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Predifferentiated Adult Stem Cells and Matrices for Cardiac Cell Therapy

Cardiovascular Surgery Department, University Campus BioMedico, Rome, Italy
¹ Université Pierre et Marie Curie
² Pompidou Hospital Paris, France

Jorge Genovese, MD, PhD Tel: +39 06.22541.1 Fax: +39 06.22541.456 Email: genoveseja{at}yahoo.com, Cardiovascular Surgery Specialists School, University Campus BioMedico of Rome, Via Álvaro del Portillo 21, 00128 Rome, Italy.

	ABSTRACT

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
Stem cell therapy is a major field of research worldwide, with increasing clinical application, especially in cardiovascular pathology. However, the best stem cell source and type with optimal safety for functional engraftment remains unclear. An intermediate cardiac precommitted phenotype expressing some of the key proteins of a mature cardiomyocyte would permit better integration into the cardiac environment. The predifferentiated cells would receive signals from the environment, thus achieving gradual and complete differentiation. In cell transplantation, survival and engraftment within the environment of the ischemic myocardium represents a challenge for all types of cells, regardless of their state of differentiation. An alternative strategy is to embed cells in a 3-dimensional structure replicating the extracellular matrix, which is crucial for full tissue restoration and prevention of ventricular remodeling. The clinical translation of cell therapy requires avoidance of potentially harmful drugs and cytokines, and rapid off-the-shelf availability of cells. The combination of predifferentiated cells with a functionalized scaffold, locally releasing molecules tailored to promote in-situ completion of differentiation and improve homing, survival, and function, could be an exciting approach that might circumvent the potential undesired effects of growth factor administration and improve tissue restoration.

Key Words: Adult Stem Cells • Cell Transplantation • Heart Failure • Myocardial Infarction • Tissue Engineering

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
Despite ongoing debate regarding the actual endogenous regenerative potential of human myocardium, progenitor cells or stem cells constitute a new approach to the treatment of cardiac disease. Several preclinical and clinical trials have shown exciting results, but the mechanism underlying cardiac improvement after stem cell injection is still not understood. True in-situ cardiac transdifferentiation has not yet been demonstrated, and cell viability and engrafting after administration have been found to be low, weakening the hypothesis of real tissue replacement or regeneration. Recent evidence also considers the paracrine effects exerted by the injected cells as playing a pivotal role in the improvement of cardiac function.^1,2 Moreover, issues concerning the best stem cell source and type for safe and harmonious functional engraftment with adequate electromechanical integration remain unresolved. A number of factors related to the patient’s systemic condition and the microenvironment of the injection site hamper effective integration and differentiation of stem cells within the heart. Inducing partial differentiation towards a cardiac precommitted phenotype that expresses some of the key proteins of a mature cardiomyocyte could provide better integration into the cardiac environment. The predifferentiated cells would receive signals from the new environment, thus achieving gradual and complete differentiation. Our group developed a cytokine-free system to obtain stem and non-stem cell predifferentiation.^3,4 Furthermore, the combination of stem cells or predifferentiated stem cells with biomaterials organized into 3-dimensional structures replicating the extracellular matrix might obtain full tissue restoration and prevent ventricular remodeling. Biocompatible materials could be further functionalized with different growth factors to guide tissue regeneration and promote in-situ completion of the differentiation of precommitted progenitor cells.

	PREDIFFERENTIATED STEM CELLS

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
Recently, research efforts on stem cell characterization and application in cardiology have exploded.^5,6 Cellular cardiomyoplasty is a stem cell-based regenerative or reparative therapy with the goal of limiting the consequences of myocardial infarction or non-ischemic cardiomyopathy. One unanswered question is how differentiated should the stem cells be for application in a sick heart. An intermediate option might result in better cell integration, higher environmental dependence, improved survival, and more efficient functional restitution. The predifferentiation process could let cells express in-vitro cardiac proteins that are the phenotypic expression of genetic changes toward the final cardiomyocyte stage. After injection, the precommitted cells would receive signals from the new environment, thus achieving gradual and complete differentiation. Avoiding signals associated with cell transformation, as many cytokines are known to be, a safer and "clean" predifferentiation model might be defined.^3,4 Stem cell predifferentiation results in several morphological changes including multinucleated cell formation and cytoskeleton rearrangement. The expression of cardiac proteins related to cell contraction and intercellular communication is also detected early. Therefore, cardiomyocyte predifferentiation of stem cells is a potentially powerful tool to obtain cells with oriented evolutive capacity and still able to respond to environmental signals. These cells exhibit phenotypic and metabolic changes that could facilitate their homing, adaptation, survival, replication, and function, with consequently improved therapeutic effects. Such predifferentiated stem cells could be useful in the generation of in-vitro and in-vivo tissue or organ substitutes by tissue engineering. This approach might be even more effective if it employed functionalized scaffolds that release molecules contributing to the completion of the predifferentiation process.

	PREDIFFERENTIATED STEM CELLS: WHICH ONES?

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
The concept of "stemness" includes 2 main characteristics: the capability of self-renewal, and the potential to differentiate into mature cell types. Independent of the embryologic and developmental stages, stem cells can be distinguished by their age and differentiation potential. As the differentiation process progresses, cells lose evolutive capacity. It can be assumed that cells displaying major evolutive capacity will be more easily predifferentiated toward a defined phenotype.

EMBRYONIC STEM CELLS
Embryonic stem cells (ESC) derive from the morula stage or the epiblast tissue of the inner cell mass of blastocysts. They are characterized by totipotency or pluripotency, being able to give rise to all derivatives of the 3 primary germ layers, and thus to almost every mature cell type. Despite the enormous differentiation potency of ESC, the risk of tumorigenesis if the wrong differentiation pathway is taken, immunogenicity, and animal-derived pathogen transmission, limit their clinical translatability. At this point, despite their usefulness and potential benefits, ESC need to overcome numerous barriers before reaching the patient’s bedside. Until now, human ESC application has seemed too complicated to be considered for general application.

ADULT STEM CELLS
Adult stem cells are lineage-restricted (multipotent) and classified as limited to differentiating into only a few types of mature cell. Down in the differentiation potential scale, progenitor or precursor cells are considered capable of differentiating into only one specific mature cell type. Adult multipotent or progenitor cells can be isolated from a number of different sources and tissues. Recent insights highlight the possibility of finding a particular stem cell niche in almost all human organs. However, bone marrow, blood, umbilical cord, adipose tissue, muscle, heart, and skin have been considered more useful for stem cell isolation.⁷ In both the experimental and clinical fields, some beneficial effects of adult stem cell application have been reported already. In some cases, such as hematology, the use of adult stem cells has already had a definite impact on the treatment of disease. Beyond the application of hematopoietic stem cells in oncology, preliminary data have shown potential therapeutic effects of myoblasts in cardiology, and fat-derived stem cells in plastic surgery.⁸ A less conclusive but even more hopeful scenario is the use of mesenchymal stem cells in cardiac regeneration. Although the mechanisms underlying their effects have not been elucidated, adult stem cells could be beneficial in many pathological conditions, and they have been reported to improve patients’ conditions.

BONE MARROW-DERIVED MESENCHYMAL STEM CELLS
The bone marrow contains mesenchymal stem cells, multipotent cells, with proven ability to differentiate into osteoblasts, chondrocytes, and adipocytes, as well as cardiomyocytes under specific conditions in vitro.^9,10 Moreover, mesenchymal stem cells have elicited substantial attention because they can readily be made available for autologous application (by bone marrow aspiration), isolated, and expanded ex vivo, and they exhibit high plasticity. These cells have fueled an increasing amount of research and are an attractive therapeutic tool.

ADIPOCYTE-DERIVED STEM CELLS
Mesenchymal stromal cells are present in adipose tissue, in a perivascular niche. Adipocyte-derived stem cells can be easily isolated and cultured ex vivo, and express markers associated with mesenchymal and perivascular cells, including STRO-1, CD146, and 3G5, maintaining their characteristic multipotency, as shown by their capacity to form Alizarin Red-positive mineralized deposits, Oil Red O-positive lipid droplets, and Alcian Blue-positive proteoglycan-rich matrices in vitro. They also possess plasticity, under appropriate in-vitro culture conditions, to adopt cardiomyocyte and vascular cell phenotypic characteristics, and they have been shown in in-vivo preclinical studies to facilitate both myocardial repair and neovascularization.¹¹ This beneficial effect appears to be related to an indirect paracrine action rather than direct regeneration of endogenous cells by transdifferentiation, especially because current transplantation strategies achieve a quantitatively modest engraftment of cells into the host myocardium.¹²

MUSCLE STEM CELLS
Mouse skeletal muscle has been found to contain a population of non-satellite cells that can differentiate into spontaneously beating cells with cardiomyocyte features.¹³ Interestingly, the discovery of these cells arose from a few simple variations in the usual technique of skeletal muscle cell culture. It has been suggested that these cells are closer to true cardiomyocytes. Efficient homing to the heart in a model of acute ischemic injury, and the subsequent in-vivo differentiation into a cardiac phenotype at the border of an old infarct, predict possible further experimentation focusing on chronic heart failure or dilated cardiomyopathy.

MYOCARDIAL STEM CELLS
Mammalian myocardium includes a small proportion of stem cells that express the cell-surface markers Kit or Sca-1.^14,15 These cells are unable to repair the myocardium after ischemia, but they can be isolated from human myocardial samples obtained using a minimally invasive procedure. These cells can be further expanded to an adequate number in a complex culture medium, with minimal risks of immune rejection or teratoma formation. However, no clinical data on the use of these cells are available yet.

INDUCED PLURIPOTENT STEM CELLS
Another recent strategy to overcome the restricted differentiation potential of adult stem cells is genetic reprogramming by incorporating an immaturity genome (e.g. from oocytes). The concept of nuclear transfer and cellular cloning has been applied in rodents, and may result in autologous pluripotent cell lines. Byrne and colleagues^16–18 were able to reprogram adult mammalian skin cells by nuclear transfer, obtaining a pluripo-tent cell line, and the transduction of only 4 defined transcription factors (Oct3/4, Sox2, Klf4, c-Myc) could induce pluripotency in an adult human dermal fibroblast cell line. These approaches might have value in future research in the cardiovascular field, despite being hampered by economic and technical issues relative to the need for expertise and genomic facilities, and the above-mentioned problems of controlling the final differentiation of pluripotent cells.

	STEM CELLS: A TRANSLATIONAL APPROACH

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
To date, there have been 3 suitable physiological sources of stem cells identified following myocardial ischemia: the damaged myocardium itself, the peripheral circulation, and the bone marrow. Consequently, a wide variety of cell types derived from these tissues have been considered for therapeutic transplantation into humans. Bone marrow mononuclear fraction, hematopoietic and mesenchymal stem cells, cardiac stem cells, endothelial progenitor cells, adipose-derived stem cells plus embryonic stem cells, and skeletal myoblasts have been successfully employed in clinical trials.¹⁹ A comprehensive index of clinical trials is available from the American Library of Medicine at http://www.the-scientist.com/supplementary/html/24104/.

The first clinical trials were performed using skeletal myoblasts, with encouraging results in terms of increases in regional wall motion and left ventricular ejection fraction.²⁰ Myoblasts are resistant to ischemia and show ability to improve ventricular function in animal models, differentiating into myotubes in vivo. Unfortunately, a true cardiomyocyte phenotype could not be achieved, and myotubes do not integrate electrically with surviving cardiomyocytes, failing to contract synchronously with the surrounding myocardium. Several human trials using myoblasts in heart failure are ongoing, but some have been terminated because of lack of efficacy or recurrent malignant arrhythmias.²¹

Other progenitor cells have been assessed as therapeutic agents in conditions such as myocardial infarction or heart failure. Recent studies demonstrated that intracoronary infusion of bone marrow mesenchymal stem cells induces significant improvement in myocardial performance, with the most obvious benefits in patients who received the intervention more than 5 days after myocardial infarction. In addition to these positive findings, a number of additional clinical trials have yielded controversial results.²² Although cell therapy has been suggested as an effective strategy to improve the performance of human myocardium, these results are not consistent across the literature, and the lack of confirmatory results from the most recent clinical trials puts on hold the initial enthusiasm regarding the clinical use of cell therapy in heart regeneration, questioning its actual effectiveness and safety.^23–25 The wide range of delivery techniques and different methodological approaches used by various research groups make the results of these studies difficult to interpret. There is an obvious need for methodological consensus or the creation of technical guidelines to draw reliable conclusions regarding the usefulness of cell therapy in human heart regeneration. Despite the myriad of cell types in several experimental studies, the ideal cell type has not yet emerged, and few studies have directly compared different stem cell types.¹⁰ Phenomena related to cell plasticity or fusion have been claimed to be at the root of the stem cell contribution to improvement in cardiac function, but an insufficiently consistent amount of data supports this concept.

The actual beneficial effect of stem cell therapy could rely on a paracrine effect whereby growth factors, cytokines, and signaling molecules are produced and released by the infused stem cells. Considering the pharmacological potential of stem cells as a cytokine delivery system, it is also important to evaluate the characteristics and different clinical indications for each type of cardiac disease in which cellular therapy might be applied. Stem cells have been used in various clinical settings in largely nonuniform populations of patients.²⁶ This lack of standardization affects even the route of delivery, which poses another question regarding maximum survival and retention in such a specific area of the myocardium as the infraction zone. Sufficient collateral perfusion in the ischemic area is crucial for the survival of a high proportion of cells. Timing from the ischemic event to injection determines the selection of the delivery approach. Transvascular (intracoronary or intravenous) and direct injection into the ventricular wall (transepicardial, transendocardial, or transcoronary-vein infusion) have been proposed. For intracoronary infusion, a high concentration of stem cells is delivered through the central lumen of an over-the-wire balloon catheter, allowing the cells to travel directly into myocardial regions in which blood, nutrient, and oxygen supply are preserved. However, occluded coronary arteries in the ischemic area prevent progenitor cells migrating out of the vessels into the perivascular heart tissue. Intravenous infusion is hampered by issues of nonspecific and noncardiac homing, which dramatically reduce the amount of cells engrafted and their therapeutic effectiveness.

Systems based on direct injection into the ventricular wall during open heart surgery allow direct visualization of the myocardium and superior engraftment, but require associated revascularization procedures to avoid grafting in areas of poorly perfused scarred myocardium, resulting in the formation of islands of cells with minimal perfusion and poor survival.²⁷ It is also possible to perform a transcoronary-vein injection that delivers stem cells parallel to the ventricular wall through veins deeply within the myocardium. However, this system presents drawbacks related to the complexity of the injection technique and to the catheter system required.²⁸ Another approach involves transendocardial injection by means of a needle catheter advanced across the aortic valve and stabilized against the endocardial surface. Stem cells can be injected into viable regions of the myocardium detected by electrophysiological mapping of the heart.²⁹

	STEM CELL THERAPY: MANY ANSWERS, MANY QUESTIONS

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
Despite the enormous number of experimental efforts reporting positive results in terms of myocardial function and improvement in different pathological conditions, there has been no conclusive demonstration of the mechanism involved in tissue regeneration associated with adult stem cell treatment. Many different properties and presumed actions have been hypothesized, such as immunomodulation, paracrine activity, or the capacity to differentiate or transdifferentiate, but their role in the reported beneficial effects has not been clarified. No insights have been provided to dampen the dramatic loss of cells immediately after implantation. Trauma related to handling and the injection itself, together with the negative effects of the hostile environment in which the implant takes place, are most likely at the root of this phenomenon. No precise data are available on the number of cells viable after injection and effectively homing to the affected site. These unanswered questions raise concerns about the actual effects of these cells, especially when an attempt is made to match the biological uncertainties with the evident improvement in cardiac function. Undoubtedly, obstacles to translating basic research into clinical use and the need to perform meticulous clinical trials are still at the forefront. Many issues have prevented the generation of useful and reliable data that could facilitate a potential clinical translation. In spite of its feasibility and attractiveness, cell therapy requires high quality and coordinated scientific, clinical, and technical efforts to validate the technique.

To date, no practical guidelines or scientific consensus regarding the technical aspects of the application of cell therapy in patients have emerged. The widespread application of this promising therapeutic tool is also hindered by some remaining clinical and biological questions. Patient selection, contraindications, timing of treatment, effective degree of benefit, safety, cell type, cell dosage, cell engraftment, and immunogenicity are some of the issues that need to be addressed. Understanding the mechanisms of normal and post-injury cardiomyocyte development and turnover will be crucial in guiding the direction of stem cell-based therapies. Evidently, cardiac regeneration requires a complex cascade of events beyond simple injection of the right type of cell into the right place. Thus characterization of the factors present in the hostile micro-environment of the injured myocardium, which hamper the survival and functional integration of transplanted cells, is also essential. Undoubtedly, along with the biology of stem cells, the actual effectiveness of therapy and safety in patients require to be further defined. Clearly, these issues of the best stem cell source and type, their harmonious and functional engraftment, and their safety, pose an even more paradoxical and provocative question: do we really need a stem undifferentiated phenotype? On the other hand, would a totally differentiated phenotype, the beating cell, achieve the necessary electromechanical integration to survive without generating ectopic autonomous contractile foci?

An intermediate partial cardiac precommitted phenotype, expressing some of the key proteins of a cardiomyocyte, might provide better integration into the cardiac environment. The predifferentiated cells would receive signals from the new environment, thus achieving gradual and complete differentiation. Expression of cardiac-specific gap junction-related proteins (such as connexin43) or fibrillar structural proteins (such as desmin or troponin) could potentially answer issues concerning defective electromechanical integration leading to arrhythmias. Avoidance of the potentially harmful drugs and cytokines involved in cell transformation and a rapid off-the-shelf availability of cells with reduced culture time are required. Recent advances demonstrated the role of cytokine-free biophysical stimulation systems in inducting cell differentiation.^30,31 Our group described a method to induce cardiac predifferentiation in both stem and non-stem cells by controlled delivery of electrical stimuli.^3,4 The basis of this approach was the observation that electro-stimulation prevents immobilization-induced muscle atrophy. Mimicking physiological stimulation conditions provides the correct sequence of signals to maintain and improve muscle structure and function. Simulation of the cardiac cell environment via pacing and long-term electrostimulation mimics the electrical features of a beating heart, and could provide an additional stimulus to direct the cellular apparatus of stem cells toward a cardiomyocyte-type configuration. Interestingly, with this method, we described changes in the cell architecture of fibroblasts, the most abundant cell type in a myocardial scar, resulting in the formation of multinucleated myotubes. Thinking in terms of cell therapy, if these myotubes can be made to contract, systolic function could be improved. If they do not contract, as perhaps occurs with most of the cells injected into the myocardium, a passive cardiac improvement would still be expected through increased compliance and hence diastolic function of the infarcted myocardium.⁴ We were able to reproduce similar results by applying this method of stimulation to both embryonic and adult cell lines, suggesting a non-pluripotential cell-restricted phenomenon.

The possibility of inducing a reorganization of the cell biosynthesis apparatus after a short period of stimulation allowed us to speculate on the early activation of mechanisms probably related to membrane transport modifications. Ion channel aperture timing could possibly be affected by electrostimulation, leading to temporary changes in intracellular pH with further nuclear transcriptional enzyme activation.⁴ Interestingly, this system of predifferentiation could be applied to stem cell lines such as mesenchymal stem cells.³ We further investigated potential mechanisms underlying the effect of electrostimulation, indicating growth factor and molecular pathways potentially accounting for this cardiomyogenic predifferentiation. We focused on an important member of the transforming growth factor-beta superfamily, follistatin (FST), a muscle differentiation regulator and a potential candidate for this process in stem cells.³² FST is implicated in many biologic processes such as cell proliferation and differentiation, immune responses, various endocrine activities, wound repair, inflammation, and fibrosis. FST is considered to be a key modulator in muscle development, differentiation, and regeneration, and it has been implicated in the repair of mesodermal- and endodermal-derived tissues, promoting cell proliferation and hampering fibrogenesis. The mechanism of action of FST concerns neutralization of activins. Those molecules are increased in the serum of heart failure patients and in cardiomyocytes after experimental myocardial infarction, suggesting the involvement of activins in heart failure pathogenesis. We could induce the production of FST by stem cells predifferentiated towards the cardiomyogenic phenotype by electrostimulation.³² Considering the role of FST in the pathogenesis of heart failure, the possibility of producing a precommitted phenotype actively secreting a key regulator of mesodermal differentiation has potentially relevant therapeutic clinical applications.

	TISSUE-ENGINEERED PREDIFFERENTIATED CELLS

TOP ABSTRACT INTRODUCTION PREDIFFERENTIATED STEM CELLS PREDIFFERENTIATED STEM CELLS:... STEM CELLS: A TRANSLATIONAL... STEM CELL THERAPY: MANY... TISSUE-ENGINEERED... REFERENCES

 
Survival in the inflammatory environment of the infarcted myocardium presents a challenge common to all types of cells, regardless their differentiation state. Fibrotic tissue in the ischemic scar also limits cell engraftment. Survival and integration of transplanted cells can be improved by embedding them in a matrix such as collagen.³³ The concept at the root of tissue engineering is to generate a functional 3-dimensional environment suitable for cell culture to produce tissues in vitro, which are tailored in size, shape, and function, before implanting them into the body, or alternatively, using them directly at the site of damage. Several approaches have been developed and recent advances have been made in the engineering of cardiac tissue.³⁴ Biosynthetic and biodegradable materials have largely been investigated. A wide range of natural materials (collagen, fibrin, hyaluronic acid) and approaches involving tissue decellularization have been evaluated (Table 1).^35–53 A 3-dimensional collagen type I matrix, seeded with human umbilical cord blood mononuclear cells and grafted onto infarcted ventricles in an ischemic murine heart model, showed beneficial effects on left ventricular function and myocardial remodeling.³³ Recently, a similar approach was used successfully in a clinical trial (MAGNUM trial) with a bone marrow mesenchymal stem cell-seeded collagen sponge applied on top of the scarred area, with the aim of regenerating myocardial cells and restoring extracellular matrix function which is deeply altered following myocardial infarction.^54,55 The MAGNUM trial is an example of the possible association between stem cell therapy and tissue engineering.

We explored the use of tissue engineering for enhancement of cell retention and proliferation purposes. We developed a 3-dimensional extracellular-matrix-like differentiating device containing the adequate signals locally compartmentalized inside the scaffold to promote differentiation of stem cells in an environment closely resembling the natural architecture of tissues. Previously, we developed a hydroxyapatite functionalized scaffold with the aim of reproducing the native histoarchitecture and molecular signaling of osteochondral tissue to facilitate cell differentiation towards chondrocytes. Poly-L-lactic/hydroxyapatite nanocomposites induced differentiation of human mesenchymal stem cells in a chondrocyte-like phenotype, with the generation of a proteoglycan-based matrix.⁵⁷ We further developed a scaffold tailored to cardiovascular structures. Cells seeded on this scaffold are not only exposed to growth factors promoting their differentiation but also encounter pre-constituted environments that differentially guide their commitment towards the phenotypes characterizing cardiovascular tissues.⁵⁸

For myocardial repair, in-situ tissue engineering has been suggested as a potentially effective candidate; it involves injection into the infarcted wall of a mixture of biomaterials and cells.⁵⁹ As mentioned above, the electrospinning method is an attractive way of producing scaffolds that remarkably mimic the size and scale of the natural extracellular matrix. It enables the fabrication of structures with sub-micron pores and nanotopography, which is reported to be optimal for the realization of successful extracellular-matrix-like scaffolds for cardiac tissue engineering.⁶⁰

Although tissue engineering has been shown to be a promising tool for cardiac repair, a number of technical issues need to be answered. The mechanical resistance of the constructs and their immunogenicity are key factors that could hamper effectiveness and delay the actual clinical translation.⁶¹ In addition, production of a cardiac graft as well as a tissue-engineered vascular graft faces hurdles related to availability and accessibility of autologous adult differentiated cells to populate the underlying biomaterial. The possibility of combining the regenerative capacity of stem cells with the potential of polymeric scaffolds as a ventricular mechanical support, as well as a cell retaining and delivery system, has been explored. Recently, poly-co-caprolactone seeded with bone marrow-derived mononuclear cells has been used as a cardiac patch in rat myocardial infarction, achieving a reduction in left ventricular remodeling and preservation of systolic function.^38,62

Considering the current limitations of both cell therapy and tissue engineering, the most amenable alternative could be a combination of the 2 approaches. The idea of a bridge between stem cell plasticity and the biomaterials that actively guide and provide the correct sequence of signals to allow ongoing lineage-specific differentiation of these pluripotent precursor cells is attractive, and it may represent a promising answer to the problems related to cardiovascular healing. Importantly, controlled delivery systems may provide an alternative means of releasing localized doses of bioactive molecules over sustained periods, minimizing undesired systemic effects, defining a favorable environment for cell engraftment and proliferation, and improving the post-implantation management of the grafts. The combination of predifferentiated cells with a functionalized scaffold locally releasing molecules tailored to promote in-situ completion of differentiation and improve homing, survival, and function, could be an exciting approach circumventing the risk of potential undesired effects of growth factor administration, and improving tissue restoration.

Presented at the 4^th International Cardiac Bio-Assist Association Congress, March 12–13, 2008, Singapore.

	REFERENCES

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Asian Cardiovasc Thorac Ann 2010; 18:79-87
© 2010 by SAGE Publications
DOI: 10.1177/0218492309355836

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