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Review
Engineering cell platforms for myocardial regeneration

Udi Sarig & Marcelle Machluf
1. 2. 3. 4. 5. Introduction Injectable platforms Patch-based platforms Future platform technologies Expert opinion
Technion--Israel Institute of Technology, The Laboratory of Cancer, Drug Delivery & Mammalian Cell Technology, Faculty of Biotechnology & Food Engineering, Haifa, Israel
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Hebrew University on 11/28/11 For personal use only.
Introduction: Various engineered cell-platforms have been reported in recent years for the possible treatment of myocardial infarction (MI) and end-stage heart failure. These engineered platforms rely on two key factors: cells and/or biomaterial scaffolds for the regeneration of the infarcted heart tissue. Areas covered: Two major cell-platform approaches are described and broadly categorized as injectable cell platforms and patch-based cell platforms. The recent advancements in these cell-platforms in terms of their relative successes in-vivo as well as their clinical feasibility are summarized. Natural as well as synthetic scaffolds, with or without the cellular component, are compared with cell based therapy alone. This review focuses on achievements, as well as the gaps that are presently checking any progress towards producing clinically relevant panacea for myocardial regeneration. Expert opinion: Cardiac and induced pluripotent stem cells will probably be the focus of future research. The combined cell-biomaterial scaffold therapy is superior to cell therapy alone. Nevertheless, encouraging pre-clinical successes have limited translation into clinical practice due to limited cell survival post transplantation, inadequate construct thicknesses for humansized hearts and the traditional use of flat (2D) tissue culture techniques. The development of complementary dynamic 3D cultivation platforms will probably lead to improved outcomes and enable fast screening of various therapeutic approaches.
Keywords: cardiomyocyte cell sources, cell therapy, extracellular matrix (ECM), injection- and patch-based cell platforms, myocardial infarction, myocardial tissue engineering, scaffolds Expert Opin. Biol. Ther. (2011) 11(8):1055-1077
1.
Introduction
In myocardial infarction (MI) the blood supply to the cardiac muscle (myocardium) is halted, leading to profound cell necrosis, downstream of the blocked artery. As cardiomyocytes (CM, the actively contracting heart cells) have a limited regeneration capacity, rapid inflammatory response takes over, leading to the formation of a fibrotic scar tissue that is unable to actively contract and imposes an extra burden on the remaining healthy myocardium. In the USA alone, approximately 1 million individuals suffer from acute MI annually, making it the leading cause of heart failure [1]. Current pharmacological therapies and surgical interventions can slow the progression to end-stage disease, but they rarely prevent or reverse the progression of the failing state [2]. At present, the only therapeutic options available to treat patients with terminal end-stage heart failure are heart transplantations or ventricular assist devices (VADs). These options have significant cost and availability limitations, leading to vast research in emerging fields such as cell therapy and tissue engineering [3]. Such research has resulted in the development of various engineered
10.1517/14712598.2011.578574 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted
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Article highlights.
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Cell platforms for myocardial regeneration can be broadly categorized as either injection- or patch-based platforms. Combined cell--biomaterial scaffold platforms show superior results compared with cell therapy alone. Even though many reports have shown significant improvement in pre-clinical studies, the translation into clinical practice is still limited. We review the latest achievements and drawbacks of these strategies and mark the technological hurdles blocking the achievement of clinically relevant solutions. Evolving technologies that may enable improved pre-clinical outcomes are discussed.
This box summarizes key points contained in the article.
cell-platforms that rely on two key factors: cells and/or biomaterial scaffolds for the regeneration of the infarcted heart tissue. The cellular component, the more complex part of the engineered cell-platform, has to contract, remodel and ultimately regenerate the defective myocardium. The ideal cell source should be easy to obtain and cultivate in great numbers as the native cardiac tissue is densely populated (~ 5 108 cells/cm3) [4]. Furthermore, the cell source and/or its differentiated products should be biocompatible, to avoid rejection post transplantation. Last but not least, cell safety is a matter of concern, particularly when referring to genetically modified cells. The heart muscle (i.e., myocardium) consists of at least three basic cell types (i.e., 20 -- 40% CMs; 60 -- 80% cardiac fibroblasts and endothelial cells) [4]. Thus any applied tissue engineering technique should be able to combine these three cell populations resulting in CM that are electromechanically coupled to the host tissue while having a proper vascular supply and connective tissue to ensure functionality [5]. However, as only CM contribute to functional contraction, and these cannot be expanded ex-vivo, several other CM cell sources have been suggested for the replacement and regeneration of the infarcted heart, including embryonic stem cells (ESCs) [6-12], induced pluripotent stem cells (iPSCs) [11-18], mesenchymal stem cells (MSCs) [19-27], and cardiac stem cells (CSCs) [28-32]. Each of these cell sources has its own pros and cons, as discussed in recent reviews [1,3,33]. Nonetheless, cellular therapy by itself is still limited and does not yet lead to sufficient cardiac tissue regeneration [3], demonstrating the need for other supporting, yet crucial factors, such as the biomaterial scaffold. Biomaterial scaffolds serve to mimic the natural extracellular matrix (ECM) and aim to provide the physical and biological support to the grafted cells during in-vitro/ ex-vivo cultivation, as well as to the host cardiac tissue post transplantation. Ideally, these scaffolds should degrade to biocompatible byproducts in parallel to the secretion of
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a substituting ECM by the host tissue and the grafted cells. Further incorporation of growth and pro-survival factors within these scaffolds may enhance tissue regeneration by affecting both host and transplanted cells. However, the application and effect of such growth factors will be discussed briefly, as it is outside the scope of this review. Despite the importance of these two key elements (i.e., cells and biomaterial scaffolds), they cannot solely account for the performance of engineered myocardial cell platforms without mechanical stimuli in the form of the cardiac rhythm and electrical cues, which mimic the natural cardiac environment. This area of dynamic cultivation technologies is an emerging one that plays a fundamental role at the level of cell -- cell and cell -- scaffold interactions. It is also thoroughly reviewed elsewhere [34,35]. In this article, recent cell platforms that have been engineered for myocardial regeneration are addressed (Figure 1). We discuss, in detail, their relative successes in-vivo and their clinical feasibility. Furthermore, the most promising emerging technologies, particularly those that might contribute to improved outcomes in-vivo are also described.
2.
Injectable platforms
Injection of cells alone, biomaterial alone or combinations thereof, is a clinically preferred delivery technique, as it is considered to be minimally invasive and a controlled procedure. The injection can be performed either directly into the infarct area or, when cell size permits, through the coronary circulation [36]. Injected platforms can take the form of any infarct with relatively little effort, delaying subsequent wall thinning. Furthermore, the ability of injected cells and active biomaterials to attract host stem and progenitor cells into the infarct area is compelling [37]. Studies have reported the injection of cells alone or in combination with polymers such as liquidized ECM, collagen, fibrin, alginate, chitosan, hyaluronic acid (HA) hydrogels, and synthetic derivatives of poly(N-isopropylacrylamide) (PNIPAAM) and PEG (Tables 1,2,3). These two forms of injections will be discussed separately in the following sections.
Injectable cell-therapy In general, the contribution of cells to cardiac regeneration is believed to be mitigated through three independent mechanisms of action: angiogenesis, myogenesis and paracrine effects [3]. The intracoronary injection of cells has shown limited success in terms of their delivery efficiency into the myocardium and in terms of transplanted cells homing and survival, when compared with direct intramyocardial injections [36]. This review focuses on the use of the most reported and clinically relevant cell sources for CM differentiation: ESCs [10,38-51], iPSC [11-18], MSCs [52-65] and CSC [28-32]. Nevertheless, as many studies have reported the utilization of other cell types (such as skeletal myoblasts, bone marrow mononuclear cells and endothelial progenitor cells), we would
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Expert Opin. Biol. Ther. (2011) 11(8)
Sarig & Machluf
A.
D.
B.
E.
C.
F.
Injectable platforms
Patch-based platforms
Figure 1. Schematic representation of the major strategies for myocardial tissue engineering. These strategies rely on cardiogenic cell sources, which are administered either alone or in combination with various natural or synthetic scaffolds. Injectable platforms (A. -- C.) could be delivered through the vena cava (intravenous, A.), through the coronary arteries (intracoronary, B.) or into the infarcted zone or its border (intramyocardial, C.). Patch based platforms (D. -- F.) include scaffold-free or cell-sheet transplantation (D.), tissue engineered constructs made out of various cell--biomaterial scaffold combinations (E.) or patches made out of biomaterial scaffolds alone (F.).
like to direct the reader to recent in depth reviews discussing the cellular aspects of myocardial regeneration [3,66-68].
Embryonic stem cells ESCs have the ability to self renew and also to differentiate in a robust biochemical procedure into beating and contracting CM-like cells, having similar (although not identical) characteristics to that of the adult CM [6,7,11,41,69]. Injected ESC-derived CM have been shown to integrate with host myocardium and improve the electrical conduction in infarcted mouse and rat hearts [38,41,44,50]. Similar results were obtained using human-ESC-derived CM in a porcine model [39] and early cardiac progenitors (stage-specific embyonic antigen (SSEA)+) of primate ESCs transplanted in an infarcted primate model [12]. Nonetheless these cells are considered immunogenic [67] and are known to form teratomas post transplantation in-vivo [9,42,43,70]. Immunogenic rejection could be overcome with the use of autologous induced pluripotent cells (iPSCs, further discussed hereafter) and the occurrence of teratoma formation is reduced by the selection of phenotypically differentiated CM produced from ESCs [9,42,51]. Further major difficulties include collecting unadulterated hESC-derived CM (hESC-CM) in large quantities and assuring their long term survival. These represent major obstacles for cell-based therapy [10,44].
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To increase hESC-CM yield, Laflamme et al., have utilized activin- A and bone morphogenetic protein 4 (BMP4). They further incorporated a pro survival factors cocktail (PSC) to limit hESC-CM death post transplantation into a rat acute MI model [41]. The engrafted human myocardium attenuated ventricular dilation and preserved regional and global contractile function compared with controls receiving non-cardiac hESC derivatives or vehicle. However, the significance of these results has been challenged by van Laake et al. who evaluated the long-term (12 weeks) fate of hESC-CM transplanted in immunocompromised acute MI mice model. Their results exhibited rapid formation of grafts with organized CM that having matured over time, were further accompanied with significant improvement in cardiac function at 4 weeks post transplantation, yet this was not sustained at 12 weeks [44]. From these results, it was argued that there is a requirement for a minimum 3 month follow-up in studies that claim to observe improved cardiac function. We further infer that this requirement is independent of whether hESC or other (adult) cell types are used for transplantation. In a more recent study by the former group, Intramyocardial injection of hESC-CM with the same PSC into chronic rat MI models was evaluated. Histology performed at the 3 month time point revealed that human CM survived, developed increased sarcomere organization and were still proliferating. Despite successful engraftment, both echocardiography and MRI analyses showed no significant difference in left
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Disadvantages Pre-clinical studies Murine [38,40-46,49,51, 136]; Porcine [39] Clinical trials None reported Requires chronic immunosuppression Capable of teratoma formation (particularly if not fully differentiated) Relatively low yield of CM Ethical issues Requires genetic manipulation Relatively low yield of CM Murine [16]; Primate [12] None reported Murine [21,56,59,60,133,
139,157,158,166,167,175,178, 190-191]; Porcine [61-63]
Table 1. Cardiomyocyte cell sources.
Cell type
Cell source
Advantages
ESCs
Embryo
Biochemical differentiation towards CM like beating cells is well characterized Give rise to other regenerative cell types (i.e., endothelial cells and fibroblasts) Integrates with host tissue and improve conductivity and function
iPSCs
Dermal fibroblasts
Autologous cell source (no immunosuppression required) Known biochemical differentiation towards CM-like beating cells Integrate with host tissue and improve conductivity and function No long-term effect/survivability (> 6 months) Little integration with host tissue Positive effects are based on paracrine effects and angiogenesis induction rather than CM differentiation No robust and safe CM differentiation procedure reported to date No robust CM differentiation procedure in large-animal models reported to date
MSCs
Bone marrow Umbilical vein Adipose tissue Dental pulp
Autologous cell source (no immunosuppression required) Relatively easy to isolate and culture in large quantities Improve cardiac function Give rise to other regenerative cell types (i.e., endothelial cells and vascular muscle)
Phase I [72]
CSCs
Heart
Autologous cell source (no immunosuppression required) Relatively easy to isolate and culture in large quantities Improve cardiac function Secrete a cocktail of growth and prosurvival factors that probably best fit myocardial regeneration Give rise to other regenerative cell types (i.e., epithelial cells and vascular muscle)
Murine [29,31-32]
Phase I [80]
CM: Cardiomyocytes; CSCs: Cardiac stem cells; ESCs: Embryonic stem cells; iPSCs: Induced pluripotent stem cells; MSCs: Mesenchymal stem cells.
Table 2. Natural biomaterials for myocardial cell platforms.

Disadvantages Murine [41,83,84,86,87,150,157,158, 168,175]; Canine [146,148]; Porcine
[147,152]
Polymer High batch to batch variability More problematic in terms of mass production Inconvenient isolation procedures Adverse effects on angiogenesis and vasculature formation Murine [40,89-94,131,165-168,170,175, 176,178,180]; Porcine [95]
Advantages
Pre-clinical studies
Clinical trials None reported
ECM
Closest mimic to natural cell surroundings Bioactive, biodegradable and biocompatible Improves myocardial geometry and function Promotes vasculature formation Inducible gelling mechanism in situ
Collagen/ Gelatin
The major building block of natural ECM Bioactive, biodegradable and biocompatible Improves myocardial geometry and function Enhanced cell retention/survival upon transplantation Commercially available Inducible gelling mechanism in situ No significant long term effects observed in human patients
Phase I [179]
Fibrin
Biological glue and cell adhesive with controlled polymerization and degradation kinetics Bioactive, biodegradable and biocompatible Leads to improved myocardial geometry and function upon injection Low mechanical properties match with the myocardium No significant improvement in vasculature formation upon injection Adverse bioactivity Mechanism of vasculature support are poorly understood Absence of data on chitosan bioactivity and contribution in large-animal models
Murine [99,100]; Chick [101]; Sheep [98]
Phase II [102]
Alginate
Biodegradable and biocompatible Inducible gelling mechanism in situ Commercially available at relatively low cost FDA approved Improves myocardial geometry and function upon injection
Murine [104-106,172-174]; Porcine [107]
On-going Phase II [109]
Chitosan
Biodegradable and biocompatible Commercially available at relatively low cost Variable formulations allow for controlled and versatile end-product mechanical properties Inducible gelling mechanisms in situ Improves myocardial geometry and function upon injection Promotes vasculature formation
Murine [46,113]
None reported
HA
Improves myocardial geometry and function upon injection Bioactive, biodegradable and non-immunogenic Commercially available
Absent data on bioactivity in large animal models Low mechanical properties match with the myocardium Relatively expensive
Murine [116]
None reported
Sarig & Machluf
ECM: Extracellular matrix; HA: Hyaluronic acid.
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Disadvantages Lacks biodegradability (derivative-dependent) Poor physiological clearance (derivative-dependent) Lack of data in large animal models
[121,125]
Table 3. Synthetic biomaterials for myocardial cell platforms.

Pre-clinical studies Murine [126]; Rabbit Clinical trials None reported
Polymer
Advantages
PNIPAAM (and derivatives)
Inducible gelling mechanism in situ Promotes vasculature formation Cell adherence is temperature-dependent Mechanical properties could match those of the native myocardium Controlled and robust manufacturing processes Commercially available at relatively low cost Lacks biodegradability (derivative-dependent) Poor physiologic clearance (derivative-dependent) Lack of data on PEG and derivatives contribution in large animal models No significant improvement in vasculature formation upon injection Might cause local high acid concentrations leading to an inflammatory response Sudden loss of functional integrity due to bulk degradation kinetics Used primarily for patch-based platforms Low mechanical properties match with the myocardium Lack of data on polyesters contribution in large-animal models Local inflammation caused by degradation byproducts (derivative-dependent) Used primarily for patch-based platforms Lack of data on elastomers contribution in large animal models
PEG (and derivatives)
Improves myocardial geometry and function upon injection (derivative dependent) Bio-inert
Murine [128,129]; Rabbit [130]
None reported
Polyesters (e.g., poly-aHydroxy-acid; PLA; PGA; PCL; PLGA; PCLA; PLCL)
Biodegradable and biocompatible Co-polymers could allow for a more controlled degradation kinetics Controlled and robust manufacturing processes Commercially available Improves myocardial geometry and function
Murine [45,138, 139,175,176,178]
None reported
Elastomers (PU; poly (TMC) and co-polymers with PLA/ PCL; PGS
Biodegradable Biocompatible (derivative-dependent) Mechanical properties could be made to match those of the myocardium Short-term improvement in myocardial geometry and function
Murine [49,141, 142,175]
None reported
NIPAAM: Poly(N-isopropylacrylamide); PCL: Poly(e-caprolactone); PCLA: Caprolactone-co-L-lactide sponge reinforced with knitted poly-L-lactide fabric; PGA: Poly(glycolic acid); PGS: Poly(glycerol-sebacate); PLA: Poly(lactic acid); PLCL: Poly(lactide-co-e-caprolactone); PLGA: Poly (lactic co-glycolic acid); poly(TMC): Poly(1,3 trimethylene carbonate); PU: Polyurethane.
Sarig & Machluf
ventricular structure or function compared with controls. This suggested that cell transplantation should be conducted in the acute or sub-acute phases of MI and is probably not effective once the fibrotic scar tissue is already formed at the chronic phase of MI [71]. This finding contradicts previous results reported by Caspi et al., who injected hESC-CM into a similar chronic infarction rat model, revealing attenuation of left ventricular dilatation and improved cardiac function one month post tranplantation [9]. Although more preclinical studies are needed before such therapies can be implemented clinically, a recent FDA approval for ESC-based clinical trials in human patients for the treatment of spinal injuries [72] is a positive sign that ESC technology may one day be applied to clinical cardiac regeneration.
Induced pluripotent stem cells In the last few years efforts have been invested in reprogramming autologous dermal fibroblasts, both murine and human, into a pluripotent state to avoid immunogenic rejection. The phenotype of isolated iPSCs is very similar to that of ESCs and thus CM isolation from an autologous source is possible, as demonstrated by several groups [13,16-18]. In addition, the ability to create iPSCs, without the use of genome-integrated viral vectors and oncogenes, has been demonstrated recently and supports the notion of future clinical use [14,15,73]. Nelson et al., have recently reported the intramyocardial delivery of iPSCs into immune-competent adult MI mice model, yielding progeny that properly engrafted without disrupting cytoarchitecture. In contrast to parental non-reparative fibroblasts, iPSCs treatment restored post-ischemic contractile performance, ventricular wall thickness and electrical stability while achieving in situ regeneration of cardiac, smooth muscle and endothelial tissue [16]. However, as the isolation technique for iPSC-derived CM, still relies on the micromanipulation of beating areas within the embryonic body [10], the ability to produce the needed amount of CM-like cells still limits its application for clinical cell-based therapy. Furthermore, the limited understanding of the process of iPSCs senescence, may hinder their potential use in the elderly [74].
2.1.2
intramyocardial [64,65] injection of MSCs alone, yielded improved results compared with controls in mice and rat animal models. However, MSCs did not exhibit long term (6 months) survivability [55] and their positive influence is largely due to paracrine effects [24] rather than differentiation into active CM cell populations. Schuleri et al. reported the autologous intramyocardial injection of MSCs in a swine chronic MI model. High-dose (200 million cells) MSC therapy reduced infarct size and increased regional contractility in both infarct and border zones [63]. They also reported that engrafted MSCs can differentiate into three lineages: CM, endothelial and vascular muscle [62]. Poncelet et al., have shown that allogeneic porcine mesenchymal stem cell transplantation enhanced angiogenesis after myocardial infarction, yet this effect was limited to the viable myocardium in the borders of the infarct area [61]. These encouraging results have led to a recent clinical trial with allogeneic MSCs administered intravenously to MI patients. In addition to significant improvement in cardiac function, no immunosuppression was required post transplantation and no safety issues were detected, hence demonstrating the feasibility of using allogeneic MSC cell sources [54]. Unfortunately, the lack of a reproducible differentiation procedure holds back the degree of regeneration that could be expected using this cell source as non differentiated MSC do not actively contract.
Cardiac stem cells The traditional view that myocardium lacks regenerative capacity, has been questioned in the past decade with the discovery of cardiac progenitor cells [28,76-78] Several groups have since reported the successful isolation of CSC from murine [31,78], rat [28], porcine [32] and human hearts [30-32]. Oh et al., performed magnetic enrichment of stem cell antigen positive cells (Sca1+) from mice hearts and differentiated them in-vitro upon exposure to 5-azcytidine [78]. Messina et al., developed a robust technique for the isolation of CSC from cardiac explants of murine and human origin. Using a cardiosphere intermediate step, they isolated cells that are capable of self-renewal and give rise to vascular and contractile cells. While murine cells exhibited spontaneous differentiation upon formation of cardiospheres, human cells exhibited CM differentiation only upon exposure to rat neonatal CM [31]. Transplantation into infarcted murine hearts led to functional improvement which was also reported in a subsequent collaborative study with the same group using human and porcine CSC [31,32]. In a more recent study, Davis and co-workers further improved the isolation procedure by skipping the cardiosphere step altogether, while exhibiting similar results when transplanting the directly explanted crude population into a rat MI model [29]. Despite the fact that the efficiency of the cardiosphere methodology has yet to be reproduced sufficiently, with some reports challenging the stemness of these cardiospheres [79], these cells are regarded as holding great promise for future cardiac regeneration and are currently under evaluation in a clinical trial [80].
2.1.4
Mesenchymal stem cells MSCs are multipotent stem cells isolated from several adult tissues including bone marrow, adipose, umbilical vein and dental pulp [53,75]. Their ability to differentiate into CM like cells was first demonstrated a decade ago by the exposure of murine bone marrow stromal cells to 5-azacytidine [19,20]. Even though these results have not yet been reproduced with human MSCs [57,58] and are probably dependent on the MSC tissue of origin and on yet unknown signaling factors within the myocardial milieu [75], they serve as a proof of concept for the applicability of MSC in cardiac regeneration. In addition, MSCs were demonstrated to express cardiac-specific markers [23], which increase when exposed to neonatal CM-conditioned media [26,27,52]. Intracoronary [59,64], intravenous [56,60,64] and
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Biomaterial-injection-based platforms To improve cell attachment, survivability and proliferation, studies have also focused on the incorporation of liquidized biomaterials. These biomaterials can be injected to the myocardium either alone, with various homing and growth factors or in combination with various cell sources with the aim of supporting the infarcted scar tissue or recruiting endogenous stem and progenitor cells. These attractive biomaterials can be rapidly polymerized in-situ by various chemical, thermal or optical stimuli [81], thus assuring site-specific delivery in a minimally invasive technique. Different injectable biomaterials have been utilized for myocardial tissue engineering, which include both synthetic and natural biomaterials. Natural materials feature high bioactivity, biocompatibility and biodegradability. However, there is a high variability associated with their production and no exact control of their composition and physical characteristics, hence standardization and quality control in mass production becomes overly complex. In contrast, synthetic materials have a more robust manufacturing capability, allowing for a controlled biochemical composition and characteristics. Unfortunately, their bioactivity and biocompatibility qualities are less than satisfactory in comparison with the natural scaffolds.
2.2
infarcted scar, leading to a significantly increased infiltration of endothelial and smooth muscle cells 11 days post injection along with an increase in the number of arterioles. The group also reported similar results when using pericardial matrix [66]. Zhao et al. injected an emulsion made out of acellular subintestinal ECM directly into the rat myocardium following an ischemia--reperfusion event [87]. They reported an improvement in functional cardiac parameters (i.e., ejection fraction, fractional shortening and stroke volume) compared with controls given injections of saline solution. Taken together, these results raise a question as to the difference between various ECM origins, as to that which one has the optimal characteristics for promoting myocardial regeneration. As no reported data so far has compared these different materials, it is difficult to assess which would be most beneficial for long-term effect. Future experimentation may focus on elucidating this question as well as on the incorporation of various CM cell sources together with the injected acellular ECM.
Collagen As the most abundant structural protein in our bodys ECM, collagen has been widely researched for its suitability for injectable myocardial tissue engineering. So far 28 distinct types of collagen have been described [88], of which, types I, III and V are the most abundant in cardiac tissue. Most studies reported improved LV geometry and cardiac function when dissolved collagen was injected. However, a question arises as to its contribution to angiogenesis with some reporting no involvement [89] whereas others showing improved capillary density and myofibroblasts infiltration [90]. Researchers also attempted to incorporate cells into injectable collagen or its gelatinous derivatives. According to these studies this approach resulted in improved cell retention, survival and angiogenesis accompanied by a better cardiac function compared with either cell-only or collagen-only controls. Suuronen et al., have shown a significant restoration of the vascular supply to ischemic tissues upon injection of a collagen-based matrix with progenitor cells [91]. Kutschka et al., further indicated, through bioluminescence, that injection of cardiomyoblasts into pre-transplanted collagen based GelfoamTM (Pfizer) [92] or together with liquidized Gelfoam [93] enhances their survival in rat hearts compared with cell-only injections and non treated controls. Moreover the incorporation of reduced Matrigel (with no growth factors) together with the pre-transplanted Gelfoam matrix, showed an even better improvement than that of cardiomyoblast injected Gelfoam alone. This may be attributed to other ECM components within the Matrigel that contributed to cell adhesion and survival [92,93]. In another study Zhang et al., used positron-emission tomography in addition to histology to assess the bio-distribution of collagen-seeded progenitor cells. Their results showed enhanced cell retention and survival compared with cells-only-seeded controls [94]. Takehara et al., used gelatin, a collagen derivative, to deliver basic fibroblast growth factor (bFGF). This growth factor, with or without gelatin, was injected into a porcine
2.2.2
Extracellular matrix The cardiac ECM consists of a variety of structural proteins (collagen, elastin), adhesive proteins (fibronectin, laminin) and other active components (MMP, tissue inhibitors of MMP (TIMP) and plasmin) within a hydrated proteoglycan and glycosaminoglycan-rich milieu [82]. To mimic this complex environment, three distinct strategies have been employed: I. Using commercially manufactured ECM (MatrigelTM, BD Biosciences), II. Using acellular ECM and III. Using defined single ECM components or mimetics. Matrigel is a secreted ECM mixture that is commercially manufactured, exploiting mouse tumor cell ECM production. It has been proven effective in the delivery of mouse ESCs to the left ventricle (LV) either by injection into a pouch made in the left ventricle of rats [83] or by using a direct injection into the infarcted myocardium of a mouse model [84]. Both these studies have resulted in improved heart geometry and function with no apparent side effects as compared with either cells-only or Matrigel-only controls. Similar results were confirmed by Zhang et al., who mixed Matrigel, collagen and cell culture medium for the delivery of neonatal CM into a rat MI model [85]. In another study, Laflamme et al., formulated a cocktail of pro-survival factors preventing ESC-derived CM death. This formula contained Matrigel, which was shown to independently prevent anoikis and increase vasculature formation after injection into the infarcted myocardium [41]. In an attempt to regenerate the infarcted heart using acellular matrices, Singelyn et al., liquidized acellular cardiac ECM using pepsin and subsequently injected it into the myocardium via catheter in a rat MI model [86]. Gelation occurred within the
2.2.1
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MI model alone or in combination with either human cardiosphere-derived cells (CDCs) or MSCs. Their results suggested increased microvasculature formation in the gelatin--bFGF group compared with bFGF directly injected with no gelatin. They also reported a synergistic effect of bFGF-gelatin when combined with human CDCs (but not with MSCs), which included improved cardiac function, LV wall motion, CM differentiation and infarct scar size [95].
Fibrin Fibrin, the active truncated form of fibrinogen (cleaved by thrombin), is an important physiological protein that has been widely used in tissue engineering [96,97]. The attractive features of fibrin are its ability to serve as a biological glue, holding cells together in the desired site as well as its ability to stimulate angiogenesis [96]. Due to these properties, fibrin has been used both as a delivery vehicle as well as a tissue engineering bioactive scaffold [97]. Several groups have shown improvement in geometry and function of the LV when it was subjected to fibrin alone or in combinations with various cells (i.e., endothelial cells, skeletal myoblasts and bone marrow mononuclear cells) compared with controls that received injections of saline [98-100]. In addition, under physiological conditions, fibrin undergoes degradation in parallel to the formation of a healthy replacement tissue. This process can be controlled using inhibitory drugs such as aprotinin [97,101], therefore enabling the adjustment of the degradation kinetics with that of substitute ECM production by the host and seeded cells. Fibrin polymerization kinetics in situ has been characterized by Martens et al., proving its suitability for catheter-based delivery. By maintaining fibrinogen and thrombin concentrations below 5% w/v and under 20 U/ml respectively, polymerization rates can be controlled to allow adequate time for delivery [81]. Despite these encouraging pre-clinical results in animal models, and the vast use of fibrin glues in the clinical setting, little is known on the effects of fibrin on MI in human patients. In a clinical trial aimed to reduce reperfusion injury post MI, Atar et al., have shown that the intravenous bolus injection of a fibrin derivative (FX06) led to a significant short term effect on the size of the necrotic center within the infarct zone compared with controls receiving placebo injections [102]. Yet, no significant long-term effect was observed apart from that there were fewer patients with serious cardiac events in the treated groups compared with the placebo group patients.
2.2.3
of alginate injections as it had similar and in some cases better effects on the infarct size and function, compared with saline [105], neonatal CM [104] or fibrin injections [106] in rodents. Alginate has also been reported to cause reverse remodeling in swine [107]. Its positive effect on the remodeling and function of the left ventricle is probably related to its chemical and physical traits [105]. Ruvinov et al., have developed an injectable affinity-binding alginate enabling the sequential delivery of IGF-1 and hepatocyte growth factor (HGF). The affinity-binding alginate protected the proteins from proteolysis while maintaining their bioactivity as demonstrated by mass spectroscopy and several in-vitro assays respectively. Intramyocardial delivery to a rat model of acute MI, preserved scar thickness, attenuated infarct expansion and reduced scar fibrosis after 4 weeks, concomitantly with increased angiogenesis and mature blood vessel formation at the infarct. Apparently this treatment prevented cell apoptosis, induced cardiomyocyte cell cycle re-entry and increased the incidence of GATA-4+ cell clusters implying that this biomaterial has potential to induce endogenous regeneration of cardiac muscle [108]. These impressive results in animal models, are currently progressing towards a clinical trial [109].
Chitosan Chitosan is another naturally occurring polysaccharide, which is widely studied as a biocompatible scaffold for skin, bone, cartilage, liver and blood vessel tissue engineering [110]. Chitosan allows the formation of relatively hard porous structures, nanofibrous meshes or compliant hydrogels -- depending on the choice of chitosan derivative [67]. Chitosan hydrogel is formed either by temperature changes (chitosan -- glycerol phosphate [67]) or optical stimuli (acrylate conjugated [111]; or photoinitiator based hdrogels [112]), hence it is capable of polymerizing in-situ while entrapping genes, growth factors and, most attractively for tissue engineering, cells. Temperature responsive chitosan-glycerol phosphate (GP) hydrogels have been studied for the delivery of ESC and nuclear transferred ESCs to an infarcted rat myocardium, showing improvement in heart function and wall thickness compared with the injection of chitosan-GP or cells only [46,113]. Interestingly, both studies showed improvement in the microvessel density of the infarcted scar in the chitosan-GP only injected hearts. Though the exact mechanism by which this angiogenesis process occurs is unknown, it is probably related to the ability of chitosan to modulate the proliferation of vascular cells in-vitro as well as in-vivo [114].
2.2.5
Alginate The natural polysaccharide alginate forms compliant hydrogels upon exposure to divalent ions such as Ca2+ [103]. This induced gelling mechanism, the low cost of high-quantity production and most importantly the FDA approval for human use as a wound dressing material, makes alginate an ideal cell delivery platform for myocardial tissue engineering. Several recent studies [104-106], have demonstrated the effectiveness
2.2.4
Other biomaterial-injection based platforms Davis et al., have developed a self-assembling peptide-based scaffold, which was injected into healthy murine hearts either alone or in combination with neonatal CM. In both cases, recruitment of endogenous smooth muscle cells and endothelial cells was reported, particularly when CM were added to the injectable scaffold [115]. Recently, Yoon et al., reported the epicardial injection of a hyaluronic acid (HA)-based
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hydrogel for the treatment of MI in rats. They reported a significant improvement in wall thickness and cardiac functionality (assessed by histology and hemodynamic measurements respectively) of the HA-gel-treated group compared with the myocardial infarction non-treated control group [116]. Injectable platforms based on synthetic biomaterial used for the treatment of MI, include mostly variations of poly (N-isopropylacrylamide) (PNIPAAM) and PEG. PNIPAAM is a synthetic hydrogel that is in a liquid state at room temperature yet solidifies to a compliant gel form at 37oC. However, PNIPAAM is non-biodegradable and is not cleared from the body at physiological temperature [117]. Thus, several modifications were explored to enhance its biodegradability by incorporating either natural biodegradable materials (i.e., hyaluronic acid [118], collagen [119], gelatin [120], dextran [117,121] and peptide derivatives [122]) or by copolymerization with synthetic monomers having hydrolytic polyester side chains [123,124]. Upon cleavage of these side chains the polymer hydrophobicity was increased, leading to an increase in the lower critical solution temperature of the polymer above body temperature enabling polymer dissolving and physiological clearance. Recently, several variations of PNIPAAM have been utilized for the treatment of MI in rabbit [121,125] and rat models [126] showing overall improvement in cardiac function, neovascularization and ventricular remodeling. Thus the addition of a biodegradable dextran grafted to poly (e-caprolactone)-2-hydroxylethylmethacrylate (Dex-PCL-HEMA/NIPAAM) yielded a reduction in scar size and wall thinning accompanied by improvement in the left ventricular ejection fraction and overall cardiac function [121]. Li et al., also incorporated bone marrow mononuclear cells (BMMC) with this composite [125] leading to an increase in neovascular formation. Similar modification to PNIPAAM was employed by Fujimoto et al., who evaluated the copolymerization of NIPAAM, acrylic acid (AAc) and hydroxyethyl methacrylate-poly(trimethylene carbonate) (HEMAPTMC). They assessed a range of monomer ratios, poly(NIPAAm-coAAc-co-HEMAPTMC), showing that at a feed ratio of 86:4:10 the polymer formed a hydrogel at 37 C and in-vitro it gradually became soluble over more than 5 months, through hydrolytic cleavage of the PTMC residues. Injecting the hydrogel into rats with chronic infarction has led to the preservation of the LV cavity area and contractility, compared to the controls. Furthermore, tissue ingrowth was observed in the hydrogelinjected area, together with a thicker LV wall and higher capillary density. These effects were found in the hydrogel-treated group and were absent in the PBS-treated controls [126]. Another variation of PNIPAAM in combination with other natural and synthetic polymers was recently reported by Wang et al. [127]. Hydrogel composites were fabricated from Type I collagen, chondroitin sulfate (CS) and a copolymer based on NIPAAM, AAc, N-acryloxysuccinimide and the macromer poly(trimethylene carbonate)-hydroxyethyl methacrylate. These composites were capable of being injected through a 26-gauge needle and gelled within 6 sec at 37 C forming highly flexible gels with moduli matching those of the rat and human myocardium.
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Finally, PEG, well known as a commonly used bio-inert material has also been studied as a non-degradable alternative for MI injection [66]. Nevertheless, injection of PEG-vinyl sulfone in a rat MI model, showed no difference in the longterm functional benefit as compared with saline injections [128]. As it is believed that PEGs non-degradability prevents a long-term sustainable effect [66], other PEG derivatives that enable proteolytic degradation have been suggested. These include, PEGylated fibrinogen [47] and PEGylated fibrin biomatrix [129] and a hydrogel made out of a-cyclodextrin (a-CD) combined with PEG-b-polycaprolactone-(dodecanedioic acid)-polycaprolactone-PEG (MPEG-PCL-MPEG) triblock polymer [130]. Both these biomaterials have shown improved heart geometry and function upon injection into mouse [129] or rabbit [130] MI models. The effect of these materials in larger animal models has yet to be reported.
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Patch-based platforms
Patch-based platforms, are extensively studied for cardiac regeneration. They are composed of biomaterial either with or without cells, and can be transplanted on the epicardial surface of the infarct. They serve as a bag pack that contains biological therapeutics and growth factors needed for the support of the transplanted cells and host tissue, until angiogenesis occurs and the natural ECM is remodeled by the cells, eventually replacing the artificial patch platform. It is suggested that with time, the recruitment of endogenous stem and progenitor cells may enable a mechanical and electrical integration of such a patch with the host myocardium hopefully enabling the regeneration of the infarcted scar tissue. Patch deposition requires a more invasive surgical intervention in contrast to the relative ease of employment of the injection-based platforms. Nevertheless, a recent side by side comparison between intra-myocardial injections of skeletal myoblasts, and a patch of skeletal myoblasts in collagen foams showed that the latter is much more effective in terms of improved cardiac functionality as determined by echocardiography and histological assessment one month post transplantation in rats [131]. Two major patch-based platform groups have been discussed in literature; patch-based platforms that are composed of solely cells forming cell sheets and patch-based platforms composed of synthetic or natural polymers with or without the combination of cells.
Cells only-patch based platforms Shimizu et al., were the first to report, in 2002, the construction of a cell patch stacked out of four CM monolayers [132]. They utilized poly-NIPAAM-coated dishes for the cultivation of neonatal rat cardiomyocytes which were later detached using temperature reduction (below 32 C). The engineered constructs were macroscopically observed to pulse spontaneously, in-vitro, and morphologically communicate via connexin43. In-vivo, layered cardiomyocyte sheets were transplanted into
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subcutaneous tissues of nude rats, leading to the formation of neovasculature. Miyahara et al. transplanted stacked monolayers of adipose tissue-derived mesenchymal stem cells onto hearts of myocardial infarcted rats [133]. One of their most impressive findings was that the monolayers expended, in situ, producing a 600-m-thick tissue over the infarct scar. This finding contradicted the previously held view that myocardial patches could not grow beyond ~ 100 m due to diffusion limitation. Nevertheless, given the thickness of human left ventricles (circa 10 -- 15 mm), this diffusion limitation still needs to be surpassed to achieve human cardiac restoration [134]. In a more recent work published by the same group, the promotion of neovascularization by increasing densities of co-cultured endothelial cells within the same cell-sheet-based platform was studied. In-vivo studies, performed in a MI rat model, revealed that the cardiac function was significantly improved in correlation with the increase in endothelial cell densities of the original co-cultured constructs. They also reported a significant increase in the number of capillaries in the grafts which connected with the host tissue vasculature and functionally supplied the grafted construct [135]. A different novel and scalable method, which is a scaffold free-based platform but in a shape of a patch was lately developed by Stevens et al. [48,136]. A rotating orbital shaker was used to form macroscopic disc-shaped 300 -- 600 m thick beating ESCderived CM patches having diameters directly proportional to input cell number [48]. CMs were concentrated around the patch edges and exhibited increased purity and maturation with time (determined by the abundance of b-MHC transcript using quantitative reverstranscription (RT)-PCR and immunohistological staining). Patches also exhibited automaticity and synchronous calcium transients, indications of electromechanical coupling. A recent study published by this group, demonstrated that these patches, composed only of enriched CM, did not survive to form significant grafts after implantation in-vivo in rats [136]. Thus, second-generation prevascularized, and entirely human patches from CM, endothelial cells and fibroblasts were created. Implantation of these patches resulted in 10-fold larger cell grafts compared with patches composed of CM only. Despite the great interest these new technologies have generated, they still need to be tested in large-animal models.
Synthetic patch-based platforms Two classes of synthetic materials have been proposed in the last decade for the generation of patch based platforms: polyesters and elastomers. Each of these materials has unique characteristics in terms of structure and composition. They can give rise to a family of derivatives with distinct properties thorough structural and biochemical modifications, such as different toxicity profiles of their degradation byproducts and various mechanical elasticities and strengths.
3.2
Although this term is applicable to many types of materials, we will focus on the ones that are the most commonly used for myocardial tissue engineering such as polylactic acid (PLA), polyglycolic acid (PGA) (or their copolymer (PLGA)), poly(a-Hydroxy acid) and poly-e-caprolactone (PCL) [134,137]. These polymers are of great interest because their degradation products are mostly biocompatible (lactic, glycolic and hydroxycarboxylic acids respectively). However, these polymers are not without flaws. Their degradation products may cause a local high acidic concentration leading to an inflammatory response, which will impair the targeted tissue [2]. Furthermore, due to their bulk degradation kinetics, a sudden loss of structural integrity is expected and might be less desirable. Nonetheless, seeing as their mechanical properties, morphology and degradation kinetics are relatively easy to control, they are simple to manufacture and so became a most useful tool for patch-based myocardial tissue engineering research. Combinations of PLA and PCL (PCLA) were first produced by Ozawa et al., who employed PCLA patches, seeded with syngeneic rat aortic smooth muscle cells to replace a surgically created defect in the right ventricular outflow tract (RVOT) of rats. Compared with gelatin- and PGA-seeded patches, PCLA patches permitted better cellular penetration in-vitro, did not thin or dilate in vivo and did not produce an inflammatory response [138]. Jin et al., have investigated the effect of transplanted poly(lactide-co-e-caprolactone) (PLCL) scaffolds pre seeded with MSC in a rat MI model. Ten days post implantation, some transplanted MSCs survived and differentiated into cardiomyocytes in the injured region, which was accompanied with some 20 -- 30% improvement in cardiac function [139]. Notable work using PLGA-based patch platforms was recently published by the Levenberg group [9,45], who used a tri-culture of ESC-derived CM, endothelial cells and embryonic fibroblasts to form a synchronously contracting engineered human cardiac-like tissue containing endothelial vessel networks [9]. This tissue construct was further assessed in terms of its ability to engraft in-vivo into the rat heart and was found to promote functional vascularization [45]. Interestingly, in common with the scaffold free technique discussed above, the patch thickness did not develop to more than 600 m. Even though, this thickness represents almost half the wall thickness of the rat heart, it is far from that required for clinical application. In addition, there is increasing evidence that successful scaffold materials must have elastic properties that resemble those of the native heart, allowing them to move in synchrony with every heart beat, thus maintaining their structural integrity. Unfotunately, polyesters, are generally less flexible than the heart tissue, hence there is a requirement for more elastic synthetic materials such as elastomers.
Elastomers Elastomers are synthetic mimics of natural rubber. Accordingly they are defined as synthetic materials that have the ability to
3.2.2
Polyesters The general definition Polyester refers to a group of polymers, which contain the ester functional group in their main chain.
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undergo elastic deformation under the influence of a force and regain their original shape once the force has been removed. A 3D cardiac construct made out of polyurethane (PU), exhibited, good cell adhesion in-vitro [140]. Further in-vitro and in-vivo evaluations showed no tissue inflammation and transient inhibition in the progression towards heart failure. This transient inhibition was no surprise, as transplanted cells were undetectable one year post treatment [141,142]. These promising observations are flawed by the fact that PU release a toxic byproduct -- disocyanate that hampers its clinical use [2]. Other elastomeric materials that were reported as suitable for myocardial patch engineering are 1,3 trimethylene carbonate [143] and PEG, which was suggested as a screening method for the optimization of tri-culture conditions [144]. A more biocompatible and absorbable elastomer - poly(glycerol sebacate) (PGS) was developed by the renowned Langer group in 2002 [145]. The same material was recently reported by Chen et al., to support the LVs of transplanted rat hearts while serving as an efficient delivery vehicle for ESC-derived CM [49]. The promise of these materials cannot yet be fully judged as to date no large-animal studies have been performed.
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3.3.1
Natural patch-based platforms Extracellular matrix
The use of ECM, as a patch platform for myocardial tissue engineering was introduced by the Badylak group in 2005. They reported the successful implementation of urinary bladder ECM multilaminate as an acellular myocardial patch that significantly augmented ventricular function in canine and porcine models [146,147]. Further work on a canine RV defect model, demonstrated that the mechanical function generated in the patch region correlates to the quantity of local tissue myocytes infiltration [148]. Nevertheless, when using the natural ECM, its origin needs to be considered as different tissues exhibit different ECM compositions and ultrastructure that may affect the formation of the desired tissue [66,149-151]. Therefore, a suitable ECM for cardiac regeneration may be the one that originates from heart tissue. Such ECM has the potential of better preserving the unique strutted, woven and coiled structures that enable efficient CM contraction. It may also allow the appropriate cell guidance and differentiation signals, while avoiding non-favorable remodeling towards other tissue types as also discussed by Badylak et al. [152]. Four groups (including our own) independently reported the successful isolation of cardiac ECM scaffold from rat [150] and from a more human relevant porcine xenogeneic origin [153-155]. An in-vivo application was reported in a rat MI model by Ott et al., who utilized an impressive perfusion decellularization technique to obtain an ECM template of an entire rat heart. This ECM was further re-seeded with rat neonatal CM and cultivated in a modified Langendorff apparatus to yield active beating synchronous heart. Heterotopic transplantation of this cardiac ECM platform in rats, demonstrated that functional connectivity of the vascular
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infrastructure is preserved during the decellularization procedure [150]. As the scale up from rat to porcine myocardium decellularization is a challenging one, the isolation of porcine acellular myocaridal ECM was achieved either from thin (2 -- 3 mm) left ventricular slices [153,155] or by using perfusion through the inherent vasculature [154]. Nonetheless, the yellowish appearance of the acellular perfused cardiac tissue in the latter report indicates the need for a more extensive decellularization technique as demonstrated by Ott et al. [150]. Indeed, large scale acellularity that is supported by an immunogenic evaluation has yet to be achieved to allow large animal model studies to be performed. Our own group has recently reported a procedure for isolating porcine myocardial acellular ECM (1 -- 2 mm in thickness) [153]. We demonstrated the effectiveness of such decellularization procedure, which selectively removes all inflammation-inducing cellular components while preserving much of the ECM (Figure 2). In our studies the decellularization process produced yellow areas on the ECM that were indicative of a non-complete decellularization (Figure 2B). As remaining cellular debris is potentially immunogenic [149] and may possibly affect cell attachment and viability, any decellularization process, particularly one of thick tissue, should produce complete acellular ECM and also address the remains of the coronary adipose tissue. Moreover when isolating a thick heart tissue, the left ventricular tissue can be advantageous, as the inherent vasculature can be used for the perfusion cleaning process and can also be proven to be essential for future construction of functional inherent blood vessels by seeding with endothelial cells (Figure 2C). Once having ensured that the ECM is totally acellular, it was evident that it encourages cell growth and cellular remodeling both in-vitro as well as in-vivo (Figure 3). Current work in our lab focuses on the optimization of this procedure for thicker tissue specimens that could be potentially used as patch material and also for transmural replacement therapies (Figure 4). Another approach for obtaining an acellular patch material is by the decellularization of pericardial matrix as reported by Chang et al., [156]. In more recent studies performed by the same group, mesenchymal stromal cell [157] or MSC monolayers [158] were sandwiched together with pre cut acellular pericardia to form a multilaminate. This tissue graft (sandwiched patch) was used not only as a patch, but also as a replacement for the infarcted wall in a syngeneic Lewis rat model with chronic MI. At retrieval, the area of the empty patch serving as control, was relatively enlarged, suggesting reduced structural support while that of the sandwiched patch remained about the same. The sandwiched patches, were populated with both host and originally seeded cells [157]. Whether these results could be scaled up in larger animal models where infarct sizes are by far bigger, remains to be proven.
Collagen The casting of liquid collagen with neonatal CM into circular molds yielded in-vitro engineered heart like tissue (EHT) that
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B.
C.
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D.
Figure 2. Isolation of porcine cardiac extracellular matrix (ECM). Slices of fresh porcine heart (A.), incomplete decellularized slices showing retention of cellular debris (Yellow/Brown) marked by an arrow (B.) and clean acellular ECM preserving structural properties such as blood vessel ECM marked by arrows (C.). Masson tri-chrome staining of representative cross sections shows high cellular density (red -- cytoplasms) adjacent to ECM fibers (Blue) in native porcine cardiac tissue (D.). No cellular remains are apparent in the acellular scaffold (E.). Scale bars in D. and E.: 100 m.
A. B. C.
D.
E.
Figure 3. Cellular remodeling of porcine cardiac extracellular matrix (ECM). Rat neonatal cardiomyocytes (CM) seeded on porcine cardiac ECM. Immunological staining for a-cardiac actinin (A.) and connexin-43 (B.) at day 7 shows CM organization. Seeded CM secretes ECM fibers and remodels the acellular scaffold visualized by scanning electron microscopy (C.). Subcutaneous transplantation in mice shows the transplanted ECM at one-week post transplantation (D.) and 8 weeks post transplantation (E.). Arrows mark cell penetration and remodeling. Scale bars: A. -- C. 20 m; D. -- E. 200 m.
was subsequently electrically [159-161] or mechanically [160,162] stimulated to create synchronous beating hoops. This EHT was used for both patch implantation on infarcted immunesuppressed rat hearts [163] as well as to create a novel biological assist device out of a pouch like EHT [164]. Other groups have also attempted to use collagen-patch-based platforms [40,165-166]. Kofidis et al., transplanted a collagen
patch, which was solidified with human ESC-derived CM on an athymic nude rat heterotopic heart transplant model. Their results indicated that the three dimensional matrix prevented myocardial wall thinning and improved contractility [40]. In a recent study by Pozzoboen et al., a porous collagen patch was transplanted on a cryoinjured rat model, and allowed to neo-vascularize for a period of two weeks after
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B.
C.
Figure 4. Perfusion -- decellularization of thick porcine cardiac extracellular matrix ECM. Porcine left ventricle is perfused through the coronary artery while immersed in the same decellularization solutions for a week. Note the difference in cellular remains in the perfused area (black arrows) compared with the non-perfused parts that were exposed to the decellularization solutions only from submerging the tissue in solution (A., white arrows). Hematoxylin and eosin staining of cryosectioned slices obtained from the dashed square in A. (effective decellularization, B.) and from the continuous square in A. (ineffective decellularization, C.).
which, human CD133+ cells were injected directly into the patch. Their results indicated that this strategy, though suitable for angiogenesis and arteriogenesis, does not produce better results, in terms of selective homing and differentiation into CMs, than the traditional method of cell injection into the myocardium [166]. Simpson et al., have also attempted to assess whether an epicardially applied, tissue-engineered cardiac patch containing progenitor cells would result in enhanced exogenous cell engraftment [167]. They have embedded human MSC (hMSC) into patches made of rat tail type I collagen and glued this patch to rat MI model hearts using fibrin glue. Although hMSC engraftment, one week post implantation was about 23 4% of the original seeded quantity, long-term cell survival was not detected at 4 weeks. A patch composed of collagen and Matrigel, was used by Giraud et al., to deliver skeletal myoblasts into the infarcted myocardium of rats [168]. The patch was glued onto the myocardium using fibrin glue leading to some functional benefits, namely improved systolic heart function, however, skeletal myoblasts survival rate was low and the arrhythmogenic effect was not properly monitored [169].
Gelatin Several studies have reported the use of gelatin for the production of cardiac patch platforms [137]. Li et al. seeded fetal ventricular muscle cells onto a gelatin mesh in-vitro and cultivated it for 7 days prior to implantation on a cryoinjured
3.3.3
rat heart. Although cells survived the transplantation and interconnected with the native tissue, no analysis of cardiac function was made [170]. In a follow-up study, they reported on increased cellular proliferation and better performance when mechanical stress regimes were applied on the gelatin foam [171].
Alginate Alginate patches were first proposed by Leor et al., who isolated and grew fetal rat cardiac cells within 3D porous alginate scaffolds. Scaffolds were manufactured using a freeze drying technique and had 90% porosity with an average pore size of 100 m. Intensive neovascularization was revealed post transplantation of the alginate scaffold in a rat MI model [172]. In more recent studies performed by the same group, similar alginate scaffolds were implanted for 7 days on the omentum to achieve pre-vascularization and than transplanted on a MI rat model. The vascularized cardiac patch showed structural and electrical integration into host myocardium and induced thicker scars that prevented further dilatation of the chamber and ventricular dysfunction [173]. In a heterotopic heart transplant model similar alginate patches also exhibited improved left ventricular function [174].
3.3.4
Synthetic-and-natural-combined patch platforms Different studies exploited the advantages of both the synthetic as well as the natural biomaterials producing bio-synthetic chimeras for the construction of cardiac patches. The in-vitro
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fate of such scaffolds has been reviewed by [2,33,137]. The current review addresses the combined-patch platforms which demonstrated in-vivo success. Krupnick et al., seeded a mixture of bone marrow-derived MSC, collagen types I and IV and Matrigel onto a nonwoven polymer mesh made out of PLA reinforced with poly(tetrafluoroethylene) (PTFE). This construct was then heterotopically implanted into infarcted syngenic rat hearts. Their results suggested that this construct is non-immunogenic and biocompatible as minimal intracardiac inflammation was observed [175]. Similarly, Fukuhara et al., seeded crude bone marrow cells (cBMC) onto a bioengineered polyglycolic acid cloth (PGAC) impregnated with collagen type I and bFGF. The patch was subsequently sutured on top of a MI scar tissue of rat hearts demonstrating improved cardiac function, and a distinct cellular population within the patch material including infiltrating inflammatory cells at 4 weeks post implantation [176]. Siepe et al., produced another interesting combination of synthetic and natural polymers, which is composed of polyurethane (PU) patches coated with laminin, a natural adhesion and signaling glycoprotein of the ECM [141]. When implanted with myoblasts, this type of patch led to significantly improved heart function in a MI rat model as assessed by echocardiography. It was concluded that myoblast-seeded PU scaffolds prevented post--myocardial infarction progression toward heart failure with the same efficiency as direct intramyocardial injection. In a follow-up study by the same group, a progression toward heart failure was significantly prevented for up to 6 months after injection of myoblasts and for up to 9 months following biograft implantation [177]. This effect vanished after 12 months, with immunohistological examinations revealing the absence of transplanted myoblasts within the scaffold, thus, suggesting once more that polymer-based platforms in combination with cells are superior to cell therapy alone. However, the beneficial effects were transient and are probably dependent on the survival and viability of the seeded cells [177]. Finally, Miyagi et al., developed a new, biodegradable patch made of a sponge like inner core (modified Gelfoam, MGF) that encourages cell engraftment and an outer coating of PCL providing sufficient strength to permit ventricular repair. Using rat MI model, they demonstrated that animals whose hearts were repaired with untreated Gelfoam died of ventricular rupture, while the MGF-treated group had significantly improved myocardial systolic function. Further incorporation of cytokines (stem cell factor, stromal cell-derived factor-1a) and/or bone-marrow-derived MSC within the patched platform, promoted a-smooth muscle actin-positive cells, greater capillary formation, increased wall thickness and better preserved systolic elastance than MGF alone [178].
4.
Future platform technologies
Several emerging technologies in tissue regeneration have been recently reported to improve cell survival, differentiation, spatial organization and/or biomechanical integration with host myocardial tissue upon transplantation. These
include combinations of injection- and patch-based platforms [179,180] and the use of either physical (mechanical stretching [162,171], perfusion [181,182] and electrical [183-189]) or biochemical (hypoxic pre-conditioning [190,191]) stimulation procedures. Combinations of patch and injectable platforms exploit both the mechanical support of the patch with the ease of delivery accompanying intramyocardial injections. Cortes-Morichetti et al., tested in a mouse MI model, the effect of intrainfarct injection of human umbilical cord blood mononuclear cells (HUCBS) followed by fixation onto the epicardium of a collagen matrix seeded with the same cell type. This treatment proved to be advantageous in terms of post-ischemic ventricular dilation and remodeling, over cell-only or non-seeded-patch-only controls [180]. Further proof of concept was later provided using a similar approach in a Phase I controlled clinical feasibility study performed in 20 patients, half of whom received the combined treatment (using bone marrow cells) while the rest received only the intra-infarct cell injection. The results indicated that the combined strategy appears to improve the efficiency of cellular cardiomyoplasty as the collagen scaffold increased the wall thickness with viable tissue while limiting remodeling and normalizing cardiac wall stress in the injured regions [179]. Effective bio-mechanical pre-conditioning for collagen (and gelatin) stretching has already been discussed with respect to patch-based platforms above [160,162,171]. Perfusion offers another approach to exert mechanical stimuli on cells and constructs. This methodology confers increased mass transfer efficiency for nutrient supply and waste products removal [181,182]. Radisic et al., employed a custom built bioreactor system to demonstrate that the interstitial flow of culture medium through rat neonatal CM-seeded porous collagen scaffold, yielded thick, compact and contractile cardiac constructs. These constructs exhibited physiological densities of metabolically active and viable cells containing cardiac markers [182]. Similar observation were also reported by Dvir et al., who also developed a perfusion bioreactor prototype for alginate based constructs seeded with the same cell type [181]. The in-vivo evaluation of these perfusion end-products compared with non-perfused controls is still to be reported. Another physical condition associated with cardiac cell differentiation and maturation is that of electro-stimulation. Electrical stimuli have shown increased pre-commitment towards cardiac lineages of neonatal cadiomyocytes [185-188], fibroblasts [186], hMSCs [184], skeletal myoblasts [183] and hESC [188,189]. Genovese and colleagues have recently shown that electrostimulation induces cardiomyocyte precommitment of hMSCs in-vitro and is an effective alternative to cytokine-induced differentiation. Electrostimulated hMSCs have exhibited both morphological and biochemical changes, resulting in a shift toward a striated muscle cell phenotype expressing cardiac specific markers. This partially differentiated phenotype may allow a gradual, ongoing differentiation within the cardiac environment thus conveying potential advantages in clinical applications. The same group
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further evaluated the effects of in-situ electrostimulation (using biventricular pacemakers) on skeletal myoblasts transplanted in sheep MI model. Stimulation resulted in a significant improvement in the ejection fraction and limitation of left ventricular dilatation compared with controls. Even though the arrhythmogenic nature of skeletal myoblasts is still a matter of controversy [192], no evident arrhythmias were observed in this study, rendering the strategy of skeletal myoblast electrical stimulation prior to transplantation clinically relevant. Tandon et al., have recently reported the development of a set of electrical stimulation procedures enabling the use of modified culture dishes [188]. Such reports would hopefully lead to the establishment of standard procedures that in turn would enable a better understanding of the electrical stimulation mechanism of action. Finally, hypoxia (i.e., oxygen starvation) had long been known to have a considerable effect on the proliferation, differentiation and maintenance of the cardiovascular system during development [193]. As such, the use of hypoxia to precondition transplanted cells lead to improved results in terms of increased cell survival and angiogenesis [190,191]. Hu et al., reported that mouse MSCs grown under hypoxic conditions (0.5% oxygen for 24 h) displayed increased expression of pro-survival and pro-angiogenic factors (hypoxia inducible facor 1, angiopoietin-1, vascular endothelial growth factor and its receptor fetal liver kinase 1 (Flk-1), erythropoietin, B cell lymphoma 2 (Bcl-2) and Bcl-2-like X (Bcl-xL)). When transplanted to the peri-infarct region of a rat MI model, hypoxia preconditioned MSC exhibited higher survival rates leading to an increase in angiogenesis and to other morphologic and functional benefits, most probably associated with MSC paracrine effects [190]. Similar results were also recently reported by Wang et al., who demonstrated that anoxia preconditioning enhanced the capacity of MSCs to repair the infarcted myocardium. This was attributed to increased cell survival and angiogenesis [191]. This study together with other in-vitro evidence concerning the effectiveness of sub-physiological oxygen concentrations on ex-vivo cell proliferation, gene expression and commitment [193-198] may call into question the traditional and standard laboratory cell conditions (i.e., 5% CO2, 95% air) used in most of the studies mentioned in this report. Taken together, these evolving technologies, offer genuine opportunity to new discoveries as boundaries of traditional cell therapy and tissue engineering techniques are being challenged to create platforms having actual bio-mimetic properties in an ex-vivo environment.
5.
due to their autologous origin and their overall tendency to spontaneously give rise to several supporting cell types including beating CM and endothelial cells. Nonetheless, major research still needs to be performed towards achieving a robust and time effective differentiation procedure(s) that enhances the yield of CM production with as little genetic interference as possible being introduced. It is expected that clinical trials of in-situinjectable biomaterial platforms would produce similar results to those already obtained in large-animal models, showing improved cardiac function, with the hope of total cardiac regeneration. Nonetheless, one cannot ignore the promising results with cardiac patches. This potential should motivate further basic in-vitro research aiming to deal with several major limitations still hampering the production of relevant thick and organized tissue constructs for patch or infarct replacement therapies. These limitations include the lack of cell survival post transplantation, as it is evident from the data presented herein that without cells populating these tissue engineered constructs, no long-term effect can be achieved. Most importantly, the oxygen diffusion limitation in culture media, which dictates that in-vitro produced constructs exhibit inadequate thicknesses for human sized hearts. Finally, the static flat tissue culture is inappropriate for 3D tissue constructs. Hence a Biomimetic approach to cardiac tissue engineering would probably direct the assembly of functional tissues using biologically derived design requirements [34]. Several pivotal reports, reviewed elsewhere [34], have already been published in recent years by leading researchers in this field, such prestigious groups include those of Robert Langer, Smadar Cohen, Gordana VunjakNovakovic, Milica Radisic, Thomas Eschenhagen, Wolfram Hubertus Zimmerman, Juan Carlos Chachques and Alain F Carpentier [35,159,162,163,182-185,187,199-200]. In summary, our growing understanding and ability to supply pulsed-flow feeding as well as timely mechanical, electrical and biochemical stimuli would enable the establishment of complementary dynamic cultivation platforms. These will in turn produce more densely populated, thicker and aligned constructs prior to implantation. Furthermore, in-vitro-developed tissue constructs, serving as cardiac mimetics, would also enable rapid screening of novel therapeutics and their effects on cardiac regeneration.
Acknowledgments
The authors would like to sincerely thank S Irvine and Y Eitan for their help in the preparation of this manuscript. We would like to acknowledge D Machlouf for contributing Figure 1.
Expert opinion Declaration of interest

The authors state no conflict of interest and have received no payment in preparation of this manuscript.
The future choice of the cell or cell combinations for heart regenerative cell-based platforms are probably autologous iPSCs and CSCs. These cells offer a genuine clinical alternative mainly
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Udi Sarig & Marcelle Machluf Author for correspondence Technion--Israel Institute of Technology, The Laboratory of Cancer, Drug Delivery & Mammalian Cell Technology, Faculty of Biotechnology & Food Engineering, Haifa 32000, Israel Tel: +972 4 8294916/3079; Fax: +972 4 8293399; E-mail: machlufm@tx.technion.ac.il
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Review

Engineering cell platforms for myocardial regeneration

Engineering cell platforms for myocardial regeneration

This box summarizes key points contained in the article.

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Table 1. Cardiomyocyte cell sources.

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Bone marrow Umbilical vein Adipose tissue Dental pulp

Table 2. Natural biomaterials for myocardial cell platforms.

Clinical trials None reported

Murine [99,100]; Chick [101]; Sheep [98]

Expert Opin. Biol. Ther. (2011) 11(8)

Murine [104-106,172-174]; Porcine [107]

On-going Phase II [109]

Sarig & Machluf

ECM: Extracellular matrix; HA: Hyaluronic acid.

Table 3. Synthetic biomaterials for myocardial cell platforms.

PNIPAAM (and derivatives)

Engineering cell platforms for myocardial regeneration

PEG (and derivatives)

Murine [128,129]; Rabbit [130]

Expert Opin. Biol. Ther. (2011) 11(8)

Polyesters (e.g., poly-aHydroxy-acid; PLA; PGA; PCL; PLGA; PCLA; PLCL)

Murine [45,138, 139,175,176,178]

Murine [49,141, 142,175]

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Natural patch-based platforms Extracellular matrix

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Future platform technologies

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert opinion Declaration of interest

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8)

Engineering cell platforms for myocardial regeneration

Expert Opin. Biol. Ther. (2011) 11(8)

Sarig & Machluf