Renovation of the Injured Heart with MyocardialTissueEngineering 1

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Tissue engineering is a growing area that aims to create, repair, and/or replace tissues and organs by using combinations of cells, scaffolds, biologically active molecules, and physiologic signals. It is an interdisciplinary field that integrates aspects of engineering, chemistry, biology, and medicine.

One of the most challenging goals in the field of cardiovascular tissue engineering is the creation of an engineered heart muscle. Unlike heart valves or blood vessels, heart muscle has no replacement alternatives. New discoveries in stem cell biology suggest that stem cells are a potential source of heart muscle cells and blood vessels and can be used to rebuild or replace damaged heart tissue.

Recent advances in methods of stem cell isolation, expansion, and culture and the synthesis of new bioactive materials show promise to contribute to the creation of engineered contractile cardiac tissue in vitro and in vivo.

This chapter introduces the basic structural features of myocardium, elucidating the challenges in tissue engineering of a cardiac muscle.

It describes the principles of myocardial tissue engineering and reviews various approaches to achieve the ambitious goal of creating contractile heart muscle to treat myocardial infarction and heart failure patients.

The Myocardium

The myocardium is composed mainly of cardiomyocytes, fibroblasts, and the elements of

blood vessels: endothelial and smooth muscle cells, macrophages, and extracellular matrix (ECM) (Figure 1.1).^1,2 Cardiomyocytes constitute only one-third of the total cardiac cell number. However, they occupy more than 70% of cardiac volume. Fibroblasts are the dominant cardiac cell and account for 90% to 95% of non- myocyte cell mass.^3,4

Unlike other somatic tissues, the heart has been viewed as an organ composed of terminally differentiated cardiomyocytes and incapable of regeneration. Recent studies challenge these pre- existing notions regarding cardiac repair/regeneration and suggest that the heart is capable of limited regeneration through the activation and recruitment of a stem/progenitor cell population that is resident in the adult heart.⁵

Cardiomyocytes are tethered in an extensive extracellular network of collagen and other structural proteins, including fibronectins and proteo- glycans [Figure 1.2 (see color section)]. The extracellular and intracellular myofibrillar scaf- folding is a critical determinant of cardiac shape during normal and abnormal cardiac growth.

Collagen is synthesized principally by fibroblasts but also by vascular smooth muscle cells in response to a variety of pathologic stimuli, including increased oxidative and mechanical stress, ischemia, and inflammation.

Of the many collagen types, the major fibrillar collagens are types I (approximately 85%) and III (11%), which constitute the bulk of cardiac ECM. Collagen type I is associated mainly with thick fibers that confer tensile strength and

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Renovation of the Injured Heart with Myocardial Tissue Engineering

Jonathan Leor, Natali Landa, and Smadar Cohen

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resistance to stretch and deformation, whereas collagen type III is associated with thin fibers that confer resilience.²

Cardiomyocytes are tethered to the ECM by membrane-spanning proteins called integrins [Figure 1.2 (see color section)]. The extracellular portion of these molecules binds to fibronectins in the ECM. Perimyocyte extracellular proteins such as dystrophin and dystrophin-related proteins contribute to normal cardiogenesis. When altered in abundance, they can produce a cardiomyopathy.⁶

Myocardial Infarction and Remodeling

Heart failure after myocardial infarction can result from the substantial loss of cardiomyocytes in the infarct zone but more often is pre- cipitated by the delayed and progressive pathologic remodeling of the left ventricle. Cell death in the infarct zone is large in magnitude but short in duration.

When myocardial tissue is injured, normal healing response is initiated through a series of complex events that include acute inflammation, the formation of granulation tissue, and eventual scar formation.^7,8 Cytokines and growth factors are released to recruit white blood cells, mainly neutrophils. Monocytes are then called to the wound site where they differentiate into macrophages. The macrophages

are responsible for cleaning the infarcted zone and also for recruiting cells such as fibroblasts, endothelial cells, and stem/progenitor cells creating granulation tissue. The formation of blood vessels is essential to the healing of the infarcted myocardium. The granulation tissue is subse- quently replaced by an ECM deposited primarily by fibroblasts. The degree of ECM depends on the extent and location (e.g., anterior or apical) of infarction. In most cases, the granulation tissue is remodeled into scar tissue.

Most of the molecules and signal transduction pathways operant in cardiomyocyte growth have a role in hyperplasia of fibroblasts and in the elab- oration of collagen. The resultant fibrosis produces altered myocardial stiffness and arrhythmogenesis in ischemic heart disease, cardiac hypertrophy, and congestive heart failure.

Collagen synthesis is continuously and variably offset by ECM resorption mediated by matrix metalloproteinases. The activity of these enzymes is increased in ischemic and dilated cardiomyopathy.² Conversely, the activity of a class of enzymes known as tissue inhibitors of matrix metalloproteinases is reduced in this setting. The resultant excessive collagenolyses may induce myofibrillar slippage and contribute to the dilated thin-walled chamber geometry that characterizes acute and chronic heart failure. This process has been termed left ventricular (LV) remodeling.

Myocardial Regeneration

Myocardial regeneration is an exciting novel therapeutic concept.⁹ One approach that has received recent attention focuses on repopulation of the injured myocardium by transplantation of healthy cells.¹⁰ Several cell types that might replace necrotic tissue and minimize scarring have been considered (Table 1.1). Fetal cardiomyocytes, skeletal myoblasts, and bone marrow stem cells have all shown limited success in restoring damaged tissues and improving cardiac function. Failure to produce new myocardial fibers in clinically relevant numbers was attrib- uted to cell death occurring after engraftment and inability of engrafted myoblasts to differentiate and integrate within the host myocardium;

hence, electromechanical coupling is not likely to occur after in vivo myoblast grafting.

An alternative approach includes mobilization of progenitor or stem cells to the damaged

Endothelial, smooth muscle, etc.

Myocytes

Fibroblasts

Figure 1.1. Myocardial cells. Cardiomyocytes constitute only one-third of the total cardiac cell number. However, they occupy more than 70% of cardiac volume.²

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area or stimulation of a regenerative program within the organ.¹¹ Recent studies have sug- gested that stem cells residing within the bone marrow or peripheral blood can be mobilized and recruited to the injured heart.¹² In addition, there is now accumulating evidence that the heart contains resident stem cells that can be induced to develop into cardiac muscle and vascular tissue.^13–16However, cell transplantation approach may be of little clinical benefit when the local cardiac structure cannot support cell seeding because it is absent or seri- ously damaged. Tissue engineering approach might solve this problem by using three- dimensional (3-D) scaffolds that replace the missing or damaged infrastructure–ECM and provide a temporary support for self or implanted cells.¹⁷

Tissue Engineering

The aim of tissue engineering is to repair or replace the damaged organ or tissues by deliver- ing functional cells, supporting scaffolds, growth-promoting molecules, or DNA encoding these molecules, and electric or physiologic signals to areas in need (Figure 1.3). The field has already made headway in the synthesis of structural tissues such as skin, cartilage, bone, and bladder.¹⁸The classic tissue engineering concept is to isolate specific cells through a biopsy from a patient, to grow them on a 3-D biomimetic scaffold under precisely controlled culture conditions, to deliver the construct to the desired site in the patient’s body, and to direct new tissue

formation into the scaffold that can be degraded over time.¹⁸ To achieve successful regeneration of damaged organs or tissues based on the tissue engineering concept, several critical elements should be considered, including the biomaterial scaffold that serves as a mechanical and biological support for cell growth and differentiation, progenitor cells that can be differentiated into specific cell types, and the inductive growth factors that can modulate cellular activities.

Strategies of tissue engineering can be classi- fied as in vitro and in vivo approaches:

1. In vitro tissue engineering in culture dish or bioreactor

a. Creation of engineered cardiac graft from cell-seeded scaffold

b. Creation of cardiac graft from cell culture or expanded stem cells

2. In vivo tissue engineering (in situ generation) a. Direct cell transplantation

b. Cell-seeded scaffold implantation

c. Nonseeded scaffold implantation and recruiting endogenous cells

d. Injectable scaffold with or without cells e. Promotion of healing and self-repair by

active molecules

Table 1.1. Possible cell sources for myocardial tissue engineering 1. Skeletal myoblasts^91,92

2. Crude bone marrow⁹³ 3. Endothelial progenitor cells⁹⁴ 4. Hematopoietic stem cells⁹⁵ 5. Mesenchymal stem cells⁹⁶ 6. Smooth muscle cells⁷⁸ 7. Umbilical cord cells⁹⁷ 8. Fibroblasts^77,98

9. Human embryonic stem cells⁹⁹ 10. Fetal cardiomyocytes^73,74 11. Myocardial progenitors^13,14,16 12. Cloned cells¹⁰⁰

Cytokines Cells

Scaffold Signals

Figure 1.3. The classic paradigm of myocardial tissue engineering. The engineered heart muscle can be produced, ex vivo or in situ based on:

(1) cells, such as cardiomyocytes; (2) biomaterial scaffold, such as collagen or alginate; (3) cytokines, such as growth or survival factors; and (4) signals, electric or physiologic, such as pacing or flow. For details, see Tables 1.1, 1.3, and 1.5.

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Myocardial Tissue Engineering Versus Cell Transplantation

The promising results of cardiac cell transplantation in animal models have been partially attrib- uted to reconstruction of the ECM, which maintained the structure, thickness, and elasticity of the LV wall.¹⁹The concept of tissue engineering using 3-D scaffolds has certain advantages over the direct cell injection (Table 1.2). The 3-D scaffolds can replace the missing or damaged infrastructure (ECM) in the damaged area and provide temporary support for self or implanted cells. With tissue engineering, one can control the size, shape, strength, and composition of the graft in vitro. Tissue engineering may provide a solution to the problem of congenital or acquired heart defects and can be used to replace or reconstruct defective heart parts such as valves or vessels. Actually, these techniques can be complementary. Cellular therapy is applicable when the structure of the failing organ is relatively simple and small and when disease is localized rather than diffuse.

Biomaterials and Scaffolds

The biomaterial scaffold has a key role in most tissue engineering strategies. To guide the

organization, growth, and differentiation of cells in tissue engineered constructs, the biomaterial scaffold should be able to provide not only physical support for the cells but also the chemical and biological cues needed in forming functional tissues.²⁰The biomaterial should be able to crosstalk, on the molecular level, with the cells in a precise and controlled manner, simi- larly to the natural interactions existing between cells and the native ECM. At the same time, the basic requirements of a biomaterial should be kept; i.e., the materials and their degradation products must be nontoxic and nonimmunogenic, and their degradation rate should match the rate of new tissue formation (Table 1.3). In cardiac tissue engineering, the material properties, such as flexibility and degradability, need to be adjusted so that they will not interfere with cardiac muscle contractility.

In recent years, the trend has been to design bioactive materials, which on one hand will have the appropriate physical strength as well as the degradation kinetics of synthetic polymers and on the other hand will have the biological speci- ficity of collagen, fibronectin, and laminin – the major ECM components. Such biological resem- bling biomaterials, termed “biomimetics,”

should promote cell–matrix interactions, and elicit specific cellular responses and biomolecu- lar recognition.

The approaches for achieving biomimetic materials include synthesis of new materials from scratch or chemical modification of existing materials with bioactive molecules. This approach has the advantages of working with known materials, most of which have been tested and have been proved to be safe in human. The bioactive molecules may be whole ECM molecules or “cell-binding” domain sequences isolated from these proteins. The use of short peptides is advantageous over the whole protein because the protein tends to be ran- domly folded and the receptor binding domains are not always sterically available. In addition, the short peptide is relatively more stable during the modification process and can be massively synthesized in the laboratory. The most fre- quently used peptide sequence derived from fibronectin signaling domain is Arg-Gly-Asp (RGD).^21–25 The selection of peptide sequences for modification depends on the cell type seeded onto the matrix or the implanted site of the scaffold and its natural ECM environment and the specific required cellular responses.

Table 1.2. Comparison between myocardial tissue engineering and cell therapy for myocardial repair

Tissue Cell engineering transplantation

Cells Optional Must

Scaffold Must No

Active molecules Optional Optional Controlled drug delivery Yes No

Controlled graft Yes No

shape and size

Angiogenic Yes Yes

Myogenic Yes Yes

Replace cardiac valves Yes No or big vessels

Repair of infarcted Yes Yes myocardium

Repair of congenital Yes No defects

Injectable Yes Yes

Clinical experience No Yes

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Modification of the material can be performed either in a surface or bulk approach. Surface modification of materials with bioactive molecules is a simple way to make biomimetic materials. In most cases, the goal is to study cell attachment, spreading, proliferation, and differentiation on modified surfaces in 2-D culture, without addressing the effects of the third dimension. Bulk modification of a material, in comparison, should provide a more suitable environment for the cells as it imitates the 3-D environment of the natural ECM. Most of the bulk-modified materials are based on polymers that have been applied before as nonmodified ones for tissue engineering; for example, hyaluro- nan,²⁶ polyethylene oxide,²⁷ poly(N-isopropylacrylamide),²⁸ polylactic-co-glycolic acid,²⁹ and alginate.²³ Modification is usually performed through a chemical reaction leading to covalent bond between the polymer backbone and the bioactive peptide. Another method crosslinks the polymer to form a hydrogel using a bifunctional peptide that also has a signaling domain for interactions with cell membrane receptors.³⁰

Cell Sources

One of the major challenges of tissue engineering application in human patients is to achieve

enough cells to generate significant amount of muscle tissue. The optimal cell source for creating an engineered myocardial patch should be easy to harvest, proliferative, nonimmunogenic, and resistant to ischemia (after transplantation) and have the ability to differentiate into mature, functional cardiomyocyte. Unfortunately, no such cell currently exists. Several cell sources have been proposed (Table 1.1). Donor (allogenic) cells are relatively easier to obtain but entail risky immunosuppression. Autologous cells, however, are more difficult to obtain and to expand but have no immunologic barriers.

Table 1.4 describes the advantages and limitations of various cell sources. Theoretically, the natural electrophysiologic, structural, and contractile properties of cardiomyocytes make them the ideal donor cell type. However, cardiomyocytes are difficult to obtain and to expand, are sensitive to ischemic insults, and are allogenic, e.g., will evoke immune response in the host tissue. Thus, researchers are seeking alternative cells. Although human embryonic stem cells have been shown to have the potential to turn into cardiomyocytes,³¹no studies have demonstrated the controlled differentiation into uniform cell type.

Furthermore, unless they are derived from somatic-cell nuclear transfer, human embryonic stem cells will be rejected by a recipient.

Today, the most widely used cell types for cardiac cell therapy in human patients are skeletal

Table 1.3. Biomaterial for myocardial tissue engineering

References Natural

Porous alginate scaffolds Leor et al.,⁷⁴2000

Alginate-gelatin-PEG scaffolds Chandy et al.,¹⁰¹2003

Gelatin scaffolds Akhyari et al.,⁶⁸2002

Collagen scaffolds Zimmermann et al.,⁴⁷2002

Matrigel Zimmermann et al.⁴⁷; Kofidis et al.,⁹⁰2004; Radisic et al.,⁶⁰2004

Fibrin Christman et al.,¹⁰²2004; Ryu et al.,⁹³2005

Synthetic

PLA-PGA Stock and Mayer,¹⁰³2001

Poly-L-lactide-gelatin-PGA Ozawa et al.,¹⁰⁴2002; McDevitt et al.,⁶⁵2003 Electrically conducting membrane layers composed of PGA, gelatin, Shimizu et al.,⁴⁸2002

alginate, and/or collagen

ε-Caprolactone-co-L-lactide Matsubayashi et al.,⁷⁸2003

Polyurethanes McDevitt et al.,⁶⁵2003

TMC-co-ε-caprolactone-co-D,L-lactide Pego et al.,¹⁰⁵2003 Source: Adapted from Zammaretti and Jaconi.¹⁰⁶

PEG, polyethylene glycol; PLA, polylactic acid; PGA, polyglycolic acid; TMC, 1,3-trimethylene carbonate.

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muscle-derived progenitors, or myoblasts, and crude bone marrow mononuclear cells.¹⁰ Both cell types share advantages over other cells proposed for cardiac repair in that they are readily available, autologous, and easily expanded in vitro. A limitation of myoblasts is their appar- ent inability to transdifferentiate into cardiac or endothelial cells. In contrast, bone marrow- derived stem cells are currently gaining favor because of their seeming plasticity, which could allow them to alter their phenotype in response to cues from the target organ, and the possibility of using the patient’s own cells. A few recent clinical studies advocate the simple injection of unfractionated autologous bone marrow cells in patients with acute myocardial infarction.¹⁰ However, because such studies have been performed relatively early after the ischemic insult, their relevance to chronically infarcted myocardium remains uncertain.¹⁰

The use of autologous adult stem cells is partic- ularly restricted by their low recovery from bone marrow, fat, or circulation of elderly patients, and, therefore, it is difficult to obtain in reasonable numbers of suitable cells.³²In addition, progenitor cells from sick patients, such as type II diabet- ics, exhibit impaired proliferation, adhesion, and incorporation into vascular structures.^32–34 Furthermore, cell sources are limited by several aspects that are relevant to clinical implica- tions.^35,36 For example, safety issues have been raised regarding the use of various cells for myocardial repair: arrhythmias with skeletal myoblasts,³⁷cardiac calcifications with bone marrow mononuclear cells,³⁸ myocardial scarring with mesenchymal stem cells,³⁹and teratoma with

human embryonic stem cells.⁴⁰ In addition, the search continues for an efficient and reproducible method to control and direct differentiation of stem cells to the desired cell type in vitro.^41,42

Engineering Beating Construct In Vitro

Zimmermann et al.⁴³ proposed certain criteria for cardiac tissue construct. The constructs should be (1) contractile, (2) electrophysiologi- cally stable, (3) mechanically robust yet flexible, (4) vascularized or at least quickly vascularized after implantation, and (5) nonimmunogenic.

Today, such construct does not exist. Thus, these ambitious criteria illustrate the difficulties in engineering functional myocardial graft.^44,45

A number of groups reported encouraging results with various techniques for constructing cardiac graft for transplantation.^46–60 They showed that neonatal rat or chick embryo cardiomyocytes can be reconstituted to 3-D myocardial tissue-like constructs. For example, we have shown that cardiomyocyte seeding within porous alginate scaffolds yielded 3-D high-density cardiac constructs with a uniform cell distribution. The hydrophilic nature of the alginate scaffold, its more than 90% porosity, and interconnected pore structure, enabled efficient cell seeding onto the scaffold within a short time, up to 30 minutes. With the aid of a moder- ate centrifugal force during cell seeding, a uniform cell distribution throughout the alginate

Table 1.4. Advantages and limitations of various cell sources for myocardial tissue engineering

Easily Highly Cardiac Clinical

Autologous obtainable expandable myogenesis experience Safety concerns

Fetal cardiomyocytes No No No Yes No No

Embryonic stem cells No No Yes Yes No Yes, teratoma

Skeletal myoblasts Yes Yes Depend on age Debated Yes Yes, arrhythmias

Crude bone marrow cells Yes Yes Depend on age Debated Yes Yes, calcification

Mesenchymal stem cells Yes No Depend on age Yes No Yes, fibrosis

Hematopoietic stem cells Yes Yes Depend on age Debated Yes No

Fibroblasts Yes Yes Depend on age No No No

Smooth muscle cells Yes Yes Yes No No Unknown

Cardiac progenitors Yes No Unknown Yes No Unknown

Source: Adapted from Leor J, Amsalem Y, Cohen S. Cells, scaffolds, and molecules for myocardial tissue engineering. Pharmacol Ther 2005;105:151–163.

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scaffolds was achieved, consequently enabling the loading of many cardiomyocytes onto the 3- D scaffolds.⁵⁰

As an alternative to seeding the cells on a pre- formed scaffold, Zimmermann et al. utilized Matrigel mixed with collagen gel.43–47,61,62 The cells were mixed with the liquid material, which was solidified by casting in a cylindrical tem- plate. After a few days, they moved the tissue patch to a stretching device that simulated the heart’s contractions. They demonstrated that collagen type I and ECM proteins when mixed with freshly isolated heart cells join together to a strongly contracting and highly differentiated construct, which they named engineered heart tissue (EHT). The geometric shape of EHT could be altered by utilization of suitable casting molds (square, circular).

An alternative approach has been proposed by Shimizu et al.⁴⁸They grew rat cardiomyocytes on a thin temperature-responsive polymer, PIPAAm [poly(N-isopropylacrylamide)]. The polymer sheet promoted the thin cell layers to detach when the temperature was reduced, thus releasing cardiac myocyte sheets from the dishes without enzymatic or ethylenediaminete- traacetic acid treatment. The researchers laid four of these sheets on top of each other until they fused, and the product was implanted under the skin of rats. Six months later, the researchers observed that the engineered cardiac patches were beating and had been infiltrated by blood vessels. One of the potential advantages of this strategy is the ability to stack other necessary cell sheets between cardiomyocyte sheets as endothelial cells in attempt to cope with the perfusion limitation in thick constructs.

Another technique that may accelerate and optimize engineered myocardial assembly is

“organ printing.”⁶³ A cell printer to print gels, single cells, and cell aggregates has been developed. Layer-by-layer sequentially placed and solidified thin layers of a thermo-reversible gel served as “printing paper.” This computer- aided, jet-based 3-D tissue engineering of living human organs suggests a new strategy for growing a patch of cardiac muscle.⁶³

To achieve better control over the morphology and architecture of engineered constructs, researchers used several modifications. McDevitt et al.⁶⁴laid lanes of laminin, 5–50 micron wide, by microcontact printing onto nonadhesive (bovine serum albumin-coated) surfaces. Adherent cardiomyocytes responded to the spatial constraints

by forming elongated, rod-shaped cells whose myofibrils aligned parallel to the laminin lanes.

Similar cardiomyocyte patterns were achieved on micropatterned biodegradable polymer polylactic-co-glycolic acid, suggesting that patterned cardiomyocytes could be used in myocardial tissue engineering.⁶⁵

Radisic et al.⁵⁹ applied electrical signals, designed to mimic those in the native heart, on cardiac constructs in vitro. After 8 days, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchro- nous construct contractions, and resulted in a remarkable ultrastructural organization.⁵⁹ In another study, they used an in vitro culture system that maintains efficient oxygen supply to the cells at all times during cell seeding and construct cultivation attempting to mimic convec- tive-diffusive oxygen transport present in vivo.

Perfusion resulted in significantly higher numbers of live cells, higher cell viability, and significantly more cells in the S phase compared with dish-grown constructs.⁶⁰

The results of these pioneering experiments provide tools to investigate myocardial physiol- ogy, development, and pharmacology ex vivo. In addition, they raise hope for the use of myocardial tissue engineering to repair or replace the infarcted myocardium. Theoretically, the bioengineered cardiac tissue could be used for surgical reconstruction of the infarcted myocardium or repair of congenital cardiac defects.⁶⁶

Bioreactors

One of the major difficulties in cardiac tissue engineering is how to grow 3-D structures that contain more than a few layers of muscle cells.

To improve the results of in vitro tissue engineering, researchers have designed several bioreactors, which portray different patterns of fluid dynamics and vessel geometry. A basic fluid-dynamic cultivation vessel is the spinner flask, which is an agitated flask usually at 50 rpm.^51,53 In these vessels, the cell constructs are subjected to turbulently mixed fluid that provides a well-mixed environment around the cell constructs and minimizes the stagnant layer at their surface. It has been shown that cultivation of cardiac cell constructs in spinner flasks produces engineered tissues that are superior, in almost every aspect (e.g., aerobic

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cell metabolism, DNA content, metabolic activity, and morphologic appearance) to tissues cultivated under static conditions.^51,53,67 The spinner flask may not, however, be the optimal cultivation vessel for cardiac cells. The turbulent fluid flow at the surface of the constructs is usually characterized by eddies that destroy the seeded cells.

Bioreactors combined with mechanical signals, such as under stretching or compression modes, improved the proliferation and distribution of the seeded human heart cells throughout the scaffold volume and further stimulated the formation and organization of the ECM – all of which contributed to the improvement in the mechanical strength of the cardiac graft.^46,47,68Future bioreactors for cardiac tissue engineering should combine both perfusion and mechanical stimuli, for example, by allowing for adjustable pulsatile flow and varying levels of pressure. Such bioreactors are currently under development for engineering heart valves ex vivo.^69,70

These encouraging achievements still face significant difficulties. Most bioreactors cannot supply enough nutrients and oxygen to a growing thick tissue. Whereas adult heart muscle is more than 1 cm thick, growth in a bioreactor typically stops once the tissue is about 100 µm, or less than 10 cell layers thick.⁷¹ Beyond this thickness, the innermost cells are too far from the supply of fresh growth medium to thrive.

Furthermore, after transplantation, rapid vascularization, adequate perfusion, cell survival, integration, and function of the engineered cardiac patch remain critical steps in the transla- tion of in vitro achievements into effective therapeutic tools.^44,72

Transplantation of Engineered Myocardial Construct

In their pioneering study, Li et al.⁷³reported that bioengineered cardiac grafts can be made of fetal cardiac cells and 3-D gelatin mesh. The cells in the graft formed cardiac-like tissue and contracted spontaneously. However, after transplantation on infarcted myocardium of rat, compared with a control, LV-developed pressure was lower in hearts into which either a cell- seeded or unseeded graft had been implanted.

The authors proposed that inappropriate sizing

of the grafts interferes with the contractility of the viable myocardium.⁷³

We reported successful seeding of rat fetal cardiomyocytes into porous scaffolds composed of alginate sponges.⁷⁴ We found that the seeded fetal cardiac cells retained viability within the scaffolds and within 24 hours formed multicellu- lar beating cell clusters. After implantation of the cellular constructs into the infarcted myocardium, some of the cells appeared to differentiate into mature myocardial fibers. The implanted cardiac grafts were supplied by inten- sive neovascularization, which evidently contributed to the survival of the cells in the grafts.

The biografts attenuated LV dilatation and dete- rioration of heart function. The mechanism behind this beneficial effect remains unclear. A direct contribution of the biograft to contractility is unlikely because only a relatively small frac- tion of the biograft was composed of myocardial tissue. Attenuation of infarct expansion by virtue of the elastic properties of bioartificial grafts is possible. Restraining the expansion of the left ventricle by a mesh placed over the infarcted myocardium, preserves left ventricle geometry and resting function in a sheep model of myocardial infarction⁷⁵ and has now tested in clinical trial.⁷⁶ Angiogenesis induced by growth factors secreted from the seeded cells, resulting in improved collateral flow and augmentation of contractility, is also a possible mechanism.⁷⁷

Zimmermann et al.⁴⁶ created EHT by mixing cardiac myocytes from neonatal Fischer 344 rats with liquid collagen type I, Matrigel, and serum- containing culture medium. EHTs were designed in circular shapes to fit around the circumference of hearts from syngeneic rats. After 12 days in culture, they were implanted on uninjured hearts.

Fourteen days after implantation, EHTs were heavily vascularized and retained a well-organ- ized heart muscle structure as indicated by immunolabeling of actinin, connexin 43, and cad- herins. Ultrastructural analysis demonstrated that implanted EHTs surpassed the degree of differentiation reached before implantation. Contractile function of EHT grafts was preserved in vivo, but, compared with baseline values, did not improve LV function as indicated by serial echocardiogra- phy studies.⁴⁶ In addition, the transplantation results were limited by immune response of the host animal against the biomaterial mixture and the need for continuous immunosuppression.⁴⁶

In another study, Matsubayashi et al.⁷⁸showed that surgical repair with smooth muscle cell-seeded

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grafts reduced abnormal chamber distensibility and improved LV function after myocardial infarction as compared with unseeded grafts. The authors proposed that bioengineered muscle grafts may be superior to synthetic materials for the surgical repair of LV scar.⁷⁸

Construct Vascularization

The 3-D cell constructs that are developed ex vivo usually lack the vascular network that exists in normal tissues. One of the most important requirements from a tissue engineering scaffold is its ability to support vascular infiltration.⁷⁹ Implanted cardiomyocytes are very sensitive to prolonged ischemia and may die by necrosis and apoptosis. Thus, to become clinically relevant, a myocardial tissue engineered graft requires per- sistent neovascularization, or angiogenesis, for its growth and survival. The extent of angiogenesis is determined by the regulating molecules that grafted cells and host cells release into the microenvironment of the engineered tissue.

Recent advances in our understanding of the process of blood vessel growth have provided significant tools for the neovascularization of bioengineered tissues. Several growth factors serve as stimuli for endothelial cell proliferation and migration as well as the formation of new blood vessels. Vascular epithelial growth factor (VEGF) is a major regulator of neovascularization. VEGF has a major role in the early development of blood cell progenitors.^80,81 Basic fibroblast growth factor (bFGF) is a potent inducer of endothelial cell proliferation and blood vessel growth in vitro and in vivo. VEGF and bFGF have been injected into under-vascu-

larized ischemic myocardial tissues, resulting in new blood vessel formation and tissue perfusion.^80,81Additional potential therapeutic angiogenic factors are listed in Table 1.5.

Site-specific delivery of angiogenic growth factors from tissue engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would enhance cell survival and function. Local growth factors delivery will avoid serious adverse effects such as hyperpermeability, edema, hypotension, and accelerated atherosclerosis.⁸² Angiogenic factors have been incorporated into bioengineered tissues and have facilitated blood vessel growth.^83–85Richardson et al.⁸³moved one step forward by creating a new polymeric system that delivers two or more growth factors, with controlled dose and rate of delivery. The utility of this system was investigated in the context of therapeutic angiogenesis. They showed that dual delivery of VEGF-165 and platelet-derived growth factor-BB, each with distinct kinetics, from a single, structural polymer scaffold results in the rapid formation of a mature vascular network.⁸³

Other approaches such as prevascularization of the implanted scaffold before cell seeding⁸⁶ and incorporation of endothelial cells into the bioengineered tissues have produced encouraging results⁸⁷and could be applied to myocardial tissue engineering.

In Situ Tissue Engineering

Although in vitro tissue engineering to create an engineered muscle patch in a bioreactor is fasci- nating and exciting, it faces significant difficulties, such as constructing significant cardiac

Table 1.5. Bioactive molecules to enhance self-repair, neoangiogenesis, and regeneration in animal models Stem cell

mobilization or

Factor recruitment Myogenesis Angiogenesis Anti-apoptosis

Erythropoietin (EPO)¹⁰⁷ Yes No Yes Yes

Granulocyte colony-stimulating factor (G-CSF)^11,108 Yes No Yes No

Hepatocyte growth factor (HGF)^109,110 Yes Yes Yes Yes

Insulin-like growth factor (IGF-1)¹¹¹ Yes Yes Yes Yes

Leukemia inhibitory factor (LIF)¹¹² Yes Yes Yes Yes

Stromal-derived growth factor (SDF-1)¹¹³ Yes No Yes Yes

Thymosin β4¹¹⁴ Yes Yes Yes Yes

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muscle from scaffold and cells in vitro, and poor graft survival. An alternative to the in vitro tissue engineering is the in situ tissue engineering approach. In this approach, unseeded alginate scaffolds are implanted on the damaged myocardium and, after their vascularization, they create a friendly environment and space for the implanted cells. To accelerate angiogenesis and engraftment, the implanted scaffold may be impregnated with bioactive molecules that improve viability and survival and may enhance stem cell homing and self-repair.

This strategy could be enhanced by bioactive materials (Table 1.3). With this approach, the biomaterial itself or its degradation/dissolution products are used to stimulate local tissue repair.

Bioactive materials release chemicals in the form of ionic dissolution products, or growth factors, at controlled rates, by diffusion or network breakdown, that activate the cells in contact with the stimuli. The cells produce additional growth factors that in turn stimulate multiple genera- tions of growing cells to self-assemble into tissues in situ along the biochemical and biomechanical gradients that are present. These materials, once implanted, will help the body heal itself.⁸⁸

Molecular modifications of the biomaterial are intend to elicit specific interactions with cell integrins and thereby direct cell proliferation, differentiation, and ECM production and organization. The mechanism for in situ tissue regeneration involves up-regulation of genes that control the cell cycle, mitosis, and differentiation. Gene activation by controlled ion release provides the conceptual basis for molecular design of a third generation of biomaterials optimized for in situ tissue regeneration.⁸⁶

In situ regeneration in the injured myocardium can be enhanced by direct delivery of several cytokines that potentially stimulate myocardial healing and repair in the setting of myocardial infarction (Table 1.5). Those cytokines may induce recruitment of stem/progenitor cells into the healing infarct, which may differentiate into endothelial cells and even lead to myocardial regeneration.

Injectable Tissue Engineering

Most of the efforts in cardiac tissue engineering focus on the use of implantable scaffolds that

deliver cells to the epicardial surface. However, many strategies of cell or gene delivery to repair the infarcted myocardium are shifting toward a catheter-based approach. This semi-invasive approach avoids the risk of open chest surgery and anesthetics and is favored by both patients and physicians. The injectable scaffold facili- tates repair after infarction by providing a matrix support within which cells are retained, migrate, and neoangiogenesis takes place.^89,90 Several works suggest that injectable biomaterials can serve as a cell implantation matrix that enhances neovascularization and repair of the infarcted myocardium. An important advantage of this concept of in situ tissue engineering is its feasibility for a catheter-based approach and to avoid the need for surgical thoracotomy.

We have recently presented preliminary data that show that injection of biodegradable alginate solution into the infarcted myocardium stimulates neoangiogenesis and efficiently attenuates infarct expansion, heart dilatation, and dysfunction.⁴⁵ Our preliminary work provides a minimally invasive, catheter-based, acel- lular option to facilitate neovascularization, self-repair, and rejuvenation of the infarcted myocardium. The injectable bioactive material proposes a viable solution to the difficulties in achieving appropriate cells to treat myocardial infarction and a future strategy of catheter- based injectable tissue engineering.

Summary and Future Perspectives

The ability to engineer or regenerate lost myocardial tissue caused by injury, aging, disease, or genetic abnormality holds great promise. The vision is to generate significant mass of functional heart muscle tissue. However, the area of myocardial tissue engineering still faces significant difficulties. Scientists are still searching for cell types other than cardiomyocytes. Novel approaches are warranted for material processing to create bioactive scaffolds, which would allow composition of the evolving myocardial structure. There is a need for development of strategies to promote vascularization and/or innervations within engineered myocardial tissue. Other important goals include achievement of immunologic tol- erance for engineered constructs and increased understanding of the basic principles governing

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tissue formation, function, and failure, including the assembly of multiple cell types and biomaterials into multidimensional structures that mimic the architecture and function of native myocardial tissue.

In addition to laboratory-grown myocardial tissue, more research is warranted in the area of cardiac self-repair and regenerating functional myocardium in situ. If successful, these strategies could be used for surgical repair of the infarcted myocardium or congenital cardiac defects and would have a dramatic impact on the future of cardiovascular medicine and public health.

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