Myocardial tissue engineering: a review

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE REVIEW J Tissue Eng Regen Med 2007; 1: 327–342. Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/term.46

ARTICLE

Myocardial tissue engineering: a review H. Jawad,1 N. N. Ali,2 A.R. Lyon,2 Q. Z. Chen,1 S. E. Harding2 and A. R. Boccaccini1 * 1 2

Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK

Abstract Myocardial tissue engineering, a concept that intends to overcome the obstacles to prolonging patients’ life after myocardial infarction, is continuously improving. It comprises a biomaterial based ‘vehicle’, either a porous scaffold or dense patch, made of either natural or synthetic polymeric materials, to aid transportation of cells into the diseased region in the heart. Many different cell types have been suggested for cell therapy and myocardial tissue engineering. These include both autologous and embryonic stem cells, both having their advantages and disadvantages. Biomaterials suggested for this specific tissue-engineering application need to be biocompatible with the cardiac cells and have particular mechanical properties matching those of native myocardium, so that the delivered donor cells integrate and remain intact in vivo. Although much research is being carried out, many questions still remain unanswered requiring further research efforts. In this review, we discuss the various approaches reported in the field of myocardial tissue engineering, focusing on the achievements of combining biomaterials and cells by various techniques to repair the infarcted region, also providing an insight on clinical trials and possible cell sources in cell therapy. Alternative suggestions to myocardial tissue engineering, in situ engineering and left ventricular devices are also discussed. Copyright  2007 John Wiley & Sons, Ltd. Received 7 June 2007; Accepted 27 August 2007

Keywords

myocardial infarction; tissue engineering; biomaterials; scaffolds; cell therapy

1. Introduction Cardiovascular disease (CVD) is a major health problem and the leading cause of death in the Western world. In the UK, CVD accounts for 238 000 annual deaths, comprising 39% of all deaths per annum. Heart attacks are the main cause of death in patients with CVD. Approximately 30% of the 270 000 patients suffering from heart attacks each year die suddenly before reaching the hospital (www.bhf.org.uk). In the remaining patients who survive their initial acute event, the damage sustained by the heart may eventually develop into heart failure. A heart attack, known as a myocardial infarction (MI), occurs when one or more of the blood vessels supplying the heart suddenly occlude. These vessels are the coronary arteries and, when blocked abruptly, there is a sudden decrease in the supply of nutrients and oxygen to the portion of heart muscle supplied by the artery. If blood flow is not restored rapidly, *Correspondence to: A. R. Boccaccini, Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK. E-mail: [email protected] Copyright  2007 John Wiley & Sons, Ltd.

the result is irreversible cell death within the affected part of the heart muscle. The adult heart cannot repair the damaged tissue, as the mature contracting cardiac cells, the cardiomyocytes, are unable to divide. The result of the myocardial infarction is the formation of scar tissue which does not have contractile, mechanical and electrical properties of normal myocardium (heart muscle). The replacement of contractile myocardium with noncontracting fibrous scar reduces the pumping efficiency of the ventricles, the heart’s main pumping chambers. Various compensatory mechanisms are activated in response to the reduced cardiac output. These initially stabilize the damaged heart and maintain cardiac output at acceptable levels. Ultimately these ‘compensatory systems’ place extra burden on the weakened heart muscle. This leads to a downward spiral of cardiac function and the development of the clinical syndrome of heart failure. The deterioration of heart function accelerates as heart failure progresses. Ultimately at the end-stage of heart failure, mechanical ventricular assist devices (VADs; Birks et al., 2006) or heart transplantation are the only options. However, due to the high cost

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of VADs and the shortage of donar organs (Akins, 2002), many patients die while waiting on the shortlist. Alternatives are therefore required, with cell therapy being a potential option. Replacement of scarred tissue with skeletal muscle cells, cells derived from bone marrow (mesenchymal and haematopoietic) or embryonic stem cells (ESCs) have been proposed (Laflamme and Murry, 2005). Currently the preferred method of introducing these cells into the dead myocardium is injection of cells in suspension, either into the circulating blood or directly into the myocardium. Cell delivery by either route is inefficient, with substantial cell loss (BOOST nuclear study; Hofmann et al., 2005; Grossman et al., 2002). This has prompted a search for alternative delivery techniques for the cells, such as tissue engineering (TE). The aim of cardiac tissue engineering (CTE) is to repair or regenerate a damaged section of the heart. It has been proposed for heart valves (Zund et al., 1996) and myocardial muscle (Zimmermann et al., 2000), the latter being of interest in this review. CTE involves the synthesis of a scaffold or patch made from a biomaterial combined with cells. The main function of the biomaterial is to act as a vehicle for the delivery of cells to the damaged area, i.e. the scarred tissue. Once cells are delivered to the desired region, the hope is that the cells will integrate with the host tissue, forming new myocardium. Biomaterials research is a broad subject area, with engineers and material scientists constantly looking for improved candidates suitable for application as scaffolds in TE. To date, both synthetic and natural polymers, including collagen and alginate, have been proposed for CTE. The present review covers international research being carried out in the broad field of CTE.

2. Cardiac failure The inhibition of the heart to deliver sufficient blood to meet the body’s metabolic requirements will lead to cardiac failure. It is a major cause of death in industrialized nations, and can result from any disease that damages the myocardium, resulting in the reduced ability to pump blood. The most common causes are coronary artery disease and hypertension (high blood pressure), but damage to any part of the heart’s intricate structure can impair cardiac performance and result in heart failure. This includes disease of the heart valves, the electrical conduction system of the heart, or external pressure around the heart, due to constriction of the pericardial sac in which the heart is sited. MI is caused by a significant reduction coronary blood supply to an area of the heart over a sustained period, eventually forming non-contractile fibrous scar tissue with reduced or absent contractile ability compared to the rest of the healthy heart. Heart failure may occur immediately in severe cases, or over a longer duration if the initial insult is milder. Figure 1 shows a schematic diagram Copyright  2007 John Wiley & Sons, Ltd.

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Infarcted area

Figure 1. Schematic diagram illustrating the damage caused by MI in human heart. Source: www.heartpoint.com (accessed 25 January 2007)

representing the damaged area after a heart attack due to a blocked coronary artery. The myocardium is a terminally differentiated tissue that is unable to regenerate (Anversa et al., 2002). It cannot compensate for the cell loss which occurs during MI, eventually leading to maladaptive left ventricular remodelling and end-stage heart failure (Leor et al., 2006). For this reason, there is much interest in developing new methods to repair and regenerate an infarcted area of the myocardium. Other suggested alternatives to heart transplantation are summarized in Table 1.

3. Cardiac regeneration or repair strategies for heart failure treatment 3.1. Regeneration of lost tissue from cardiac stem cells resident in the heart Cells with the potential to self-renew and differentiate along specific lineages are called stem cells. Depending on their origin, they are referred to as either ESCs or adult stem cells. ESCs are formed from early embryos, having the ability to differentiate into every cell type in the body, and are therefore referred to as pluripotent (see section 3.4.3). Adult stem cells, which reside in tissues/organs, are referred to as multipotent, as they have the capacity to differentiate into a restricted number of cell lineages. Examples of adult stem cells include haematopoietic and mesenchymal stem cells of the bone marrow, liver and brain, and cardiac stem cells. Anversa and co-workers have described a group of cells resident in adult hearts with markers such as Lin− , c-Kit+ , Ki-67 (Beltrami et al., 2001, 2003; Smits et al., 2005) and transcription markers such as GATA-4 and Nkx2.5, and referred to these as cardiac progenitor stem cells. However, their origin is still unclear, and hypotheses vary – these cells may home to the heart from the bone marrow; alternatively, they may reside in the heart from fetal life. They may contribute to cell turnover and heart repair on a small scale through normal life, but this capacity is overwhelmed by the injury to the myocardium from acquired heart diseases, such as myocardial infarction. However, their low population and J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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Myocardial tissue engineering: a review

Table 1. Summary of the advantages and disadvantages of MTE approaches Approach

Advantages

Cellular cardiomyoplasty (injection of cells only, either directly or intravenously)

Minimally invasive surgery if injection is intravenous

In situ engineering (injection of cells and an injectable biomaterial

Biomaterial acts as a supporting matrix while cells will simultaneously regenerate infarction Matrix for homing autologous progenitor cells Does not involve cell injection

Injection of a biomaterial alone Left ventricular restraints (’wrapping’ the ventricles with a biopolymer) Tissue engineering

Ensures cells are delivered to desired area with minimal cell loss

the limited evidence for myocardial regeneration hinders their immediate use for clinical trials, even though these cells have been shown to have the ability to differentiate into cardiomyocytes and vascular cells when isolated from human myocardium (Messina et al., 2004; Smits et al., 2005). Myocardial tissue repair and regeneration has been attempted using cells from a number of sources, including bone marrow (Kocher et al., 2001), skeletal muscle (Taylor et al., 1998) and ESCs (Heng, 2005), or primary cardiomyocytes derived from neonatal rat hearts (Zimmermann et al., 2000). Although primary myocytes implant well, limitations such as their low yield and poor proliferative capacity have encouraged tissue engineers to overcome this problem by using stem cells as an alternative cell source (Kofidis et al., 2002b).

3.2. Regeneration by the bone marrow stem cells 3.2.1. Progenitor cell mobilization from bone marrow Cytokines, such as granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF), are known to stimulate progenitor cell release from the bone marrow. After mobilization, i.e. releasing a pool of stem cells into the peripheral circulation, it has been hypothesized that these undifferentiated stem cells may home to the infarcted region of the heart. Much debate exists as to whether this phenomenon occurs in humans, and if so whether the homing bone marrow cells have any capacity to differentiate into cardiomyocytes, contributing to myocardial regeneration (Smits et al., 2005). Research is being carried out to investigate this route further, with some reports on an improvement in the infarcted area and cardiac function (Orlic et al., 2001b), while other studies have reported that G-SCF and SCF had no effect on the infarct size but encouraged vessel formation (Noral et al., 2003). Quaini et al. (2002) reported that 9% of cells express stem-cell antigens; however, this percentage has Copyright  2007 John Wiley & Sons, Ltd.

Disadvantages Lack of knowledge as to how cells contribute to myocardial regeneration or repair. Much concern revolved about direct injection only affecting endocardium and not epicardium. Concern regarding cell loss Involves open chest surgery and this suggestion is at infancy Immunogenicity, as only natural polymers have been suggested Prevents remodelling, but does not repair or regenerate damaged area Involves open chest surgery and more work is required to determine suitable cell type and material

not been reproduced by others (0.016% according to Smits et al., 2005). Debate exists as to whether bone marrow progenitor cells do indeed migrate to the infarcted region in the adult human. If they do, then further questions arise: what fraction of cells is lost whilst in the blood circulation, what is the fate of the mobilized cells, and do these cells differentiate into cardiomyocytes? Most experiments have been carried out using young healthy mice (Kawada et al., 2004) and applicability to the human clinical situation is limited (see below).

3.2.2. Bone marrow stem cell (BSMC) injection Over the years, much attention has been focused on bone marrow as a source of stem cells for cell therapy. It contains two components, stromal and haematopoietic, the former producing mesenchymal stem cells while the latter is involved with new blood cell (both red and white) formation. Advantages of BMSCs include their easy access; they are autologous and therefore there are no issues with cell rejection. Moreover, Wang et al. (2000) and Hassink et al. (2003) reported their ability to differentiate into cardiomyocytes in vivo. Jackson et al. (2001) demonstrated a 26% survival rate of mice that had been injected with haematopoietic stem cells into their circulation following MI. Results suggested that the transplanted stem cells responded to signals in the infarcted region of the myocardium, causing them to migrate to the damaged area (cell homing) and differentiate into the cells required for cardiac repair, contributing to neovascularization. Orlic et al. (2001a, 2002) used a florescence-activated cell sorting technique to select Lin− c-kit+ bone marrow cells from mice; the cells were then injected directly into the infarcted area, creating new cardiomyocytes, smooth muscle cells and vascular endothelium, forming de novo myocardium with living tissue 9 days after cell transplantation. The results were promising, as the regenerated myocardium occupied 68% of the infarcted area and the mice that received cell transplantation survived in greater numbers than the J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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mice that did not. Analysis of the region surrounding the damaged tissue suggests that the stem cells responded to signals in the infarcted area, causing them to multiply into ‘specialized’ cardiomyocytes (Orlic et al., 2001a). In another study, BMSCs were shown to be capable of developing vascular endothelial cells when transplanted into rat hearts, forming new blood vessels in the damaged area (Kocher et al., 2001). These findings have only been demonstrated in rodent models, with young animals receiving young healthy bone marrow. Reproduction of these findings by others groups has been unsuccessful (Nygren et al., 2004; Murry et al., 2004; Balsam et al., 2004), and publication bias towards positive studies may skew the true extent of bone marrow progenitor cells to regenerate the infarcted myocardium. However, in elderly patients with cardiac disease the bone marrow has greatly reduced function (Heeschen et al., 2004), and similarly for older rats in comparison with younger ones (Lehrke et al., 2006). Therefore, the likelihood of clinically significant reparative or regenerative qualities of their own bone marrow progenitor cells is minimal. The current dogma is that BMSCs do not transdifferentiate into contractile cardiomyocytes. This has brought about suggestions that the marginal improvements (e.g. left ventricular ejection fraction) in clinical trials may be be due to other factors, such as increased cytokine release and/or formation of more blood vessels. The latter could provide significant benefit, as angiogenesis would supply more oxygen and nutrients to support native cells in ischaemic tissue (Takahashi et al., 2006). Critical issues to be addressed include the possible formation of nonmuscle tissue types, i.e. bone and cartilage (known to be formed from BMSCs), and the need for more long-term studies. Recently, Rosenzweig (2006) reported on the mixed results achieved from BMSC transplantation into patients with infarcted hearts; with Schachinger et al. (2006) reporting the best evidence so far on the success of BMSCs. However, other trials suggesting only mild or no improvement have lessened enthusiasm for the therapeutic use of BMSCs.

3.3. Repair of myocardium with skeletal myoblasts A subpopulation of skeletal muscle cells have the ability to become active, self-renew and differentiate, permitting muscle regeneration upon muscle injury (in vivo) (Al Attar et al., 2002). Due to the ability to contract and continuously repair the damaged muscle as well as being easily cultured, multiplied and recognized in vitro, these autologous cells were introduced into the heart to repair the infarcted area (Hassink et al., 2003). The results of studies attempting to restore cardiac function have been mixed, with some being successful (Taylor et al., 1998; Hutcheson et al., 2000; Chiu et al., 1995; Menasche et al., 2001), and somewhat encouraging clinical trials have been reported (Menasche et al., 2001), while Copyright  2007 John Wiley & Sons, Ltd.

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others reported no sign of physiological improvements and/or cardiac differentiation (Murry et al., 1996; Scorsin et al., 1997, 2000). Key obstacles for the use of these cells for myocardial regeneration are their apparent inability to transdifferentiate into cardiomyocytes. This prevents electrically coupling with native cardiomyocytes, creating a pro-arrhythmic substrate, as the impulse cannot propagate evenly across the myocardium and transplanted cells. This reflects the absence of gap junctional protein connexin43 expression in skeletal myoblasts. Patients require pacemakers or defibrillators to ensure synchronous beating, and treatment of any dangerous heart rhythms (arrhythmias) which may be triggered. Introduction of connexin43 by genetic modification has not been successful because it has been shown to affect cell life span (Reinecke et al., 2004). However, despite these drawbacks, it has been suggested that fibrin glue and skeletal myoblasts together can preserve cardiac function when injected into a myocardial infarcted region (Christman et al., 2004a).

3.4. Implantation of differentiated cardiomyocytes to increase cardiac muscle mass 3.4.1. Primary cultures of cardiomyocytes The first cell types to be investigated for cell transplantation were cardiomyocytes, as they are the cells that contribute to the majority of the cells that makes up the heart. The use of isolated adult and neonatal cardiomyocytes have been pivotal for our understanding of the many aspects of cardiac cell biology, electrophysiology and pharmacology (Menasche and Desnos, 2002). The main advantage of isolated adult cardiomyocytes is their true morphological representation and perseveration of cardiac function in vitro. Adult ventricular and atrial cardiomyocytes have been obtained successfully from both human (Harding et al., 2007) and animal hearts. Unfortunately, their clinical application is not ideal, for a number of reasons. First, it has been difficult to demonstrate survival of adult cardiomyocytes in culture, without losing their characteristic features, for more than 48 h. They are also reported to dedifferentiate in culture, with their phenotype eventually verging on the neonatal cardiomyocyte phenotype, which is less structured (Harding et al., 2007). More importantly, adult cardiomyocytes provide limited supply in culture, as they lack the ability to differentiate, proliferate and self-renew. Rat neonatal cardiac myocytes are easy to prepare in large quantities, last longer in culture (1 week) and can be genetically modified. For these reasons they have been used as a standard model for many years (Zimmermann et al., 2000). However, these too provide limited supply and require the involvement of animals, hindering their use in CTE in humans (Kofidis et al., 2002b). Nevertheless, researchers (Zimmermann et al., 2004) have used neonatal cardiomyocytes in tissue engineering in animals. J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

Myocardial tissue engineering: a review

3.4.2. Bone marrow-derived cardiomyocytes An alternative method for obtaining cardiomyocytes is by culturing the BMSCs in vitro, for example by treating them with 5-azacytidine, to differentiate BMSCs into cardiomyocytes and introduce into the injured heart. Fukuda (2001), Makino et al. (1999) and Tomita et al. (1999) have reported differentiated myogenic cells from BMSCs to improve myocardial function. This work, however, has been difficult to reproduce.

3.4.3. Embryonic stem cell-derived cardiomyocytes (ESCMs) For all the limitations listed above, the idea of using embryonic stem cells (ESCs) for both research and therapeutics is being more accepted, despite the ethical implications (Polak and Bishop, 2006). Mouse embryonic stem cells (mESCs) were isolated from the inner cell mass of a mouse blastocyst-stage embryos just over two decades ago (Evans and Kaufman, 1981; Polak and Bishop, 2006). However, derivation of human embryonic stem cells (hESCs) are more recent and were first reported in 1998 by Thomson et al. (1998), opening a new era in therapeutics. This is promising, since ESCs have the potential to be propagated in culture for unlimited periods without karyotypic changes, allowing a good supply of cells for potential use. Furthermore, they are able to differentiate into all cell types in the body, because all three primary germ layers that arise during embryonic development – ectoderm, endoderm and mesoderm (Bradley et al., 1984) – are developed during ESC differentiation in vitro. The outermost layer, ectoderm, forms the epidermis and nervous system, while the endoderm layer forms the gastrointestinal and respiratory tracts, as well as the endocrine glands. Cardiac tissue is formed from the mesoderm layer, which is the embryonic origin of cardiomyocytes. Mesoderm also makes blood, bone, cartilage, endothelial and mesenchymal cells (Van Laake, 2005). As well as ESCs, two other different types of pluripotent stem-cell lines from mammalian embryos are possible, embryonic carcinoma (EC) and embryonic germ (EG) cells. The defining feature of ESCs that distinguishes them from the other two is the fact that when reincorporated into normal embryonic development, i.e. a host blastocyst, they have the full potential to develop along all lineages of the embryo proper. EC cells are derived from undifferentiated embryonic components of germ cell tumors, whereas EG cells are derived from primordial germ cells in the genital ridge (Boheler, 2002), making ESCs more attractive for TE applications. Despite immunological and ethical constraints, the use of cardiomyocytes derived from ESCs is an expanding field in cell therapy and TE (Kumar, 2005; Harding et al., 2007). The differentiation of ESCs into all cardiac cell types (pacemaker, atrial, nodal, Purkinje-like and ventricular cells) have been well characterized in Copyright  2007 John Wiley & Sons, Ltd.

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many laboratories (Ali et al., 2004; Harding et al., 2007; Kehat et al., 2001, 2004; Mummery, 2002, 2003). Initially, the cardiomyocytes formed at the early stages resemble pacemaker and primary myocardial cells. It is not until later on during the differentiation process that atrial, nodal, Purkinje-like and ventricular cells are developed (Laake et al., 2005; Wobus et al., 1997). The rate at which cardiac expression factors are expressed is also evident during different differentiation stages, with cardiac-specific myosin heavy chains occurring in mature cardiomyocytes (Laake et al., 2005). Klug et al. (1996) were the first to demonstrate the potential use of ESCMs for cardiac repair, by treating dystrophic mouse hearts with mESCMs. Min et al. (2002) demonstrated that ESCMs can survive in the cardiomyocyte phenotype in vivo and repair myocardial damage in animal models, making ESCs a strong candidate for cardiac repair. Interestingly, the transplantation of undifferentiated mESCs, treated with specific growth factors (TGFβ- or BMP2; Behfar et al., 2002) or cardiaccommitted cells (Menrad et al., 2005), into infarcted hearts was successful in that cells were integrated and myocardial function was improved; however, others reported cell rejection and teratoma development (Behfar et al., 2002). With much research successfully reporting the potential of mESCMs (Caspi and Gepstein, 2006; Guo et al., 2006; Messina et al., 2004; Min, 2002; Mummery, 2002; Sachinidis et al., 2003; Stojkovic et al., 2005; Wei, 2005), Human embryonic stem cell derived cardiomyocytes (hESCM) are now receiving much attention for their possibilities for cell therapy (Laflamme et al., 2005). To date, researchers have established characteristics of many aspects of the cardiac phenotype of the hSCMs, confirming the presence of cardiac-specific structural genes, proteins and transcription factors. Also identified were calcium transients, action potentials, extracellular electrical activity, cardiac structures, such as intercalated discs and sarcomeric organization (Caspi and Gepstein, 2006; Kehat et al., 2001, 2004; Mummery, 2003; Xu et al., 2002), providing further proof of successful cardiomyogenesis from hESCs. Moreover, electromechanical integration of hESCMs with the host myocardium both in vitro and in vivo has successfully demonstrated the potential use of hESCMs in cell therapy. Laflamme et al. (2005) reported the formation of human cardiac tissue in athymic rat hearts by injecting an enriched population of hESCMs by the fourth week. Leor et al. (2005) compared undifferentiated hESCs and differentiated hESCMs and showed that both can survive when transplanted into either normal and infarcted hearts of nude rats. However, neither type contributed to new myocardium formation, and some possible teratoma formation was observed. They also reported the importance of eliminating undifferentiated cells prior to transplantation. For further and more detailed reading regarding cell types for cell therapy, see Lyon and Harding (2007). J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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3.5. Cell delivery routes Important drawbacks of cell therapy include the lack of sufficient published data on the optimal timing for cellular transplantation, the ideal number of cells required for transplantation, and their long-term survival and terminal differentiation post-implantation (Al Attar et al., 2002). Moreover, cell delivery technique is of major concern. The two different routes proposed for cell delivery to the damaged area are injection of cells either directly into the infarcted region (Orlic et al., 2001a) or via the coronary circulation (Jackson et al., 2001). Both routes have advantages and disadvantages. Directly injecting cells into the infarcted area ensures the delivery of cells directly to damaged area, but is hampered by significant cell loss after needle withdrawal from the myocardium. Cell implantation can be achieved via open chest surgery (thoracotomy), which carries risks associated with surgical operations, or percutaneously via needle injection from a catheter directed to the inner surface of the ventricle (endocardial injection). At present, the preferred method of introducing cells is by injection into the coronary vessels, with the hope that they will home to the infarcted area. In an attempt to increase the contact time of cells with the myocardium, blood flow in the coronary vessels can be temporarily occluded by angioplasty balloon inflation. This technique is also inefficient at cell delivery, with only a small fraction of the delivered cells being retained in the heart (5 µm (Pham et al., 2006). The electrospinning method is an attractive way of producing scaffolds that remarkably mimic the size and scale of the natural extracellular matrix, as it enables the fabrication of structures with sub-micron pores and nano-topography, which many have reported being optimal in structures for successful extracellular matrix-like scaffolds in CTE (Courtney et al., 2006; Ishii et al., 2006; Shin et al., 2004; Stankus et al., 2004; Zhong et al., 2005). Ishii et al. (2006) have cultured primary cardiomyocytes harvested from neonatal rats onto biodegradable electrospun nanofibrous poly(ε-caprolactone) meshes with an average fibre diameter of 250 nm, using the celllayering technique (Shimizu et al., 2001). The unique procedure carried out by this group was the layering of individual grafts after 5–7 days of cell seeding, constructing a 3D cardiac graft. The results demonstrated strongly beating cardiomyocytes, well attached to the meshes throughout the 14 day experimental period. Histological and immunohistochemical tests confirmed that morphological and electrical communications were established in constructs with up to five layers of mesh, proving strong adherence between the individual layers and cells (Haraguchi et al., 2006). However, long-term studies have Copyright  2007 John Wiley & Sons, Ltd.

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yet to determine the cells’ lifespan in vitro as well as in vivo. Bioreactor devices or systems that encourage the growth and development of biological cells or tissues are widely used in TE, their main advantage being the ability to cultivate cells on biomaterials, producing contractile constructs of cell–polymer origin (Bursac et al., 1999; Carrier et al., 1999, 2001, 2002; Kofidis et al., 2003b; Papadaki et al., 2001; Park et al., 2005). Researchers are constantly striving to design an improved bioreactor to make it possible to grow 3D myocardial tissue comprised of more than a few layers of muscles. Many different geometries, portraying various patterns of fluid dynamics, have been suggested (Leor, 2005). Currently, the negative aspect of bioreactors is their restricted ability to supply an adequate amount of nutrients to a tissue of thickness greater than approximately 100 µm (some reports suggesting 80 µm; Birla et al., 2005), or less than 10 cell layers thick (Leor, 2005). This limitation causes weak cellular integrity and short duration of contractility, as well as nonhomogeneous seeding, as a result affecting tissue function and cellular viability (Vunjak-Novakovic et al., 2006). Although the purpose of these bioartificial tissues is primarily for scientific studies, researchers also intend to improve their in vitro designs and eventually use them for cardiac repair. Carrier et al. (2001) and Radisic et al. (2006, 2005) demonstrated the importance of oxygen on engineered cardiac grafts to overcome the limitation of producing a 100 µm thick new tissue. They found that, by increasing the oxygen concentration supply to the in vitro construct, an improved engineered cardiac muscle was produced. They then went on to improve the engineered cardiac muscle further, by allowing the medium to directly perfuse the construct (Carrier et al., 2002). Additional studies are required to evaluate the specific properties of these engineered constructs, including the mechanical behaviour, biodegradability and eventually their in vivo performance. Primary neonatal rat ventricular cells were cultured on PGA scaffolds by Bursac et al. (1999), using bioreactors to form cardiac muscle constructs. The polymer scaffold provided the 3D substrate for cell attachment and consequently tissue formation, whereas the bioreactor promotes mass transfer of nutrients and gases to the forming tissue. Within a week, cardiac myocytes were organized in multiple layers in a 3D configuration, and this was used for in vitro impulse studies at a macroscopic (tissue) level, rather than at a cellular level. The above studies show the role of fetal or neonatal cardiomyocytes in bioengineered constructs. However, as mentioned earlier, the inability or limited ability of these cells to proliferate (Wu et al., 2006) and the success of directly injecting embryonic stem cells (Sabbah, 2003) into an infarcted area of the heart provoked novel approaches. For example, Ke et al. (2005) have investigated the effectiveness of grafting a commercially available PGA biodegradable patch with mESCs (in an undifferentiated state). The patch was then sutured onto J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

Myocardial tissue engineering: a review

the hearts of rats that were MI induced by ligation of the left coronary artery. Cardiac function and haemodynamics were evaluated 8 weeks following patch implantation, showing a significant improvement in ventricular function and blood pressure, with a survival rate of 82.1%. On the other hand, 46.2% of the mice that did not receive the cardiac patch with ESCs died within 8 weeks. This novel approach of combining biomaterials and ESCs is still in its infancy; however, ongoing research will provide a more solid foundation to questions that need answering for advancement of the field. Poly-co-caprolactone (PGCL) seeded with bone marrowderived mononuclear cells was recently suggested as a cardiac patch for rat myocardial infarcted hearts (Piao et al., 2007), reporting reduced left ventricle (LV) remodelling, preserved systolic function of the LV and some presence of myosin heavy chains, indicating transdifferentiation of the bone marrow cells into cardiac cells. However, this study was carried out for only 4 weeks, and the current dogma that BMSCs do not transdifferentiate in contractile cardiomyocytes suggests that much more work is needed for this idea to be accepted by many investigators. It has also been reported that the size and location of the infarct determines the success of an engineered cardiac patch (Cui et al., 2005). The strategy was found to be more successful with smaller infarct size and when the infarct was anteriorly positioned, preventing unnecessary manipulation of heart during surgery. This study does provide information for researchers who are currently investigating the success of their engineered scaffolds or patches; however, one cannot control the size and position of infarcts that occur in humans. MTE based on natural materials. Over the past years, Zimmermann et al. (2000) have developed a novel technique to engineer a cardiac muscle construct, better known as engineered heart tissue (EHT), with a combination of neonatal cardiomyocytes with an artificial extracellular matrix, made mainly from collagen type I and Matrigel in circular moulds while being subjected to mechanical strain. The all-natural EHT construct developed by Zimmerman et al. has both advantages and disadvantages. Advantages include: EHT has been reported to contract for 8 weeks in vivo; newly formed myocardium of ∼450 µm thickness has been observed; functional and morphological properties of differentiated heart muscle have been observed; and heart muscle shape and size can be manipulated accordingly. Unfortunately, several disadvantages were also found. For example, upon implantation, left ventricular function was not improved, according to echocardiography studies, and there is a need for immunosuppression for the survival of the EHT in vivo (Zimmermann, 2002). Recently, three modifications were implemented to the original EHT to improve in vivo performance: first, culture was conducted under elevated ambient oxygen; second, culture was done under auxotonic load (EHT contracts against load-adjusted coils); and finally, the culture medium was supplemented with Copyright  2007 John Wiley & Sons, Ltd.

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insulin (Zimmermann et al., 2006). 28 days after implantation, electrical coupling to the native myocardium was observed, as well as systolic wall thickening of the infarcted region. The results show that large contractile grafts may be constructed in vitro, implanted in vivo and eventually support infarcted myocardial muscle. As exciting as these results are, further investigations are needed on potential clinical use, a suitable cell source, the immunogenicity of the EHTs and finally the graft size (Zimmermann et al., 2006), although it was recently reported that mixing heart cell populations in serum- and Matrigel-free conditions is possible and may reduce the immunogenicity of the EHTs (Naito et al., 2006). Li et al. (2000, 1999) seeded fetal rat ventricular cells onto commercially available 3D gelatin mesh (Gelafoam), forming cardiac tissue that contracted spontaneously both in vitro and in vivo for 5 weeks after implantation onto rat myocardial scar tissue, forming junctions with the recipient heart. However, post-implantation results showed no sign of cardiac function improvement. Zhong et al. (2005) developed a scaffold by electrospinning collagen and glycosaminoglycan (GAG), both abundant proteins in the extracellular matrix of the body, forming a nanofibrous scaffold, reporting a favourable environment for cell proliferation upon the incorporation of collagen. Other studies have reported the successful combination of collagen and GAG, where bone-marrow derived mesenchymal stem cells were implanted into a scaffold and onto the infarcted region, resulting in neovascularization in the infarcted region (Xiang et al., 2006). Kofidis et al. (2002a) went on to design an experimental reactor with multiple chambers for the production of bioartificial tissue, where neonatal rat cardiomyocytes were inoculated into collagen ground matrix; obtaining an 8 mm graft, compared to the 2 mm previously achieved (Carrier et al., 1999), to allow the ventricular wall to restore transmurally. More recently, Kofidis et al. (2005a) combined undifferentiated mESCs with collagen type I matrix and reported the benefits of ESCs in a 3D collagen matrix when implanted in the infarcted area, introduced by ligation of the left anterior descending artery in nude rat and surgically forming an intramural pouch. Stable intramyocardial grafts were observed without altering the myocardial geometry and increasing ventricular wall thickness (1.4 ± 0.1 mm), as well as expressing gap junctional protein connexin43 in vivo. This study highlights the benefit of both the controversial ESCs and the continually questioned immunogenicity of collagen when used in TE. Ongoing studies are required in this area, as there are many more aspects to be considered, including endothelialization of the graft’s inner surface, as well as mechanical properties. Attempts have been made to improve the mechanical function of bioartificial tissues grown on natural polymers. For example, Akhyari et al. (2002) seeded heart cells on Gelafoam and cyclical mechanical stress was applied at a frequency of 80 cycles/min for 14 days, using a bio-stretch apparatus as described by Liu et al. (1999). The important effect of J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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mechanical stress during the development of bioartificial tissue was demonstrated, as the proliferation and distribution of cells improved upon incorporation of mechanical stress. Recently, Guo et al. (2006) demonstrated that ESCs can be used for cell seeding in CTE. Cardiomyocytes derived from ESCs were mixed with type I collagen supplemented with Matrigel, and pipetted into circular casting moulds, then incubated for 30–45 min to allow hardening of the mixture, making the engineered cardiac tissue (ECT). After 7 days in culture, the ECT was placed into a stretch device to undergo unidirectional cyclic stretch for an additional 7 days. However, this study is still in its infancy, and additional work is required to determine in vivo suitability. Collagen haemostatic scaffold, also called tissue fleece, was first suggested for MTE by Koofidis et al. (2003a). They developed a contractile bioartificial myocardial tissue from collagen tissue fleece and cardiomyocytes in vitro, with homogeneous cellular distribution. More recently, Gaballa et al. (2006) reported a reduction in cardiac remodelling and increased neo-angiogenesis when grafted with acellular 3D collagen scaffold onto infarcted rat hearts (625 neo-vessel/cm2 from 75 neo-vessel/cm2 in rats without scaffolds). Further neo-angiogenesis (978 neo-vessel/cm2 ) was reported when rats implanted with collagen scaffold received subcutaneous injections of granulocyte-colony stimulating factor (G-CSF), which has been reported to mobilize progenitor cells, for 5 days. Although the collagen scaffold is initially there to provide initial mechanical support and reduce remodelling, immunogenicity of the scaffold is still an obstacle. The conflicting results as to whether G-CSF does actually mobilize resident cells to home to the infarct region are still questionable (Noral et al., 2003; Orlic et al., 2001b; Quaini et al., 2002; Smits et al., 2005). Alginate, a natural biomaterial, is a negatively charged linear co-polymer produced by brown seaweed, although certain bacteria also produce alginates. Leor et al. (2000) grew fetal rat cardiac cells within 3D porous sodium alginate scaffolds made by a freezedrying technique (Shapiro and Cohen, 1997). Biograft transplantation of the cardiac graft took place 7 days post MI. Visual and histological examinations revealed intensive neovascularization from the neighbouring coronary network and limited myofibres embedded in the collagen matrix. In conclusion, alginate scaffolds were shown to provide a supporting and conducive environment to encourage cardiac cell culture, as well as promising results for the regeneration and healing of the infarcted myocardium. Leor et al. (2005) then went on to seed hESCs on alginate scaffolds and found human ES cells to have no significant contribution to myocardial regeneration. However, because no experiments were carried out previously for alginate scaffolds without cell incorporation, it is yet to be determined whether it is the alginate alone or the combination of the two that encourages neovascularization. Dar et al. (2002) achieved 3D high-density cardiac constructs with a uniform cellular Copyright  2007 John Wiley & Sons, Ltd.

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distribution. This was achieved by applying a moderate centrifugal force during cell seeding, which in turn permitted the loading of a large number of cells onto the 3D alginate scaffolds. Robinson et al. (2005) have reported successful implantation of an engineered cardiac patch made from a fourlayer multilaminate urinary bladder-derived extracellular matrix in the infarcted left ventricular walls of pigs. Results from 1 week and 1 one month revealed thrombus formation and inflammation. However, 3 months post-implantation fibrocellular tissue was revealed with contractile cells and biodegradation of the matrix, when compared to the implantation of polytetrafluoroethylene (PTFE), where necrosis and calcification was observed. Kochupura et al. (2005) found that cardiomyocyte populations due to a myocardial patch derived from extracellular matrix provided mechanical benefit in the myocardium; however, this was not carried out for infarcted hearts. Combination of natural and synthetic materials. Collagen is the major constituent of the extracellular matrix, and it enhances cell attachment when used as a scaffold (Kofidis et al., 2002b) or matrix (Kutschka et al., 2006). However, the mechanical properties of collagen are poor, which is a disadvantage. Researchers have attempted to combine synthetic polymeric materials with collagen to engineer a suitable scaffold using the electrospinning technique, improving cellular adhesion although the mechanical properties are still not optimized. An example of this was reported by Stankus et al. (2004), with a combination of poly(ester urethane)urea with type I collagen. Cardiomyocytes from neonatal Lewis rats were cultured on electrospun, nanofibrous polycaprolactone (PCL) meshes coated with type I collagen to enhance cellular attachment, forming cardiac nanofibrous meshes (CNMs) (Shin et al., 2004). Formation of contractile cardiac grafts in vitro was achieved. However, this is still in its infancy. The CNM consisted of a wire ring that acted as a passive load for the beating cells, permitting contractions at natural frequency. Advantages of this electrospinning method include topography similar to that of the extracellular matrix of the heart, and the collagen coating permitting cellular adhesion and scaffold contraction in synchronization with beating cells. Researchers have also attempted to combine polymeric materials with collagen, using bioreactors to produce a potentially suitable engineered cardiac tissue. Biodegradable, hydrophobic PGA, poly(DL-lactide-co-caprolactone) and commercially available hydrophilic collagen sponge (Ultrafoam ) were mixed and made into a composite scaffold. Neonatal heart cells were seeded onto it, using bioreactor cartridges (Radisic et al., 2003). Improved cellularity as well as an increase in the presence of cardiac markers was observed. Krupnick et al. (2002) evaluated the transplantation of multipotent bone marrow-derived mesenchymal progenitor cells into infarcted syngenic rat hearts. Instead of directly injecting these cells into the infarcted region, J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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they were seeded within a collagenous matrix made from collagen type I and IV and Matrigel. The mixture of cells and collagenase was then seeded onto a nonwoven polymer mesh [poly-(L-lactic acid) reinforced with PTFE) and implanted in vivo. The results were promising, as minimal intracardiac inflammation occurred, the cells retained their fibroblastic shape and immunohistochemistry revealed activity at the junction of native myocardium and the engineered construct in three of the four rats. Some transplanted cells stained for myosin, indicating that cell differentiation had occurred. Cardiac function appeared to be normal, with no arrhythmias occurring. Natural materials used in in situ-engineered myocardial tissue. Although TE has been shown to have successful outcomes, the reality is that there is a need for further investigations until it can be clinically applied, due to the disadvantages already mentioned as well as the unanswered questions. In situ engineering has also been suggested for myocardial repair; it involves the injection of a mixture of biomaterials and cells. The biomaterials will in some way act as a supporting matrix for the cells, which will attempt to repair or regenerate the infarcted region, depending on the cell type used. Recently, acellular alginate biomaterial has been implanted in situ with bioactive molecules into the infarcted heart with the hope that the reported ‘resident cardiac progenitor cells’ will home to the damaged region (Leor, 2005). Fibrin glue has been reported to induce angiogenesis (Christman et al., 2004b; Thompson et al., 1992). Birla et al. (2005) engineered a contractile 3D cardiac tissue in an exciting new method, in which neonatal cardiac myocytes seeded onto fibrin glue were cultured in vivo in silicone chambers while being in close proximity to a vascular pedicle, therefore having an intrinsic vascular supply. Within 3 weeks, the chamber was full of living tissue. This exciting methodology is a different approach for engineering 3D cardiac tissue in vivo. Christman et al. (2004b, 2005) demonstrated the success of transplanting skeletal myoblasts in fibrin glue, a biopolymer formed by polymerization of fibrinogen monomers, via direct injection into the infarcted region, where infarct size decreased and fibrin glue increased arteriole density and cell survival in vivo. Injecting fibrin alone was reported to preserve cardiac function and reduce cardiac remodelling when left ventricular geometry was unchanged (Christman et al., 2004a). Ryu et al. (2005) reported similar results with implantation of bone marrow mononuclear cells with and without fibrin matrix. The combination of the bone marrow mononuclear cells and fibrin matrix produced approximately 350 microvessels/mm2 density in the infarcted region, compared to 262 and 76 microvessels/mm2 achieved without fibrin matrix and with medium injection only, respectively. Implantation of both cells and matrix also produced microvessels with larger diameters, also indicting enhanced neovascularization. Injection of endothelial Copyright  2007 John Wiley & Sons, Ltd.

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cells with fibrin glue also resulted in improved left ventricular function, myocardial blood flow and neovascularization (Chekanov et al., 2003). Collagen has also been suggested as an injectable biopolymer, albeit with many conflicting results. Huang et al. (2005), for example, reported the effect of three different biopolymers, fibrin, collagen and Matrigel, injected into the infarcted region separately with endothelial cells. The results demonstrated increased capillary formation for all three biopolymers, although collagen showed greater infiltration of myofibroblasts into the region than fibrin and Matrigel. Ischaemic myocardium injected with progenitor cells in a collagenbased matrix was reported to significantly restore vascular supply in the region (Suuronen et al., 2006). However, Dai et al. (2005) reported a decrease in infarct wall thickness without neovascularization in the collagen implant, suggesting that the improvement in left ventricular function, increased scar thickness and remodelling might have been due to the decreased wall thickness, and that introducing biomaterials is a vital factor in preserving cardiac function. On the other hand, it is yet to be determined why fibrin glue improves cardiac function, whether it may be due to neovascularization or to preserved left ventricular geometry. More research is required to determine whether collagen does induce neovascularization, as some report that it does (Huang et al., 2005) and others report no signs of it (Dai et al., 2005). Thompson et al. (1992) injected a mixture of autologous bone marrow cells and collagen hydrogel into the anterior interventricular coronary vein via the coronary sinus, using a composite catheter system (TransAcess), which consists of an ultrasound tip to give guidance, to allow the transvascular myocardial access via a sheathed extendable nitinol needle for the implantation of the collagen–cell suspension. Since injection was done only on uninjured hearts, it is difficult to determine whether this method of injection is feasible for injured hearts, since inflammation and remodelling may obstruct percutaneous intermyocardial access. However, this study does provide advancements for minimally invasive surgical approaches. Implantation of a bioartificial liquid made of Matrigel and murine endothelial ESCs into the ischaemic myocardium of mice by Kofidis et al. (2005b) improved cardiac function as well as attenuated left ventricular remodelling, and expression of gap junctional protein connexin43 at intercellular sites were observed, indicating possible connections with the native myocardium. Interestingly, Matrigel alone was effective, in that cardiac function deterioration was inhibited 2 weeks after injury and left ventricular thickness was retained throughout the experiment. Interestingly, Zhang et al. (2006) suggested injecting the EHT (Zimmerman’s group) before it solidifies, into the injured region. Rats that were injected with a similar EHT solution showed far better histological configuration and appeared to be more stable than rats that only received injectable artificial matrix and neonatal cardiomyocytes. This study is in its infancy and has obvious J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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limitations, such as the immunogenicity of the Matrigel and hindered transmural repair of the infarcted region. A different biomaterial was introduced by Davis et al. (2005); ‘self-assembling’ peptide nanofibres were injected into the myocardium, creating microenvironments to recruit progenitor cells and induce vessel growth. Immunohistochemical tests revealed that progenitor vascular cells and cells expressing endothelial markers were homed to the nanofibres. Separately, neonatal cardiomyocytes were injected with the peptides, and they were reported to not only survive but also to augment endogenous cell recruitment. However, implantation of Matrigel did not recruit endothelial cells until 28 days later, when some capillary formation was observed. Interestingly, researchers have attempted to deliver injectable biomaterials along with growth factors. Gene-activated matrix composed of Pleiotrophin plasmid in commercially available fibrin glue was reported to increase neovascularization (Christman et al., 2005). Other studies reporting the success of injecting biomaterials with peptides and growth factors are Iwakura et al. (2003) and Hsieh et al. (2006). This approach is relatively new in the regeneration for myocardial tissue and has only been explored by limited number of researchers.

4.3. Left ventricular restraint devices Unlike TE, the attempt to restrain the left ventricular remodelling and dilatation caused by infarction, using a polymeric material as a restraint, does not require the incorporation of cells. Briefly, this has been suggested as either a ‘wrap’ for both ventricles (Schroder et al., 2006; Walsh, 2006) or solely the left ventricle (Enomoto et al., 2005), or as a suture in the region of infarction (Kelley et al., 1999). Results showed this approach to be controversial, with some reporting improved cardiac function by preserving left ventricular geometry (Christman and Randall, 2006; Kelley et al., 1999) and others reporting marginal improvements in suturing materials onto the infarcted area in comparison with the improvements with sheep, who received a wrap around the ventricles (Moainie et al., 2002). A commercially available cardiac support device (CSD) from Acorn Cardiovascular Inc. (St. Paul, MN, USA), manufactured from a common very slowly degrading polymer, PTFE, has been used as a wrap around the cardiac ventricles to prevent deterioration of the left ventricle (Walsh, 2006; Sabbah, 2003). This has been clinically tried with a total of 300 patients on different occasions (Christman and Randall, 2006), all reported to show an improvement in left ventricular end-diastolic volume. However, due to the lack of long-term studies and knowledge about precisely how the CSD improves cardiac function, as well as the conflicting results on the improvement of cardiac function (Christman and Randall, 2006; Schroder et al., 2006), approval from the FDA has Copyright  2007 John Wiley & Sons, Ltd.

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been hindered. Table 1 summarizes the advantages and disadvantages of the topics covered in this review.

5. Discussion and concluding remarks The findings discussed in this review contribute to a potential breakthrough in the field of MTE, highlighting several important points of discussion. First, there is a need to identify which cell type is suitable for human use to help regenerate the infarcted region. The lack of a suitable human cell source remains the major setback in regenerating the human myocardium, either by cell injection or by developing cardiac tissue via TE. Until recently, adult cardiomyocytes have been thought to be terminally differentiated and unable to proliferate. Hence, research has focused on alternative cell sources, such as skeletal myoblasts and bone marrow stem cells, which have shown marginal improvements clinically. Although progenitor cells have been reported to be resident in the heart, limitations include the controversial results reported, with different research teams reporting varying subpopulations present in the heart. It requires further investigations to determine whether these progenitor cells are present in human biopsies in adequate numbers (Freed et al., 2006). Research should focus on cells with greater potential to regenerate the heart and with unlimited supply, e.g. ESCMs. Further research is required to enhance the potential for hESCs, to overcome the key issues, such as immunogenicity, teratoma formation, animal product involvement and, last but not least, ethical constraints. MTE is an expanding field with many biomaterials, both synthetic and natural, and their contributions are currently being investigated. This review of the specialized literature has demonstrated the relevance of biomaterials and their importance in future cardiac regeneration. Several issues still need to be addressed for the success of MTE. Firts, electrical coupling between the cells is required, to ensure that cells on the graft or patch beat in synchrony. Second, electrical coupling between the construct and native myocardium for simultaneous beating is still of concern. It has been reported that cell sheeting has overcome this problem (Eschenhagen et al., 2006; Furuta et al., 2006), where graft integration and no arrhythmias was reported. There is concern whether a dense ‘patch’ is useful. It could be argued that for the cells to survive and carry out their full potential functions, they must be embedded in a 3D scaffold containing pores. In this scenario, one must consider the required functions of the engineered construct. If the construct is to act solely as a ‘vehicle’ to transport the cells into the patient, and then degrade over a short period (e.g. within 3 months), it may be that a patch would be suitable (Ke et al., 2005). Alternatively, if the construct is to support the damaged area for a sustained period, then it is vital, amongst many other factors, that it provides a porous structure, to ensure cell survival for the time they are in contact J Tissue Eng Regen Med 2007; 1: 327–342. DOI: 10.1002/term

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with the scaffold. Optimal cell density and scaffold/patch composition still need to be addressed, as well as the timing of implantation. How long after infarction should implantation be carried out? For what duration should the cells be seeded onto the construct before implantation? Which cell or cells should be used? In conclusion, the ideal myocardial construct should mimic the morphological, physiological and functional properties of the native cardiac muscle it intends to replace and remain viable after implantation. MTE will hopefully lead to improvement in function of the diseased myocardium as it integrates with the heart, reducing the morbidity and mortality of patients with heart failure.

Acknowledgements The authors acknowledge financial support from the UK Biotechnology and Biological Sciences Research Council (BBSRC), Grant No. BB/D011027/1.

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