Download Myocardial tissue engineering - Oxford Academic

Myocardial tissue engineering
Hedeer Jawad†, Alex R. Lyon‡, Sian E. Harding*‡, Nadire N. Ali‡,
and Aldo R. Boccaccini†
†
Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK,
and ‡National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3
6LY, UK
Introduction: Regeneration of the infarcted myocardium after a heart attack is
one of the most challenging aspects in tissue engineering. Suitable cell sources
and optimized biocompatible materials must be identified.
Sources of data: In this review, we briefly discuss the current therapeutic options
available to patients with heart failure post-myocardial infarction. We describe
the various strategies currently proposed to encourage myocardial regeneration,
with focus on the achievements in myocardial tissue engineering (MTE). We
report on the current cell types, materials and methods being investigated for
developing a tissue-engineered myocardial construct.
Areas of agreement: Generally, there is agreement that a ‘vehicle’ is required to
transport cells to the infarcted heart to help myocardial repair and regeneration.
Areas of controversy: Suitable cell source, biomaterials, cell environment and
implantation time post-infarction remain obstacles in the field of MTE.
Growing points: Research is being focused on optimizing natural and synthetic
biomaterials for tissue engineering. The type of cell and its origin (autologous
or derived from embryonic stem cells), cell density and method of cell delivery
are also being explored.
Areas timely for developing research: The possibility is being explored that
materials may not only act as a support for the delivered cell implants, but may
also add value by changing cell survival, maturation or integration, or by
prevention of mechanical and electrical remodelling of the failing heart.
Keywords: myocardium/infarction/tissue engineering/biomaterials
Accepted: July 28, 2008
*Correspondence to:
Sian E. Harding,
National Heart and Lung
Institute, Imperial College
London, Dovehouse
Street, London SW3 6LY,
UK. E-mail: sian.harding@
imperial.ac.uk
British Medical Bulletin 2008; 87: 31–47
DOI:10.1093/bmb/ldn026
& The Author 2008. Published by Oxford University Press.
All rights reserved. For permissions, please e-mail: [email protected]
H. Jawad et al.
Heart disease
Cardiovascular disease is the leading cause of death in the UK, comprising 39% of all deaths per annum (www.bhf.org). Myocardial
infarction (MI), commonly known as a heart attack, is caused by the
abrupt occlusion of one or more of the blood vessels (coronary arteries)
supplying blood to heart. This reduces the supply of nutrients and
oxygen to the heart muscle (myocardium). If blood flow is not restored
rapidly, irreversible cell death occurs within the blood-deprived myocardium, eventually impairing cardiac performance. Patients who
survive the acute event may eventually develop heart failure, which is
defined as the clinical state resulting from the inability of the heart to
pump enough blood to meet the body’s metabolic requirements. The
adult human heart is unable to self-regenerate to a significant degree,
and a fibrous non-contractile scar tissue is formed in the infarcted myocardial territory (Fig. 1). The replacement of the healthy myocardium
after infarction with a non-contractile fibrous scar tissue, which also
does not effectively conduct the electrical wavefront, reduces the heart
contractile efficiency. Compensatory mechanisms are activated to assist
the heart to maintain cardiac output. This ultimately places an extra
burden on the weakened myocardium, eventually leading to end-stage
heart failure, with a progressive fall in cardiac output, development of
multi-organ failure from reduced perfusion and ultimately death.
Pharmacological therapies can slow the progression to end-stage
disease, but rarely prevent or reverse progression of the failing state.
Currently, the only therapeutic options available to treat patients with
Fig. 1 Schematic diagram illustrating the damage caused by an MI in human heart.
Source: http://www.uptodate.com (accessed 20 June 2008).
32
British Medical Bulletin 2008;87
Myocardial tissue engineering
terminal end-stage heart failure are heart transplantation or left ventricular assist devices (VADs). However these two options are not widely
available, and have significant limitations of cost. While heart transplantation involves the replacement of the whole organ, VADs aim to
prevent remodelling and dilation of the left ventricle via unloading the
dilated chamber. Alternatives for advanced, but not terminal, heart
failure are reverse remodelling strategies using either biventricular pacemakers where electrical dyssynchrony is present or external constraining mesh ‘jackets’ wrapped around the failing heart to prevent ongoing
ventricular dilatation. Owing to mixed results achieved1 with some
reporting improved cardiac function while preserving ventricular geometry and others reporting marginal improvements, alternative treatments are required. Below, we discuss the current suggested potential
options to treat infarcted hearts, with focus on tissue engineering.
Suggested options for myocardial repair and regeneration
‘Resident Cardiac Progenitor Cells’
Traditionally the heart has always been thought to have no regenerative
capability, with cardiomyocytes viewed as terminally differentiated
cells that were unable to self-renew. Recently, this paradigm has been
challenged. Anversa and co-workers have reported the presence of a
group of ‘cardiac progenitor cells’ (CPCs) resident in the heart, with
specific stem cell markers, Lin-, c-kitþ and Ki-67 and early cardiac
marker such as transcription factors GATA-4 and Nkx2.5. Other
groups have identified other potential resident CPCs using a variety of
cell surface markers and demonstrated in vitro cell division and in vivo
myocardial regenerative capacity.2 Their origin is still unclear, and it is
not known whether these cells home to the heart from the bone
marrow or may reside in the heart from foetal life. Limited evidence on
their regenerative capacity and their low population in the elderly and
diseased human heart have tempered the enthusiasm over CPCs contributing significantly to myocardial regeneration. More recently, Marban
and co-workers3 expanded resident cardiac stem cells ex vivo and
reported myocardial regeneration and functional improvements postinjection into infarcted mouse hearts.
Cell transplantation
Repair and regeneration of the infarcted myocardium has been studied
with cellular cardiomyoplasty, which involves the injection of cells
British Medical Bulletin 2008;87
33
H. Jawad et al.
either directly into infarct or intravenously. A number of cell sources
have been explored, including bone marrow,4 skeletal muscle5 and
embryonic stem cells (ESCs).6 Table 1 summarizes the cell sources
explored for cell transplantation, which have been extensively reviewed
elsewhere.7 – 9 In general, all cell sources explored have had mixed
results. No candidate cell has been uniformly successful in the clinical
trials performed to date, though the modest reported improvements
provide encouragement. However, many hurdles still face the clinical
application of the autologous cell therapy strategies, including the ideal
cell type, cell dose, optimal timing for cell transplantation and mode of
delivery to optimize cell retention within the myocardium. Long-term
survival and terminal differentiation post-implantation are yet to be
determined.10 Whether cells are injected directly into the infarct or
adjacent myocardium by thoracotomy, or delivered via coronary circulation, the need for surgery or the inevitable cell loss, respectively,
raises doubts about cell transplantation.
Table 1 Cell sources used for cell transplantation.
Cell source
General advantages
5
Skeletal myoblasts
Progenitor cells mobilized
from bone marrow11
Autologous, contractile,
easily cultured and
expanded in vitro
Autologous
Bone marrow stem cells4
Easily accessible and
autologous
Bone-marrow-derived
cardiomyocytes12
Autologous
ESC-derived cardiomyocytes6
Spermatogonial-derived
cardiomyocytes13
Primary cardiomyocytes14
Remain in culture for
unlimited periods
Authentic cardiomyocyte
phenotype
True morphological
representation and preserve
cardiac function in vitro
CPCs2
Potential autologous source
General disadvantages
Inability to transdifferentiate into CM,
preventing electrical coupling causing
arrhythmias
Results are not reproducible, cell route
from bone marrow to infarct and bone
marrow cell transdifferentiation to CM
is questionable
Mixed results achieved. Limited
evidence on transdifferentiation.
Possible formation of other tissue
types
Results of differentiating bone marrow
into CM in vitro is difficult to
reproduce
Immunological and ethical constraints
Teratoma formation
Evidence of expandability and
reproducibility at an early stage
Do not survive in culture longer than
48 h (adult ventricular cells) and 1
week (neonatal myocytes),
dedifferentiate in culture, unable to
proliferate and self-renew, human
source problematic
Evidence of expandability and
reproducibility at an early stage
CM, cardiomyocytes.
34
British Medical Bulletin 2008;87
Myocardial tissue engineering
Tissue engineering
Tissue engineering comprises the combination of biomaterials, cells
and specific growth factors. It is a interdisciplinary field where material
scientists and cell biologists form a construct (ex vivo or in situ), eventually implanted onto the injured site by clinicians (Fig. 2). Tissue
engineering is currently receiving much attention in the field of regenerative medicine as an alternative to current treatments.15 Myocardial
tissue engineering (MTE) has been proposed as an alternative option to
replace the scarred non-contractile fibrous tissue caused post-infarction.
Fig. 2 Schematic diagram illustrating the principle of tissue engineering for myocardial
regeneration.
British Medical Bulletin 2008;87
35
H. Jawad et al.
The basic MTE paradigm is to seed cells capable of forming
cardiomyocytes onto a biocompatible material in vitro, followed by
implantation of the construct on or in the infarcted region of the
failing heart. The grafted tissue will in turn direct new tissue formation
as the cells integrate with the native tissue while the scaffold degrades
over time.16
Myocardial tissue engineering
Initially, a scaffold made from a biomaterial is designed. A selection of
cells (cardiac or non-cardiac) is expanded in vitro with additional
growth factors. Cells, with or without growth factors, are seeded onto
the scaffold to form the MTE construct and mechanical, electrical and
morphological properties optimized. This is eventually sutured onto or
into the infarcted region.
Requirements for MTE constructs
Although many biomaterials have been suggested for various
tissue-engineering purposes, all constructs share the following basic
requirements.
†
Biocompatible. Biomaterial must not be rejected or induce an inflammatory response in vivo.
†
Mechanical integrity. Biomaterial must enable handling during transplantation. More importantly, mechanical properties should match the host
tissue it intends to replace and provide mechanical support during
regeneration.
†
Biodegradable. The degradation rate of the biomaterial should match the
regeneration rate of the host tissue, and the degradation by-products must
be non-toxic and readily removed from the body.
†
Cell ‘friendly’. Enhance cell adhesion and survival both in vivo and
in vitro.
†
Biomimetic. Reflect the extracellular matrix (ECM) of the tissue it intends
to replace.
†
Fabrication. Biomaterial must be easily accessible and designed with acceptable cost.
Additional requirements for MTE constructs include the following.
†
36
It is important that the biomaterial is able to withstand, or even contribute
to the continuous stretching/relaxing motion of the myocardium that
occurs at each heartbeat.
British Medical Bulletin 2008;87
Myocardial tissue engineering
†
Biomaterial must encourage cell proliferation and differentiation into cardiomyocytes as well as supporting vascular cells.
†
Ideally, biomaterials could encourage cardiomyocyte alignment and maturation in vitro before implantation or in vivo, improving the contractile
properties of the graft.
†
Biomaterial must enable electrical integration of engineered graft with the
native tissue to allow synchronized beating between the artificial construct
and the heart. This requires matched excitability of host and grafted tissue
and support electrical of wavefront propagation.
†
Strategies to encourage vascularization of the construct to support the survival of grafted cells.
†
Suitable cell source that will not provoke arrhythmia once introduced into
the body.
In terms of the cardiac function itself, the physical characteristics of the
material should lie in a range of values that are close enough to the
natural myocardium that they do not massively enhance diastolic stiffness (and hence impede relaxation). It might be argued that a certain
degree of support of the scar would be beneficial to prevent scar expansion and the consequent deleterious remodelling of the myocardium,
and therefore stiffness slightly in excess of the scar itself would be
acceptable. For the systolic function, the material alone will confer no
benefit other than, possibly, contributing to elastic recoil: the important point is that the shape, attachment method and stiffness should not
physically hinder the full contraction of the ventricle. If the material is
combined with beating myocytes, then it should allow these cells to
exert their contractile effect. One desirable potential strategy is for the
material to support nascent new cardiomyocytes as they develop and
integrate into the host and biodegrade when assimilation is complete.
Methods adopted in MTE
Electrospinning
Collagen is the main constituent of the ECM, and therefore
tissue-engineered constructs require an ECM-like structure and topography to mimic the in vivo size and scale of the collagen fibrils in the
ECM. Electrospinning, a process patented in 1934 by Formhals, is
used to develop scaffolds, made from synthetic, natural or a combination of both biomaterials, with sub-micron pores and nanotopography surfaces.17 Electrospinning involves an electrically charged jet of a
polymer solution produced by a high voltage. A constant pressure generated by a metering pump causes the polymer mixture to flow from
the pipette, at a constant rate, onto a collector screen which eventually
dries/solidifies leaving a polymer fibre (fibre diameter can range from
British Medical Bulletin 2008;87
37
H. Jawad et al.
3 nm to 5 mm). This method is common in MTE and has been
suggested for various biomaterials (Table 2).
Table 2 Examples of the biopolymers, the processing used to produce the engineered
construct and cell types used in MTE.
Scaffold material
Natural materials
Collagen
Method used for construct
processing
Collagen gel
Gelatin mesh (Gelafoam)
Commercially available 3D
scaffold
Bioreactor
Commercially available patches
Collagen þ glycosaminoglycan
Biostretch/bioreactor
Two cross-linking methods
Collagen type I matrix
Collagen type I
sponge þ matrigel
Sodium alginate
Synthetic materials
Polyerethane
Elastomeric 1,3-trimethylene
carbonate and D,L-lactide and
copolymers
Vicryl mesh (Dermagraft,
Smith and Nephew)
Poly(N-isopropylacrylamide)
Poly(1-caprolactone)
Poly-glycolic acid (PGA)
Non-woven poly(lactide)- and
poly(glycolide)-based (PLGA)
Poly-co-caprolactone (PGCL)
Bioreactor
Bioreactor
Cell source
Acellular
Neonatal rat CM
Foetal rat CM
Foetal human CM
Rat stomach smooth muscle cells
Rat skin fibroblasts
Foetal human CM
Bone-marrow-derived
mesenchymal cells
Neonatal rat CM
Undifferentiated mESCs mESCMs
Freeze-drying technique
Neonatal CM
Foetal rat cardiac CM
hESC-CM
Neonatal rat CM
Solvent cast and spin coating
Salt-leaching
Neonatal rat CM
Rat cardiomyocytes cell
(CRL-1446) line and human
umbilical vein endothelial cells
Human dermal fibroblasts
Commercially available
Cell sheeting
Electrospinning
Commercially available
Electrospinning
Neonatal rat CM
Neonatal rat mesenchymal cells
Neonatal rat CM
mESC-CM
Primary rat CM
Solvent casting and particle
leaching
Rat bone-marrow-derived nuclear
cells
Combination of natural and synthetic materials
Poly(ester urethane) urea and
Electrospinning
collagen type I
Polycaprolactone (PCL) mesh
Electrospinning
coated with collagen type I
PGA, PLA and PCL polymers
Three polymers mixed and
with commercially available
pores achieved using
hydrophilic collagen sponge
gas-forming method, followed
(Ultrafoamw)
by immersion in collagen
Aortic rat smooth muscle cells
Isolated rat cardiomyocytes
Neonatal heart cells
Continued
38
British Medical Bulletin 2008;87
Myocardial tissue engineering
Table 2 Continued
Scaffold material
Collagen (types I and IV) and
Matrigel matrix mixed with
cells and seeded onto
non-woven polymer mesh
(poly-(L-lactide acid)
reinforced with PTFE)
Method used for construct
processing
Materials mixed in circular
moulds
Cell source
Multipotent
bone-marrow-derived
mesenchymal progenitor cells
3D, three-dimensional; CM, cardiomyocytes; mESC-CM, mouse embryonic stem cell-derived
cardiomyocytes; hESC-CM, human embryonic stem cell-derived cardiomyocytes.
Source: Jawad et al. 17
Bioreactors
The general concept of a bioreactor is to encourage growth and development of biological cells or tissue on biomaterials as if under in vivo conditions. The ability of producing a 3D myocardial tissue comprising
more than a few layers of muscle is the main advantage of using a bioreactor. Although the sole purpose of the construct developed with bioreactors is for scientific research, further improvements in in vitro design
and quality control may eventually allow their application for MTE.
Several different bioreactors have been suggested for cardiac constructs:
static or mixed flask bioreactors, where constructs are suspended in a
cultivation medium; rotating vessel bioreactors, where constructs are
suspended in a medium that has a constant rotational flow; and finally
perfusion cartridge bioreactors, where constructs are perfused at interstitial velocities, comparable to blood flow in native tissue.18
Cell sheeting (temperature-sensitive)
This method was initially reported by Shimizu et al. 19 in 2002 and has
been suggested for MTE. The concept of this method involves
temperature-responsive dishes made from a specific polymer, poly(Nisopropylacrylamide), which is temperature sensitive. At 378C, the
polymer is hydrophobic and cell adhesive; however, a 58C reduction in
temperature can cause the polymer to become non-cell adhesive as it
hydrates and swells because its hydrophobic nature is now hydrophilic.
Cardiomyocytes seeded onto the polymer will produce individual spontaneously beating myocardial tissue sheets. More recently, Miyahara
et al. 20 reported the success of implanting a 100 mm thick cardiac
tissue, made from six monolayered mesenchymal stem cell sheets
layered together and implanted onto the infarcted region, improving the
infarcted wall thickness. The caveats about the potential for the
bone-marrow-derived mesenchymal stem cells becoming cardiomyocytes, indicated in Table 1, also hold for these cell sheets.
British Medical Bulletin 2008;87
39
H. Jawad et al.
In situ engineering
Like tissue engineering, in situ engineering involves both biomaterials
and cells. However, this is a ‘scaffold-free’ approach in MTE. It
involves the direct injection of the biomaterial and cell mixture into the
infarcted region. Unlike prefabricated scaffolds used in MTE, the
injectable natural polymers will readily bond to the native tissue, as
they can be easily shaped or cast to the heart’s complex dimension,
simultaneously providing a good support for the cells. Different polymers such as alginate,21 fibrin glue,22 collagen23 and matrigel24 have
been suggested for in situ engineering. Acellular alginate with bioactive
molecules has also been suggested, with the hope that CPCs will home
to the infarcted region.25 More recently, biomaterials with peptides
and growth factors,26 as well as ‘self-assembling’ peptide nanofibres,27
have been suggested. None of these strategies have in fact produced
new myocardial tissue, and experience with injection of cells directly
into the heart has suggested that there is a low limit on the amount of
new material that can be introduced into either the dense and continually compressing myocardium or the stiff and avascular scar.
Biomaterials used in MTE
This section aims to highlight the achievements, to date, in producing
MTE scaffolds ( porous structure) or patches (dense structure). It must
be noted that either a porous structure or a dense patch maybe suitable,
depending on the purpose of the construct. If the engineered biomaterial is to support and possibly remould the infarcted area over a period
of time, then it is vital the construct is a scaffold that consists of interconnected pores (.90% porosity) with diameters ranging between 300
and 500 mm for cell survival. This will allow cells to exchange nutrients and remove cellular secretions, enhance cell penetration and tissue
vascularization.28 On the other hand, if the biomaterial will serve
solely as a means of cell transport, to deliver cells to the desired region
only and degrade over a given period of time (e.g. within 3 months), a
dense patch will be adequate for this purpose.17 Wide ranges of biomaterials, mainly polymers, have been suggested for MTE, being synthetic
and natural. This section will give an insight into the types of biomaterials reported specifically for myocardial regeneration.
Synthetic polymeric materials
Polyesters
The biodegradable poly(a-hydroxy acid) aliphatic polyesters such as
poly 1-caprolactone, polylactic acid (PLA), polyglycolic acid (PGA) and
their copolymers are the major classes of polymers suggested for MTE.
40
British Medical Bulletin 2008;87
Myocardial tissue engineering
The ability to tailor their mechanical properties, define their morphology and control their degradation kinetics by altering the copolymer ratio, to better suit the tissue it intends to replace, are attractive
features of these synthetic polymers.15 Furthermore, the natural metabolites of PGA and PLA and their copolymers, glycolic and lactic acid,
make these polymers more appealing as the human body is naturally
able to completely remove monomeric compounds of lactic and glycolic acids, although it has been reported that these by-products increase
acidic concentration in the body, causing inflammatory response which
in turn damages the local tissue. A further disadvantage of these polymers is their bulk degradation kinetics which causes a sudden loss of
mechanical properties of the construct.15 Freed and Vunjak-Novakovic
in 1997 were the first to report the use of polyesters (PGA) as a scaffold for cardiac tissue engineering. A spontaneously contractile 3D
engineered cardiac tissue with specific cardiac structural and electrophysiological properties was achieved using a bioreactor.28 However,
there is increasing evidence that successful scaffold materials must have
elastic properties that are similar to that of the native heart. This will
allow the construct to withstand and possibly move in synchrony with
each contraction/relaxation motion that occurs during each heartbeat,
as well as prevent cells from detaching from the bioengineered construct.29 This means that the group of polyesters, which are less flexible
than the heart tissue, will not fulfil all the requirements for an ideal
MTE scaffold or patch.
Elastomeric polymers
The key advantage of these polymers is their elastic behaviour, in that
they are able to withstand strong deformation forces and return to
their original size upon removal of stress. This can circumvent the problems related to material stiffness. Elastomeric polyurethane (PU) has
been suggested for MTE, where the 3D cardiac construct was achieved,
reporting good cell adhesion.29 Furthermore, in vitro and in vivo
studies have reported no tissue inflammation.15 However, a major
setback of PU in MTE is its toxic by-product diisocyanate, which
might be released upon degradation and known to be harmful to living
tissue. The incorporation of diisocyanate is necessary in the synthesis
of PU. Another elastomeric polymer used in MTE is 1,3-trimethylene
carbonate (TMC),30 a copolymer containing TMC, and polyester
D,L-lactide had the ability to sustain the heart’s cyclic strains under
physiological condition with no severe tissue reaction in vivo. Further
example demonstrating the success of elastomeric polymer in MTE is
polyethylene glycol (PEG), in which the spontaneous beating of PEG
discs was achieved when cardiomyocytes were cultured on the surfaces.31 More recently, Chen et al. 32 characterized a soft elastomer for
British Medical Bulletin 2008;87
41
H. Jawad et al.
MTE, made from poly(glycerol sebacate), which has already shown
promising results in soft tissue engineering for nerves and vascular
tissue engineering.32 A further group of materials being considered is
that of poly(ethylenetephatalate)/dimer fatty acid block copolymer.33
Natural polymeric materials
Extracellular derivatives
Unlike natural polymers, synthetic polymers have the key advantage of
allowing precise control of their hydrophilic/hydrophobic ratio, degradation rate and mechanical properties. However, they do not possess
the biological specifics of natural polymers.15 ECM proteins and
derivatives such as collagen type I and fibronectin are examples of
natural polymers suggested for MTE. Although they are renowned to
facilitate cell adhesion and proliferation and maintain cells in their differentiated states because of their particular adhesive properties, rapid
degradation kinetics and weak mechanical properties hinder their
success in MTE. In addition, immunogenicity and inconsistent material
properties between various batches of natural polymers are still of
major concern. Collagen has been widely investigated for both MTE 34
and in situ engineering,23 however, with many conflicting results.
Eschenhagen et al. 35 developed the all natural engineered heart tissue
(EHT) made from a combination of neonatal cardiomyocytes and
artificial matrix (collagen type I and Matrigel) in artificial moulds
under mechanical strain. Recently, the EHT has been reported to
improve the cardiac function, since 28 days post-implantation electrical
coupling to native heart and systolic wall thickening of infarction were
observed in vivo. 14 Suitable cell source and graft size are examples of
areas that need further investigation for the potential clinical use of the
EHT.17
A complex protein mixture secreted by mouse tumour cells, Matrigel
(trade name given by BD Biosciences), resembles the ECM environment. It is commonly used in laboratories as a substrate to enhance cell
adhesion on to material surfaces. Interestingly, this has been suggested
for in situ engineering as an acellular matrix and as a mixture with
endothelial ESCs.24 In both cases, Matrigel was found to be effective in
that the cardiac function improved attenuated the left ventricular function reported. Glycosaminoglycan (GAG) is also an abundant protein
found in the ECM of the body; many studies have reported the success
of incorporating GAG with collagen to form a nanofibrous scaffold.36
In 2005, Koifids et al. 37 combined undifferentiated mESCs with collagen type I in a bioreactor to form an artificial myocardial tissue.
Despite the success of the artificial myocardial tissue and other studies
forming constructs from collagen, Matrigel and GAG, immunogenicity
42
British Medical Bulletin 2008;87
Myocardial tissue engineering
and weak mechanical properties are still issues that need to be
addressed.
Gelatin is largely composed of denatured collagen, which is mainly
obtained from connective tissue found in bones, ligaments, tendons
and cartilage. A spontaneously contractile 3D cardiac tissue was
obtained in 1999 by Li and co-workers by seeding foetal ventricular
muscle cells onto a gelatin mesh in vitro. Post-implantation results
showed cell survival and interconnection with native tissue in vivo,
although no cardiac functional improvement was reported. More
recently, they reported increased cellular proliferation and better performance upon mechanical stress on the gelatin foam.38
A more extreme and more imaginative use of natural ECM material
has recently been described. Ott et al. 39 decellularized rat hearts with
detergents by coronary perfusion and obtained a perfusable acellular
vascular construct with intact chambers and valves by preserving the
myocardium matrix. This construct was then recellularized with
cardiac or endothelial cells, and showed modest but incontrovertible
contractile abilities. This approach has the potential to solve the
problem of blood supply to the graft by retaining the natural structures
guiding the formation of vessels. It also holds the vision of reconstruction of the whole organ, or large parts of it, which will be necessary if
regeneration strategies are to address complex congenital conditions in
which parts of the heart are absent or malformed.
Alginate
Although certain bacteria produce the natural negatively charged polysaccharide alginate, alginate is mainly derived from brown seaweed. Its
unique property of forming hydrogels in the presence of calcium ions
has attracted its use in MTE. Leor et al. 21 produced alginate scaffolds
with 90% porosity and pore sizes ranging between 50 and 150 mm by
freeze drying. Intensive neovascularization was revealed postimplantation of the alginate scaffold with foetal rat cardiac cells. hESC
have also been seeded onto alginate scaffolds, where no regeneration
was observed.25 Although other studies have investigated alginate for
in situ engineering where acellular alginate with bioactive molecules
was injected into the infarcted area, with hope that the reported CPCs
will home to the diseased region.25 However, due to the limited studies
on the effect of alginate, one cannot conclude whether it was the cells
alone, the alginate alone or the combination of both that contributed
to the healing of the infarction.
Fibrin glue
Fibrin glue is a biopolymer formed by the polymerization of fibrinogen
monomers. This natural polymer has only been suggested for in situ
British Medical Bulletin 2008;87
43
H. Jawad et al.
engineering. Different cell types have been investigated with this
polymer, skeletal myoblasts, bone marrow cells and endothelial cells,17
all reporting enhanced neovascularization, improved left ventricular
function and reduced cardiac remodelling. Although results are promising, this material is at infancy and requires further investigations.
Combination of synthetic and natural polymers
Owing to the poor mechanical properties of collagen, researchers have
investigated the combination of collagen and synthetic polymeric
materials. Both materials would contribute to the scaffold with equal
importance. The synthetic polymer would provide a suitable mechanical
support, whereas the natural collagen polymer would confer to the cells
a more in vivo-like environment. The combination of collagen with
various types of synthetic polymers is also summarized in Table 1. Both
electrospinning and bioreactors have been used to combine the two
materials.17 Krupnick et al. 40 mixed bone marrow progenitor cells with
a collagen and Matrigel matrix and then went on to seed the mixture
onto the synthetic non-woven polymeric mesh. In vivo implantation
onto injured hearts was successful, in that minimal intra-cardiac inflammation occurred as well as the cardiac function appeared normal, with
no arrhythmias reported. However, the immunogenicity of collagen is
still of great concern.
Closing remarks/limitations
A great effort is being made towards the development of an ‘optimal’
MTE construct; however, limitations to take this further into clinical
trials are inevitable. The choice of biomaterial, cell source and suitable
environment for cells to proliferate and differentiate in vitro before
implantation remain obstacles in the field. The support and enhancement of cardiac contractile and electrical properties need careful consideration when designing the construct. Although many biomaterials,
with varying compositions and properties, are continually being
suggested for MTE, the current challenge is to focus investigations on
the already available materials of proved biocompatibility in order to
improve their performance towards clinical trials. The proposed
materials, which use animal-derived products such as Matrigel, add
another layer of complication to the testing and approval process for
clinical application. There is little doubt about which type of cells
needs to be introduced into the infarcted region; cardiomyocytes have
shown to help regeneration and improve the cardiac function.
However, the source of cardiomyocytes, whether those resident in the
heart or derived from the bone marrow, embryonic or spermatogonial
44
British Medical Bulletin 2008;87
Myocardial tissue engineering
stem cells, to be used as the cell source, needs to be determined before
constructs can be introduced into human hearts. Furthermore, at what
stage of development cells should be used remains to be determined:
whether immature cells which could further proliferate in vivo or
mature cardiomyocytes with the properties of adult myocytes. In
addition, the question remains as to whether cardiomyocytes alone
should be introduced into the region or rather a multi-type culture consisting of cardiomyocytes, endothelial and fibroblastic cells to further
enhance neovascularization. Extreme purification strategies for cardiomyocytes may be counterproductive for this reason. Moreover, whether
the cellular constructs need to be cultured in vitro over a period before
introducing into the myocardium or implantation should occur
immediately after cell seeding remains questionable. A further possibility is the initial implantation of an acellular construct into the diseased myocardium to allow impregnation of newly formed vessels and
ECM on the scaffold to provide a suitable environment for the
implanted cells that will follow. Attachment of the material is another
challenge, given the strong and repetitive forces generated during myocardial contraction. Compressive forces upon constructs placed within
the muscle, or stretching of suture sites, will be experienced in each of
the 100 000 daily heartbeats. Although many groups have provided
evidence of the beneficial effects of MTE in vitro and in vivo (animal
models), the mechanism behind this functional improvement is yet to
be elucidated. In the meantime, it is important to overcome these
obstacles and further improve our understanding of myocardial regeneration for the design and development of the most suitable, reliable
and affordable construct.
References
1
2
3
4
5
6
7
British Medical Bulletin 2008;87
Christman KL, Randall JL (2006) Biomaterials for the treatment of myocardial infarction.
J Am Col Cardiol, 48, 907–913.
Beltrami AP, Urbanek K, Kajstura J et al. (2001) Evidence that cardiac myocytes divide after
myocardial infarction. N Engl J Med, 23, 1750–1757.
Messina E, Smith RR, Barile L et al. (2007) Regenerative potential of cardiosphere-derived
cells expanded from percutaneous endomyocardial biopsy specimens. Circulation, 115,
896–908.
Orlic D, Kajstura J, Chimenti S et al. (2001) Bone marrow cells regenerate infarcted myocardium. Nature, 410, 701–705.
Taylor DA, Atkins BZ, Hungspreugs P et al. (1998) Regenerating functional myocardium:
improved performance after skeletal myoblast transplantation. Nat Med, 4, 929– 933.
Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from
differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest, 98, 216–224.
Lyon A, Harding S (2007) The potential of cardiac stem cell therapy for heart failure. Curr
Opin Pharmacol, 7, 164–170.
45
H. Jawad et al.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
46
Dimmeler S, Zeiher AM, Schneider MD (2005) Unchain my heart: the scientific foundations
of cardiac repair. J Clin Invest, 115, 572–583.
Laflamme MA, Murry CE (2005) Regenerating the heart. Nat Biotechnol, 23, 845–856.
Al Attar N, Razak AB, Scorsin M (2002) Cellular transplantation, new horizons in the surgical management of heart failure. J R Coll Edinb, 47, 749– 752.
Orlic D, Kajstura J, Chimenti S et al. (2001) Mobilized bone marrow cells repair the
infarcted heart, improving function and survival. J Cell Mol Med, 9, 25 –36.
Fukuda K (2001) Development of regenerative cardiomyocytes from mesenchymal stem cells
for cardiovascular tissue engineering. Artif Organs, 25, 187–193.
Guan K (2007) Generation of functional cardiomyocytes from adult mouse spermatogonial
stem cells. Circ Res, 100, 1615– 1625.
Zimmermann WH, Fink C, Karlisch D, Remmers U, Weil J, Eschenhagen T (2000)
Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol
Bioeng, 68, 106– 114.
Giraud MN, Armbruster C, Carrel T, Tevaearai HT (2007) Current state of the art in myocardial tissue engineering. Tissue Eng, 13, 1825– 1836.
Shin H, Jo S, Mikos AG (2003) Biomimetic materials for tissue engineering. Biomaterials,
24, 4353–4364.
Jawad H, Ali NN, Lyon AR, Chen QZ, Harding SE, Boccaccini AR (2007) Myocardial tissue
engineering: a review. J Tissue Eng Med, 1, 327 –342.
Vunjak-Novakovic G, Radisic M, Obradovic B (2006) Review: cardiac tissue engineering:
effects of bioreactor flow environment on tissue constructs. J Chem Technol Biotechnol, 81,
485–490.
Shimizu T, Yamato M, Isoi Y et al. (2002) Fabrication of pulsatile cardiac tissue grafts using
a novel 3-dimensional cell sheet manipulation technique and temperature responsive cell
culture surfaces. Circ Res, 90, e40–e48.
Miyahara Y, Nagaya N, Kataoka M et al. (2006) Monolayered mesenchymal stem cells
repair scarred myocardium after myocardial infarction. Nat Med, 12, 459– 465.
Leor J, Aboulafia-Etzion S, Dar A et al. (2000) Bioengineered cardiac grafts—a new approach
to repair the infarcted myocardium? Circulation, 102, 56 –61.
Christman KL, Vardanian AJ, Fang Q, Sievers RE, Fok HH, Lee RJ (2004) Injectable fibrin
scaffold improves transplant survival, reduces infarct expansion, and induces neovasculture
formation in ischemic myocardium. J Am Col Cardiol, 44, 654 –660.
Dai W, Wold LE, Dow JS, Kloner RA (2005) Thickening of the infarcted wall by collagen
injection improves left ventricular function in rats. J Am Col Cardiol, 46, 714– 719.
Kofidis T, Lebl DR, Martinez EC, Hoyt G, Tanaka M, Robbins RC (2005) Novel injectable
bioartificial tissue facilitates targeted, less invasive, large-scale tissue restoration on the
beating heart after myocardial injury. Circulation, 112, 173– 177.
Leor J (2005) Cells, scaffolds, and molecules for myocardial tissue engineering. Pharmacol
Ther, 105, 151– 163.
Iwakura A, Fujita M, Kataoka K et al. (2003) Intramyocardial sustained delivery of basic
fibroblast growth factor improves angiogenesis and ventricular function in a rat infarct
model. Heart Vessels, 18, 93 –99.
Davis ME, Motion JPM, Narmoneva DA et al. (2005) Injectable self-assembling peptide
nanofibres create intramyocardial microenvironments for endothelial cells. Circulation, 111,
442–450.
Chen QZ, Harding SE, Ali NN, Jawad H, Boccaccini AR (2007) Cardiac tissue engineering.
In Boccaccini AR, Gough JE (eds) Tissue Engineering Using Ceramics and Polymers.
Woodhead Publishing in Materials, 335–356.
McDevitt T, Woodhouse KA, Hauschka SD, Murry CE (2002) Spatially organized layers of
cardiomyocytes on biodegradable polyurethane films for myocardial repair. J Biometr Res,
66A, 586– 595.
Pego AP, Van Luyn MJA, Brouwer LA et al. (2003) In vivo behavior of poly(1,3-trimethylene
carbonate) and copolymers of 1,3-trimethylene carbonate with D,L-lactide or epsiloncaprolactone: degradation and tissue response. J Biomed Res, 67A, 1044– 1054.
Iyer RK, Radisic M (2006) Microfabricated poly(ethylene glycol) templates for tri-culture in
cardiac tissue engineering. J Mol Cell Cardiol, 40, 877.
British Medical Bulletin 2008;87
Myocardial tissue engineering
32 Chen QZ, Bismarck A, Hansen U et al. (2008) Characterisation of a soft elastomer poly(glycerol sebacate) designed to match the mechanical properties of myocardial tissue.
Biomaterials, 29, 47 –57.
33 El-Fray M, Boccaccini AR (2005) Novel hybrid PET/DFA-TiO2 nanocomposites by in situ
polycondensation. Mater Lett, 59, 2300–2304.
34 Kofidis T, Akhyari P, Boublik J et al. (2002) In vitro engineering of heart muscle: artificial
myocardial tissue. J Thorac Cardiovasc Surg, 124, 63 –69.
35 Eschenhagen T, Fink C, Remmers U et al. (1997) Three-dimensional reconstitution of
embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J,
11, 683– 694.
36 Xiang Z, Liao R, Kelly MS, Spector M (2006) Collagen-GAG scaffolds grafted onto myocardial infarcts in a rat model: a delivery vehicle for mesenchymal stem cells. Tissue Eng, 12,
2467–2478.
37 Kofidis T, de Bruin JL, Hoyt G et al. (2005) Myocardial restoration with embryonic stem cell
bioartificial tissue transplantation. J Heart Lung Transplant, 24, 737– 744.
38 Akhyari P, Fedak PWM, Weisel RD et al. (2002) Mechanical stretch regimen enhances the
formation of bioengineered autologous cardiac muscle grafts. Circulation, 106, I137– I142.
39 Ott HC, Matthiesen TS, Goh SK et al. (2008) Perfusion-decellularized matrix: using nature’s
platform to engineer a bioartificial heart. Nat Med, 14, 213– 221.
40 Krupnick AS, Kreisel D, Engels FH et al. (2002) A novel small animal model of left ventricular tissue engineering. J Heart Lung Transplant, 21, 233–243.
British Medical Bulletin 2008;87
47