart As previously said, cell route of delivery to damaged heart represents the major topic in the setup of efficient, minimally invasive techniques to treat cardiac pathologies. Recently, a number of techniques to deliver stem cells to the injured site have been proposed but questions remain regarding the optimal approach able to favor high cell retention, differentiation rate and clinically relevant improvement in cardiac performance. a) Direct injection Stem cell direct intramural injection, including trans-epicardial and trans-endocardial cell injection, is the elective strategy for patients with severe occlusion of coronary vessels. In particular, trans-epicardial approach consists in the direct injection of a high number of cells into the infarcted area or around the border zone. Endocardial stem cell injection is performed using catheters such as MyoStarTM injection catheter (Biosense Webster) integrated with imaging systems like NOGA® system (Cordis Corp., Warren, NJ, USA), which allows real-time three-dimensional reconstruction of left ventricle as well as the targeting and functional assessment of specific myocardial area [29]. Such procedures are highly invasive since they require open-heart surgery and gave contrasting results so far. For example, pre-clinical studies performed on experimental animals demonstrated that, although a certain extent of cardiac repair was achieved when bone marrow Stro-3+ perivascular cells are implanted in vivo, the cells vanished from the application site within few days [30]. In other reports, when Sca-1+ cardiac resident stem cells were injected in infarction border zone, a modest but significant improvement in cardiac function was reported, with evidence of cell engraftment and differentiation [31]. Finally, in another pre-clinical study, bone marrow-derived c-kit+ cells were shown to repair entire ventricular areas while massively engrafting and differentiating in contractile and vascular figures in vivo [32]. Of interest, independent groups already demonstrated that c- kit+ bone marrow-derived hematopoietic stem cells fail to acquire contractile phenotype when implanted in diseased myocardium [19, 20]. Such discrepancies are not surprising since different stem cell subsets or preparation protocols were probably used in these studies. Stem cells can be delivered intravenously to the heart, through coronary arteries or even through retrograde coronary sinus. The major drawback of stem cells being infused through peripheral venous system seems to be the low retention of cells into infarcted area. Results obtained in pre-clinical animal models showed that this minimally invasive approach results in a significant percentage of injected cells being sequestered in lungs, liver or spleen, due to blood flow [33]. On the other hand, intracoronary or retrograde coronary sinus infusion of the cells are mainly performed after acute myocardial infarction using an angioplasty balloon and high pressure to deliver cells to heart muscle [34]. The coronary route was proven to be free of stem cell systemic delivery, while a limited number of cells could be found in the infarcted area [35]. Finally, an interesting attempt with stem cells being injected into the pericardial cavity has been proposed. By this means, a higher number of cells could be deposited and retained in the pericardial cavity, while migration across the visceral pericardium is required (Table 1).
- 40. Tissue Engineering forTissue and Organ Regeneration 24 Table 1. Advantages and disadvantages of injecting stem cells by intravenous, intracoronary, intramyocardial, retrograde coronary sinus or intra-pericardial route b) Injectable scaffolds Injectable scaffolds are defined as materials offering the unique solution of replacing damaged myocardial ECM and/or delivering cells directly to the infarcted region while holding the potential for minimally invasive delivery [36]. Such scaffolds can be composed of biocompatible microspheres or in situ gelling materials having reasonable dimensions as to surpass capillary barrier. They are considered a promising tool for stem cell delivery to damaged myocardium. In situ gelling materials are generally made of components of extracellular matrix (ECM), which are induced to a transition after being implanted in situ. Complex injectable gelling materials have been prepared by decellularization technique out of ventricular or epicardial ECM, thus possibly avoiding animal-derived components and paving the way to the definition of patient-specific treatments. The use of injectable, synthetic microspheres has already been proven promising in the treatment of neurological diseases in vivo [37]. Recently the possibility of using injectable scaffolds in cardiac cell therapy has been explored by interfacing murine mesenchymal (mMSC) and cardiac stem cell (mCSC) lines with poly-lactic acid (PLA) microspheres having a diameter of 30 and 100 μm. Preliminary in vitro experiments demonstrated that such cells can be grown onto PLA microspheres while preserving their phenotype, but the formation of cell clumps can hamper the application of this technique [38]. The use of dynamic seeding techniques (i.e. bioreactors) would favor a more homogeneous distribution of the cells. An interesting approach has been recently proposed to deliver human mesenchymal stem cells to the injured myocardium: RGD-modified alginate microsphere (diameter: 200-700 μm) encapsulation of hMSC was setup. In vitro experiments showed that hMSC could survive, proliferate and migrate through the porous material. When intramyocardially injected in a rat model of myocardial infarction by left anterior descendant coronary (LAD) ligation, cell-loaded alginate microspheres promoted angiogenesis and prevented LV negative remodeling [39]. Nonetheless, few human cells were found in the injection area after few days, while microbead remains were still present
- 41. Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site 25 within host myocardium 10 weeks after the injection. The aspect of microbead resorption should thus be addressed before clinical perspectives could be foreseen. c) Scaffold-based technology The possibility of using biocompatible scaffolds to deliver stem cells to the injured heart has been explored by a number of independent research groups so far. The scaffolds proposed are natural of synthetic but when designing cardiac-specific constructs, a number of requirements should be fulfilled. For example, it cannot be neglected that myocardial contractile function relies on the transmission of electrical and mechanical forces throughout a functional syncytium. So, the integrity of the tissue has to be preserved. For this reason, a cardiac-specific scaffold should comply with tissue architecture and thus be deformable enough to indulge and, if possible sustain cardiac contraction. Moreover, as far as stem cell engraftment is concerned, scaffolds should be able to start at least cell alignment and commitment to favor stem cell electromechanical coupling with host tissue. In this respect, the work of Mandoli and collaborators using Cerium Oxyde nanoparticles to affect poly- lactic acid film surface and obtain a controlled nanorugosity appears intriguing [40]. In fact, far from being a noxious compound for stem cells, ceria was able to induce cardiac stem cell alignment and growth. Nonetheless, cardiac tissue is extremely complex and highly demanding in terms of blood supply and catabolite removal, so that porous scaffolds that could allow microvascular branches formation and oxygen perfusion are to be preferred. To fulfill such requirements, the first attempts were performed by the group of Thomas Eschenhagen. Neonatal cardiomyocytes were seeded in Collagen I + Matrigel to produce Engineered Heart Tissue (EHT). Continuous contractile activity up to 1 week in vitro as well as cell survival and integration in vivo in singenic rat hearts were reported [41]. In another attempt, anisotropic accordion-like honeycomb scaffolds were prepared by excimer laser microablation using poly(glycerol sebacate) as an elastomeric tool to mimic anisotropic cardiac muscle stiffness distribution [42]. Although the authors demonstrated that such scaffolds promote neonatal rat cardiomyocyte alignment and contraction, in vivo testing has not been performed so far. The same material has been utilized to produce elastomeric patches on which human embryonic stem cell-derived cardiomyocytes were grown, showing that it is indeed possible to observe spontaneous beating activity in vitro up to 3 months [43]. Such patches were shown to be suitable as delivery systems and, when sutured in the absence of cells onto healthy rat left ventricle, they did not affect cardiac contractile activity. More basic studies were also conducted to study the ability of stem cells to interface with different synthetic and natural materials. In this respect, few research groups focused on the possibility to drive a certain extent of stem cell commitment through tailoring scaffold physical and chemical properties, independently of biological cues. In this respect, a common agreement on the ability of stem cells to sense substrate rugosity and elasticity has been reached [44]. Thus, in order to rule out the occurrence of spontaneous events of differentiation in implanted cells, the possibility to induce in vitro stem cell commitment on scaffolds towards a desired phenotype is being investigated. Indeed, Engler and collaborators compellingly demonstrated that the possibility to affect stem cell fate determination by simply tuning substrate elasticity as to match tissue-specific stiffness, exists. Recently, this concept has been corroborated by other research groups, showing that cardiac resident progenitors (Sca-1+ CPC) can be committed to cardiac phenotype by the physico-chemical signals arising from matrix, but biological factors are needed to complete the differentiation process [45, 46].
- 42. Tissue Engineering forTissue and Organ Regeneration 26 d) Preparation of thick cardiac substitutes by Scaffold-free technology To overcome the problem of poor cell retention reported in cell injection experiments in the heart [30] and avoid the release of possibly harmful scaffold byproducts, scaffold-free technology has been developed, in which cells are grown in a monolayer onto thermo- responsive surfaces and easily detached in the form of cell sheet by lowering the temperature [47]. Such technology takes advantage of the ability of polymers like poly-N- isopropylacrylamide (PNIPAAm) to shift between hydrophobic and hydrophilic status when the temperature ranges from 37ºC to 32ºC. Cell sheets can be serially stacked to obtain multilayered scaffoldless constructs (Figure 3). Such an approach has already been applied to obtain cell sheets composed of rodent [48, 49] and human [50] cells. Given the need for thick cardiac substitutes suited to comply with cardiac muscle continuous contractility, thermo-responsive technology has been envisaged as a possible answer to the lack of heart donors. Pre-clinical trials performed onto experimentally infarcted animals demonstrated that when a murine adipose-derived monolayer sheet is leant onto injured myocardium, it can be retained and help tissue repair [48]. Similarly, striking results are obtained when a Sca-1+ cardiac progenitor cell-derived sheet is used [49]. Finally, an interesting approach has been recently proposed to deliver cardiac stem cells cultured in the form of cardiospheres to the injured heart: cardiospheres were embedded into a cardiac stromal cell-derived sheet obtained by using poly-lysine/ collagen IV-coated dishes [51]. The formation of mature vessels as well as new cardiomyocytes in vivo was reported after 3 weeks. Fig. 3. Generation of scaffoldless multilayered bio-constructs by means of thermo- responsive technology: cells grown in a monolayer onto thermo-responsive poly-N- isopropilacrylamide (PNIPAAm)-coated dishes can be detached by lowering the temperature below 32°C. At 37°C the surface is highly hydrophobic and allows cell adhesion. When the temperature is lowered, PNIPAAm becomes hydrophilic, the cell sheet is detached and extracellular matrix (ECM) preserved. Multilayered cell sheets can be obtained by serially stacking monolayered sheets
- 43. Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site 27 4. Clinical trials In the attempt to transfer bench experience to bedside, a number of clinical trials in which different stem cell or progenitor subsets are used have been approved (see http://www.clinicaltrials.gov). Most of them are still in the recruitment phase while some already gave indications and preliminary results. Since most of the ongoing trials are based on the injection of raw stem cell preparations (mostly bone marrow-derived cells), the time and route of cell application remain the key problems to be addressed before proceeding to routine clinical practice. In this respect, recent animal experiments demonstrated that the acute phase of myocardial infarction is probably not suitable for stem cell engraftment and differentiation [52]. Therefore, the right moment in which stem cells should be delivered is to be studied. An overview on some of the ongoing clinical trials is given below. 1. MAGIC (Myoblast Autologous Grafting in Ischemic Cardiomyopathy). In one of the first phase II clinical trials setup to study the possibility to use stem cells to treat cardiac pathologies, ninety-seven (97) patients undergoing coronary artery bypass grafting (CABG) were enrolled. 400-800 X 106 autologous myoblasts harvested from patient muscle biopsy were implanted in the akinetic area of ventricular wall 21 days after in vitro culture. The follow-up after 30 days and 6 months demonstrated the arising of arrhythmia events, thus requiring the implantation of pacemaker. Moreover, no cardiac function improvement was reported. Such negative results were ascribed to the inability of skeletal myoblasts to balance cell death and achieve complete electromechanical integration with the recipient myocardium. Finally, skeletal myoblast administration was reported to determine no enhancement in major cardiac adverse events and mild effects on left ventricular remodeling process [53, 54]. More recently, final results from SEISMIC [Safety and Effects of Implanted (Autologous) Skeletal Myoblasts (MyoCell) Using an Injection Catheter] Trial, a phase II-a study encompassing 40 patients experiencing congestive heart failure and receiving percutaneous intramyocardial injection of autologous skeletal myoblasts, reported the feasibility and safety of this procedure without significant arrhythmogenic events recorded at 6-month follow-up with respect to control groups, although left ventricular ejection fraction did not result significantly improve. These encouraging results suggest that myoblast cell therapy could be considered as a potential effective treatment when associated with standard medical therapy in patients with previously implanted cardiac defibrillators [55]. 2. TOPCARE-CHD, -AMI, -DCM (Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction, Chronic Stable Ischemic Heart disease or Dilated Cardiomyopathy). In this complex clinical trial, a total of 346 patients were classified to CHD, AMI or DCM pathologies and infused either with bone marrow cells (BMCs), blood-derived stem cells, or no infusion. In TOPCARE-CHD, 121 patients (mean age: 59) with chronic stable ischemic heart disease (CHD) were treated. Although complications occurred in 21% of the patients during 3 months follow-up, BMC intracoronary administration was related with a reduction of both brain and atrial natriuretic peptide (NTP) serum levels (indicators of LV remodelling process) in the remaining population (79%), especially in patients with higher NTP levels at baseline and receiving a greater BMC number with a high functional capacity. Moreover, these results were also correlated with a left ventricular ejection fraction (LVEF) increase and better survival during the further follow-up, suggesting that cell therapy could be
- 44. Tissue Engineering forTissue and Organ Regeneration 28 associated with cardiac function enhancements in patients with advanced chronic post- infarction heart failure [56]. Similarly, two hundred and four (204) patients were treated using bone-marrow-derived progenitor cells directly into the infarct artery three to seven days after an acute myocardial infarction (AMI). A statistically significant 2.5% improvement in left ventricular ejection fraction at four months was reported for patients randomized to the bone marrow injection [57]. Finally, intracoronary infusion of bone marrow cells was performed in 33 patients with dilated cardiomyopathy (DCM) by using an over-the-wire balloon catheter. Three month follow-up demonstrated an improvement in left ventricular pump function while a modest improvement in Brain Natriuretic Peptide (BNP) levels was reported after 1 year [6]. Importantly, the conditions chosen in the present clinical trial were representative of different conditions (acute, chronic phase) encountered in the clinic. Unfortunately, no clear indication on stem cell characterization or on their actual ability to regenerate contractile cells is available. 3. TRACIA STUDY (Intracoronary Autologous Stem Cell Transplantation in ST Elevation Myocardial Infarction). The phase II/ III clinical trial aimed at evaluating the effects of intracoronary administration of adult stem cells on LV ejection fraction and major adverse cardiovascular events (MACE) after 6 months follow-up. For this reason, 1-2 million CD34+ cells were injected through the infarct-related artery few days after post-infarct angioplasty using an "over-the-wire" catheter in 80 patients aging from 20 to 75 years. The results of this study are still to be published. 4. Combined CABG and Stem-Cell Transplantation for Heart Failure. Intramyocardial delivery of autologous bone marrow cells extracted from iliac crest and purified by Ficoll centrifugation, during cardiac surgery for CABG intervention in 30 patients, as compared to 30 patients undergoing CABG without cell infusion. Although information on the number and characteristics of cells to be injected has not been given, the trial is currently ongoing and the follow-up is scheduled in 6-12 months (http://clinicaltrials.gov). 5. POSEIDON-Pilot Study (The Percutaneous Stem Cell Injection Delivery Effects on Neomyogenesis Pilot Study) Poseidon-pilot Study is a phase I/ II multi-center trial in which the trans-endocardial injection of autologous Mesenchymal Stem Cells (20-, 100-, 200 X 106) is compared to autologous non-purified bone marrow cells and to allogeneic human Mesenchymal Stem Cells. The implant is performed during cardiac catheterization using the Biocardia Helical Infusion Catheter in fifty (50) patients suffering from chronic ischemic left ventricular dysfunction secondary to myocardial infarction. The data collection is currently ongoing. 6. SCIPIO (Cardiac Stem Cell Infusion in Patients With Ischemic CardiOmyopathy). This phase I clinical trial is aimed at assessing the safety and effectiveness of intracoronary autologous cardiac stem cell therapy. As such, forty (40) patients suffering from ischemic cardiomyopathy are exposed to intracoronary injection of cardiac resident stem cells (CSC). Cardiac stem cells are harvested from right atrial appendages and selected for c-kit expression, cultured and expanded in vitro prior to injecting them via intracoronary route, three to five months after CABG surgery. The hypothesis is that CSC infused into nonviable myocardial segments will regenerate infarcted myocardium by differentiating into cardiomyocytes and vascular cells. The preliminary results are encouraging: in the nine patients treated at four months after
- 45. Cardiac Muscle Engineering:Strategies to Deliver Stem Cells to the Damaged Site 29 CSC infusion, LVEF increased from 31.3 + 2.5 percent before CSC infusion to 38.8 + 3.2 percent four months after CSC infusion. Moreover, in the five patients in whom data are available at 12 months after stem cell infusion, the improvement in LVEF observed at four months was even greater, averaging 15% at 12 months. The follow-up is scheduled in 1,5 years. 7. ALCADIA (AutoLogous Human CArdiac-Derived Stem Cell to Treat Ischemic cArdiomyopathy). In this phase I, multicenter clinical trial, a rather different approach is followed. In fact, patients’ own cardiac stem cells obtained by endo-myocardial biopsies are delivered by a single intramyocardial injection. The cells injected are 0.5 million cells/kg (patient body weight) and their engraftment should be favored by the concomitant implantation of gelatin hydrogel sheet releasing human recombinant beta Fibroblast Growth Factor (bFGF), during CABG surgery. The study has been designed to treat refractory heart failure, ischemic cardiomyopathy or ventricular dysfunction cases. Importantly, this is the first clinical trial, to our knowledge, in which a human recombinant growth factor is used. Unfortunately, the number of enrolled patients is limited to six (6). 8. REGEN-IHD (Bone Marrow Derived Adult Stem Cells for Chronic Heart Failure). In this phase II/ III study, granulocyte-colony stimulating factor (G-CSF) is subcutaneously administered for 5 days to patients with heart failure secondary to ischemic heart disease to mobilize CD34+ bone marrow stem cells. A concomitant intracoronary or intramyocardial administration of bone marrow derived stem cells is performed. The number of enrolled patients is high (165) and the aim of the study is to compare the effects of G-CSF and autologous bone marrow progenitor cell infusion on the quality of life and left ventricular function in the patients. The follow-up timepoint is scheduled in 6-12 months. A number of papers reporting statistical analyses and comparisons among the clinical trials in which stem and progenitor cells have been adopted are currently available. [For further information, please refer to www.clinicaltrials.gov]. 5. Conclusions The possibility to treat cardiac diseases by cell therapy techniques is an extraordinary promise. While a number of different approaches has been so far proposed to setup minimally invasive techniques for cardiac repair, few of them being already in the clinical experimental phase, basic questions still need to be addressed. In fact, the molecular processes leading to cardiac differentiation still need to be fully clarified, while the impact of novel, genetically modified cell types obtained from adult differentiated cells on cardiac microenvironment deserve further investigations. More importantly, the seek to identify suitable delivery systems (i.e. scaffolds) able to foster stem cell survival, growth and differentiation, while degrading without negative effects as the formation of new tissue occurs is still open. A look at the literature reveals that an impressive effort to translate the information obtained by in vitro and pre-clinical studies to the bedside is being produced. In particular, a number of stem cell subsets, which have been previously tested in vitro and in animal models, are currently being tested in phase I, II clinical trials. As expected, the predominant delivery system used in the ongoing clinical trials is intracoronary or intramural injection of stem cells. The possibility to adopt tissue engineering techniques to
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- 51. 3 Cardiovascular Tissue Engineering Basedon Fibrin-Gel-Scaffolds Stefan Jockenhoevel1 and Thomas C. Flanagan2 1Department of Tissue Engineering & Textile Implants, AME-Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen, 2School of Medicine & Medical Science, Health Sciences Centre, University College Dublin, Dublin 1Germany 2Ireland 1. Introduction Cardiovascular disease is a major cause of death in the Western World. Novel drugs and innovative devices have enhanced the quality of life for patients with cardiovascular disease, but such treatments are not without limitations and complications. The major constraint with these current treatments is the inability for growth, repair and remodeling of the structure. The emergence of tissue engineering as an alternative therapy for cardiovascular disease has generated an intensity of research into the development of many components of the cardiovascular system, including heart valves, small-calibre vascular grafts and biological stent materials. The composition of the biomaterial used as a support for the developing cardiovascular structure is a key mediator of cell behaviour and function in the tissue, and the ideal scaffold biomaterial for development of a successful end-product continues to be a matter of debate. Fibrin, a major structural protein involved in wound healing, represents an ideal scaffold for the rapid synthesis of autologous tissue-engineered cardiovascular grafts, as its primary scaffold constituents (fibrinogen and thrombin) can be isolated directly from a blood sample of the patient requiring the graft. Fibrin gel scaffolds offer immediate high cell seeding efficiency and homogenous cell distribution by gelation entrapment, and have a degradation rate that can be controlled by protease inhibitors, e.g. tranexamic acid or aprotinin. Fibrin is also known to stimulate the secretion of reinforcing extracellular matrix (ECM) proteins by seeded cells. The potential to control the fibrin polymerisation process also offers the opportunity to produce complex 3-D structures, like heart valve prostheses and to embed porous, textile or metal (stent) structures. This book chapter reviews the properties of fibrin that make it an ideal scaffold candidate for applications in the area of cardiovascular tissue engineering, and documents the successful development of fibrin-based heart valves, vascular grafts and biostents for clinical application. 2. Scaffold materials Scaffolds play a central role in cardiovascular tissue engineering. Essential requirements for the ideal cardiovascular scaffold are easy handling properties and the ability to mould
- 52. Tissue Engineering forTissue and Organ Regeneration 36 complex 3-D structures from the material, such as aortic roots or vessels with complex side branches. The scaffold material should neither be toxic, nor elicit any immunological side effects. The diffusion barrier of the scaffold material should have the lowest possible resistivity in order to guarantee an optimal nutrition supply in thicker tissues. Furthermore, both the mechanical and the chemical properties (e.g. the integration of growth factors) of the scaffold material should be modifiable. Controllable degradation of the material is also important in order to adapt the structural support of the scaffold with regard to the developing tissue. A multitude of scaffolds are currently employed in the field of tissue engineering, e.g. synthetic polymers (polyurethanes, polyglyolic acid, polylactic acid, polyhydroxybutyrate, copolymers of lactic and glycolic acids, polyanhydrides, polyorthoesters) and natural polymers (chitosan, glycosaminoglycans, collagen), or biological scaffolds such as acellularised porcine aortic conduits (Bader et al., 1998; Chevallay & Herbage, 2000; Flanagan et al., 2006; Freed et al., 1994; Grande et al., 1997). Scaffold-related problems including cytotoxic degradation products, fixed degradation times, limited mechanical properties and the absence of growth modulation, etc. necessitate further extensive investigations in developing the ideal cardiovascular scaffold. 3. Fibrin as scaffold material? Based on the assumption that successful tissue engineering should mimic the process of tissue regeneration, and that regeneration is closely related to haemostatis, fibrin (gel) seems to be an ideal candidate as a tissue engineering scaffold by virtue of its role as a “physiological scaffold” in tissue regeneration. Several influences of fibrin gel on tissue development have been described in the literature: it is known that fibrin gel is one of the major ligands for ß3 integrins, which leads to cell migration into a wound/tissue-engineered construct (Ikari et al., 2000; Nomura et al., 1999). Thrombin, fibrinogen, fibrin monomers and fibrinopepide B all increase DNA synthesis in smooth muscle cells (SMCs) and consequently the proliferation of the cell (Pakala et al., 2001). 3.1 Physiology of fibrin Fibrin is the end-product of the coagulation cascade following the conversion of fibrinogen in the presence of thrombin and calcium (Figure 1). Fibrinogen is a soluble plasma glycoprotein, which is produced by the liver. Fibrinogen is an acute phase protein with a normal blood concentration of 1.4 – 3.5 g/l. The fibrinogen molecule has a length of 45 nm, a molecular weight of 340 KDa and consists of 2 subunits and 3 polypeptides chains - α, β and γ. During the polymerisation process, the fibrinopeptide A of the α-chain and the fibrinopeptide B of the ß-chain are cleaved by thrombin. The exposed N-terminal positions of the fibrinopeptides bind to the γ-chain of the fibrinogen and produce the so-called proteofibrils. In the subsequent step, the lateral association leads to apposition of the proteofibrils to form a 3-D fibrin network structure (Meyer, 2004). FXIIIa stabilises fibrin further by incorporation of the fibrinolysis receptors, alpha-2-antiplasmin and TAFI (thrombin activatable fibrinolysis inhibitor, procarboxypeptidase B), and binding to several adhesive proteins of various cells (Muszbek et al., 2008). The polymerised fibrin gel matrix is a hydro-gel, which contains ~95-98% water. The water content can easily be exchanged against a buffer solution or a cell culture medium, allowing an optimal nutrition supply of the cells that are embedded after the gelation process.
- 53. Cardiovascular Tissue EngineeringBased on Fibrin-Gel-Scaffolds 37 Fig. 1. Coagulation cascade: the conversion of fibrinogen into fibrin is triggered by thrombin and calcium 3.2 Production of autologous fibrin The classical approach for production of autologous fibrin is the cryoprecipitation method: after the production of platelet-poor plasma (PPP), the plasma is frozen at -80°C and thawed overnight at +4°C. The precipitate formed contains ~60-70% of fibrinogen. After centrifugation, the supernatant is decanted and the precipitate is subsequently washed twice in rinsed water. After the precipitate is dissolved in water, overnight dialysis against calcium-free TRIS buffer solution is necessary to provide optimal conditions for the embedded cells. The cryoprecipitation method has two major disadvantages: (1) the efficiency of fibrinogen isolation is relatively low with only ~20-25% of the total fibrinogen content removed, and (2) the production process is time-consuming (~2 days). The low isolation efficiency is particularly problematic regarding the use of autologous fibrin gel scaffolds in paediatric
- 54. Tissue Engineering forTissue and Organ Regeneration 38 patients, as the volume of sampled blood needs to be kept to an absolute minimum. Therefore, the use of alternative precipitation methods with different chemicals has been evaluated: (1) ethanol (Kjaergard et al., 1992; Weis-Fogh, 1988), (2) ammonium sulphate alone, and (3) in combination with the cryoprecipitation method (Wolf, 1983), (4) albumin plus cryoprecipitation, and (5) polyethylene glycol (PEG) plus cryoprecipitation (Epstein et al., 1986). Heselhaus investigated each of these different precipitation methods with regard to their efficiency and their use in the development of fibrin scaffold materials for cardiovascular applications (Heselhaus, 2011): Fig. 2. [A] Fibrin gel after polymerisation in a 6-well plate. [B-D] Scanning electron microscopy (SEM) images demonstrating the nano-fibre network structure of the fibrin, which enables the gentle embedding of cells, with a vascular smooth muscle cell (SMC) shown in [D] immediately after the gelation of the fibrin within a web-like network surrounded by cell culture medium Figure 3 demonstrates that all of the reported alternative methods are more efficient than the standard cryoprecipitation method. Here, the technique using ethanol as the precipitation reagent is observed as the most efficient method, with an isolation efficiency of ~80%, ~4-times higher than the efficiency of the standard method. The technique applying both albumin and cryoprecipitation indicates a false positive high result due to contamination of the precipitate with albumin (shown by a significant band in SDS gel electrophoreses) (Heselhaus, 2011).
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- 57. 126 Hermin afedett Eliz a fedett 127 tudott dükösködni tudott dühösködni 129 ne történjék! ne történjék!“ 131 IX. Különböző fogadtatások X. Különböző fogadtatások 131 lehessen vvnni lehessen venni 133 egyib jobbról egyik jobbról 136 Bizonyosan tévedész Bizonyosan tévedés 141 a ha-hazajövetelét a hazajövetelét 146 Önt azt mondta Ön azt mondta 148 jövök jöbbet jövök többet 156 olyan azszonyra olyan asszonyra 159 ki iudott ki tudott 167 már többet. már többet.“ 170 arrál álmodik arról álmodik 171 megasszony fog lenni menyasszony fog lenni 173 meg nem gyulnék meg nem gyógyulnék
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