Reviews - Circulation Researchcircres.ahajournals.org/content/circresaha/97/12/1220.full.pdf · create 3-dimensional heart muscle equivalents that may not only serve as experimental

This Review is part of a thematic series on Cardiovascular Tissue Engineering, which includes the followingarticles:

Custom Design of the Cardiac Microenvironment with BiomaterialsHeart Valve Tissue Engineering

Engineering Myocardial Tissue

Small-Diameter Artificial Arteries Engineered In Vitro

Regenerative Cardiomyocytes for Cardiovascular Tissue Engineering Richard T. Lee, Guest Editor

Engineering Myocardial TissueThomas Eschenhagen, Wolfram H. Zimmermann

Abstract—To create an artificial heart is one of the most ambitious dreams of the young field of tissue engineering, a dreamthat, when publicly announced in 1999 (LIFE initiative around M. Sefton), provoked as much compassion as scepticismin the scientific and lay press. Today, it is fair to state that the field is still far away from having built the “bioartificialheart.” Nevertheless, substantial progress has been made over the past 10 years, and a realistic perspective exists tocreate 3-dimensional heart muscle equivalents that may not only serve as experimental models but could also be usefulfor cardiac regeneration. (Circ Res. 2005;97:1220-1231.)

Key Words: tissue engineering � heart � stem cells � regeneration

The term “Tissue Engineering” was introduced in 1987 bymembers of the US National Science Foundation (NSF)

in Washington, D.C. and defined a year later at an NSF-organized conference on tissue engineering in Lake Tahoe,California as “Application of principles and methods ofengineering and life sciences toward fundamental under-standing of structure–function relationship in normal andpathological mammalian tissues and the development ofbiological substitutes to restore, maintain, or improvefunctions.”

The “repair” part of tissue engineering overlaps, but is notsynonymous with, “cell therapy” which intends to promotethe formation of new tissue or to improve the function of anexisting tissue by injecting or infusing suspensions of isolatedcells. This concept has gained much attraction over the pastyears and is currently tested in controlled clinical trials (forreview see references 1–6). Tissue engineering aims at gener-ating functional 3D tissues outside of the body that can bytailored in size, shape, and function according to the respec-tive needs before implanting them into the body. First clinicalexperiences have been published using bioengineered skin,cartilage, and vascular grafts,7–9 but the present data are still

preliminary (for review of the “bioartificial heart,” see alsoref 10). This review will give an overview on the evolution ofcardiac tissue engineering and today’s state-of-the art con-cepts. Tissue engineering heart valves and blood vessels willnot be covered here because these are topics of other reviewsin this Series.

History of Cardiac Tissue EngineeringThe spontaneous beating of heart tissue explants and theircellular outgrowth when placed in culture dishes undersuitable conditions have fascinated researchers for genera-tions.11–15 Cardiac tissue engineering has several roots: de-velopmental biology, cardiac muscle cell biology, and mate-rial science. The first man-made 3D heart tissues have beengenerated long before the term tissue engineering was intro-duced.11–15 In the late 1950s, Moscona generated spheroidaggregates from embryonic chick heart cells by cultivatingfreshly isolated cells in Erlenmeyer flasks under continuousgyration (Figure 1A).16 After 18 hours in this simple biore-actor aggregates formed containing up to 200 cells. Manyresearchers adapted Moscona’s model and found that theaggregates were functionally more similar to intact heart

Original received July 5, 2005; revision received September 20, 2005; accepted November 3, 2005.From the Institute of Experimental and Clinical Pharmacology, University Medical Center Hamburg-Eppendorf, Germany.Correspondence to Thomas Eschenhagen, Martinistrasse 52, D-20246 Hamburg, Germany. E-mail [email protected]© 2005 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000196562.73231.7d

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tissue than standard 2D-monolayer cultures (Figure 1A).17

This early work proved that isolated cells from immaturehearts retain the capacity to reform heart-like tissues undercell culture conditions.

Another fascinating finding supports the notion that car-diac myocytes have an inherent preference to aggregate.When cultured at high density for extended time periods inserum-containing culture medium researchers often see thatentire rhythmically contracting monolayers start to detachfrom the culture surface forming spontaneously beatingcardiac sheets floating in the dish. Because these sheets,devoid of mechanical load, quickly retract and stop to beat,the detachment has been considered a shortcoming of cellculture experiments rather than an exploitable feature untilShimizu and colleagues have used free floating monolayer

sheets to generate essentially exogenous matrix free cardiactissue constructs.18 In fact, in the late 1980s, Vandenburghand colleagues searched for a solution to overcome thedetachment problem of differentiated skeletal myotubes(showing a similar contractile behavior as cardiac myocytes)and came up with the idea to cover the cultured myotubeswith a layer of freshly neutralized collagen type I to chroni-cally impose load on the cells.19 This led to an improveddifferentiation state of the myotubes. Similar observationshad actually been made in various other cell types before,either by placing collagen on top of or below cellularmonolayers or by embedding cells in collagen gels. Forexample, epithelial thyroid cells cultured in collagen orga-nized into follicles,20 and kidney and mammary gland epithe-lial cell lines formed lumina resembling those of the tissues

Figure 1. Strategies to generate 3D striated muscle tissues in vitro. A, Phase contrast microscopic view of aggregates generated bygyratory shaking of embryonic chick heart cells in an Erlenmeyer flask.16 B, Macroscopic view of a device to stretch skeletal myocytemonolayers to generate thin strips of 3D muscle (top) and immunostaining of myosin heavy chain (bottom).23 C, Scheme of a method togenerate fibroblast-populated collagen matrices from chick fibroblasts.30 D, Macroscopic (top) and microscopic view (bottom) of syn-chronously beating and force developing cardiac myocyte-populated collagen matrices anchored between two Velcro-coated glasstubes.31 E, Scheme to prepare and culture polyglycolic acid-based cardiac constructs in a bioreactor (top) and microphotograph (bot-tom) of construct.37 F, Electron microscopic photograph of alginate matrix (top) and microphotograph (bottom) of an H&E stainedalginate-based cardiac construct.39 G, Macroscopic view of the setup to cast, culture and phasically stretch engineered heart tissue(EHT) from neonatal rat heart cells and to determine contractile force (top and middle) and microphotograph of H&E stained EHT (bot-tom).35 H, Macroscopic (top) and microscopic view (H&E stain bottom) of stacked monolayers forming a beating 3D tissue.18 I, Tissueformation (H&E stain, top) and sarcomere structure (bottom) in electrically stimulated collagen�matrigel-based constructs from neona-tal rat heart cells.42

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from which they were derived.21 Collectively, these datanicely demonstrate that the 3D environment in a collagen gelpromotes tissue formation and cellular differentiation ofvarious cell types in vitro.

Besides cell composition and differentiation, tissues arealso defined by cell orientation. In this respect, two factorshave been shown to be important: Mechanical strain andorientation of the growth substratum. The groups of Vanden-burgh and Terracio independently designed motorizedcomputer-controlled devices to impose stretch on culturedskeletal and cardiac myocytes and showed that mechanicalstimulation had a favorable effect on muscle cell orientationand differentiation.19,22 When repetitive cycles of stretch andrelaxation were imposed on skeletal myotubes cultured atvery high density, thin, 3D strips of longitudinally alignedand well developed skeletal myofibers developed.23The stripseven initiated tendon development and performed work inresponse to depolarization, and therefore may be consideredto be the first engineered 3D skeletal muscle (Figure 1B). Animportant conclusion from this work was that mechanicalload is crucial for the orientation and differentiation of(skeletal) muscle cells and muscle tissue development.

Others showed that cell growth can be influenced by theunderlying matrix substrate. For example, when a scraper wasdrawn across the surface of culture dishes coated with freshlyneutralized collagen type I before gelation, Simpson andcolleagues observed that neonatal rat cardiac myocytesaligned themselves in parallel with the collagen and adopteda tissue-like organization.24 High magnetic fields have beenused as an alternative means to orient collagen,25 andTranquillo and colleagues showed that this method can beused to improve longitudinal orientation of fibroblasts26 andsmooth muscle cells.27 Others used photolithographic pattern-ing of culture surfaces to govern the growth of cardiacmyocytes in specific patterns28 or biodegradable elastomericpolyurethane films patterned by microcontact printing withlaminin lanes.29

Another approach to cardiac tissue engineering originatedin the search for an improved in vitro heart model for targetvalidation that would allow both the measurement of contrac-tile force and genetic, pharmacological, and mechanicalmanipulations under controlled conditions in vitro. The solu-tion was an adaptation of a method previously developed forembryonic fibroblasts by Kolodney and Elson in St. Louis(Figure 1C).30 Essentially, cardiac myocytes were cultured incollagen gels as described above,19–21 but between twoVelcro covered glass tubes that were positioned in a rectan-gular well and held at a fixed distance with a metal spacer.Under these culture conditions the cells formed spontane-ously contracting biconcaval lattices that were anchored onthe Velcro covered glass tubes (Figure 1D) and could beattached to mechanical force transducers to record contractileforce.31 Neonatal rat cardiac myocytes initially failed toexhibit differentiation, growth, and tissue formation insidecollagen I gels as described previously.32 Only after additionof extracellular matrix from Engelbrecht Swarm tumors(Matrigel) to the initial reconstitution mix, engineered hearttissue (EHT) could be generated also from rat heart cells.33

Interestingly, chronic cyclic stretch of the EHTs improved

contractile force by a factor of 3 and induced better cardiactissue development.34 A further improvement was the intro-duction of circular casting molds that (1) allow large scaleproduction with minimal handling and are reusable, (2) canbe easily miniaturized for high through-put screening, and (3)lead to better tissue formation than the original lattice designbecause the circular geometry causes a homogeneous forcedistribution throughout the tissue (Figure 1G).35 A mainconclusion from these observations is that, similar to what hasbeen found before by Vandenburgh and Terracio and col-leagues, mechanical load is crucial for a proper heart tissuedevelopment.

Two other, principally different tissue engineering ap-proaches were developed in parallel. One originated fromthe material sciences field and can be viewed as anadaptation of “classical” tissue engineering principles36 tocardiac cells. One of the pioneering groups (Vunjak-Novakovic and colleagues at the MIT in Cambridge, MA)initially used polyglycolic acid as a scaffold in combina-tion with bioreactor cultures (Figure 1E).37 Alternatively,the group of Li seeded fetal rat ventricular cells ontogelatin scaffolds, cultured them for 7 days in vitro andimplanted them onto cryo-infarcted rat hearts.38 Spontane-ous contractile activity, both in vitro and in vivo, wasreported, but the histological microphotographs showedmainly cells of unknown identity embedded in the scaffoldmaterial and poor sarcomere development in the fewcardiac myocytes present in the construct. Leor and col-leagues generated alginate scaffolds seeded with fetalcardiac cells and implanted these graft onto rat hearts(Figure 1F).39 The constructs were highly vascularized, butdid not exhibit true integration into the host myocardium.The conceptionally important advantage of the classicaltissue engineering approach using preformed matrices ascompared with the liquid matrix cell entrapment approachis that technically fabricated, solid materials can be shapedin any 3D geometric form, on a macroscopic, but also, atleast in theory, at a microscopic level. Thus, one couldfabricate the ideal patch by computer design and then seedit with cardiac myocytes and potentially other cell types,hoping that they would use the matrix as a scaffold togenerate a tissue similar to the native heart. In essence, thisstrategy either uses the natural organ as a blueprint(exemplified in the famous “ear-mouse”40) or decellular-ized grafts that are already successfully utilized in clinicaluse such as porcine heart valves. One could term this latterapproach “biologization” of tissue grafts. The great prom-ise of these strategies with regard to cardiac muscle tissueengineering remains unproven, so far. Limitations includeunfavourable matrix properties such as limited diffusioncapacity, low mechanical compliance, liberation of poten-tially toxic substances during degradation, and incompat-ibility with physiological cell growth. Essentially, cardiacmyocytes appear to survive for some time and start to beatinside such artificial scaffolds, but remain largely isolatedand do not form coherently beating cardiac tissue. Newmaterials, the molecular refinement of surfaces and cre-ation of microstructures that promote spreading of cellsmay offer solutions to this problem (review in reference

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41). Alternatively, Vunjak-Novakovic and colleagues suc-cessfully developed a somewhat mixed approach by seed-ing preformed collagen sponges/foam with neonatal ratheart cells suspended in Matrigel together with electricalstimulation for extended times.42 This led to the generationof cardiac muscle constructs with improved cardiac tissuemorphology, contractile function, and molecular markerexpression when compared with nonstimulated cultures(Figure 1I). Hence, electric stimulation seems to induce asimilar degree of cardiac myocyte differentiation as me-chanically stimulation.34,35 Whether electrical activity perse is a stimulus for advanced differentiation and tissueformation or the resulting contractile activity is not clearand may be difficult to experimentally separate. Yet, thework by Radisic et al also supports the notion thatbiological extracellular matrices mainly composed of col-lagen and laminin (present in high concentration in matri-gel) are presently the optimal scaffold material in cardiactissue engineering.

The third approach can be termed tissue engineeringwithout matrix and is a refinement and systematization ofthe phenomenon of detachment of cellular monolayersafter prolonged culture time (see above). Shimizu andcolleagues used a thermo-sensitive surface coating forculture dishes which allows cardiac myocytes (or any othercell type) to attach at 37°C as in other normal dishes, butto detach under controlled conditions at room temperatureas an intact sheet.18 These beating cardiac myocyte mono-layers can be stacked on one another and grown together toform interconnected 3D tissue sheets of up to 50 to 75 �mthickness (Figure 1H). The advantages of this techniqueare its relative ease and the independence from potentiallyimmunogenic or pathogenic scaffold materials. Disadvan-tages are its fragility (leading to handling problems),restriction in terms of geometric form and difficulties toimpose mechanical load on the contractile sheets.

Other studies have described variations of the abovetechniques. One report described generation of coherentlybeating aggregates from neonatal rat heart cells by exposingcell suspensions with fibronectin coated polystyrene micro-carrier beads or oriented collagen fibers to microgravity inbioreactor cultures.43 Other studies seeded commerciallyavailable collagen sponges with neonatal rat heart cells aloneand observed cardiac tissue formation.44 Van Luyn andcolleagues experimented with liquid collagen type I matricesand used a bioreactor to improve tissue formation.45 Thegroup of Dennis developed a technique that allows sponta-neously detaching heart cell monolayers to form cylindricaltissues spanning between two laminin-coated silk sutures.45a

Finally, another strategy may be to create new cardiac tissueby injecting liquid, “cardiogenic” cell-matrix mixtures intothe heart, either with or without cells.46 This “tissue engineer-ing in situ” is based on the premise that suitable matricesprovide appropriate signals for cardiac progenitors such asc-kit,47 sca-1,48 and isl-149 positive cells to proliferate andform new cardiac tissue. The idea is interesting, but has to beproven more rigorously.

Taken together, various techniques have been developedover the past 10 years that allow engineering of beating 3D

cardiac tissues. Such tissues develop force and can be formedat different shapes and sizes in a directed manner.

How Far Has Cardiac Tissue EngineeringGone and What Are the Present Limitations?

Tissue DevelopmentIt has been questioned whether the term “tissue” may actuallybe used for engineered myocardial constructs. This argumentis based on the classical definition of a “tissue” as being “apart of an organism consisting of an aggregate of cells havinga similar structure and function.” Yet other widely useddefinitions state that a tissue is “a group of similar cells unitedto perform a specific function” or “a grouping of cells that aresimilarly characterized by their structure and function,” adefinition that applies to engineered tissue constructs. In fact,use of stretch34 or electrical stimulation42 and mixed popula-tions of heart cells including cardiac myocytes, fibroblasts,smooth muscle cells, endothelial cells, macrophages amongothers35 in cardiac tissue engineering leads to the formation ofheart muscle constructs with a high degree of organotypicdifferentiation on the single cell level and also a high nativetissue-like cellular complexity. The cellular complexity canbe further enhanced by using unpurified heart cell popula-tions instead of purified cardiac myocytes.50 Cardiac myo-cytes themselves showed many structural features of terminaldifferentiation including well-developed sarcomeres, desmo-somes, gap junctions and fasciae adherentes. Interestingly,T-tubule-sarcoplasmic reticulum junctions were found regu-larly that are not or only rarely observed in neonatal rathearts.51 Interestingly, EHTs developed approximately two-fold higher contractile force if generated from an unpurifiedcell preparation than from a relatively pure cardiac myocytefraction.50 Similarly, endothelial cells can promote cardiacmyocyte survival and spatial organization when coculturedon hydrogels.52 These in vitro data are well in line with theestablished role of the endocardial endothelium for normalcardiac development,53,54 where disruption of normal endo-thelial cell function led to severe cardiac malformation.Collectively, these data suggest that endothelial cells, smoothmuscle cells, fibroblasts, macrophages and other noncardio-myocytes play an important or even essential role in theformation of engineered cardiac tissue grafts.

Important structural characteristics of cardiac myocyteterminal differentiation are sarcomere organization in registrywith a cross-striation pattern including M-bands,55 formationof cell-cell contacts through desmosomes, gap junctions, andfasciae adherents, as well as development of T-tubule-sarcoplasmic reticulum junctions (dyads) at the Z-band level.These features have been observed in engineered cardiacconstructs that have been chronically stretched35 or electri-cally stimulated42 indicating that these conditions promoteterminal cardiac differentiation. Interestingly, M-band forma-tion was found to be enhanced after engraftment of EHTsindicating that additional differentiation factors are providedin the intact adult heart that are lacking in vitro.56

Thus, critical factors identified today for cardiac tissuedevelopment in vitro are (1) a suitable biological matrix suchas collagen I, IV and laminin (which, in case of the sandwich




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technique of Shimizu, may be entirely cell-derived), (2)mechanical load, (3) a total heart cell mix, and (4) eitherelectrically stimulated or spontaneous contractile activity. Insummary, the various techniques presently used generateconstructs with a very high degree of cardiac tissue develop-ment (Figure 1E, 1H, and 1I).

Contractile FunctionAlso functionally, artificial heart constructs resemble intactheart tissues in terms of force-frequency behavior, force-length relationship (Frank-Starling mechanism), response toextracellular calcium and the �-adrenergic agonist isoprena-line as well as its antagonism by acetylcholine-derivatives.31,33 These data support the above conclusion oftrue heart tissue-development in vitro from a functional pointof view. However, differences exist as well. For example,EHTs exhibit a higher calcium sensitivity, and absolute forcesremain lower than in the intact heart. Initially, the maximaltwitch tension was 0.5 to 1 mN, optimized EHTs today reachup to 4 mN. Others reported active forces between 0.05 mN44

and 1 to 2 mN18 (overview in reference 50). Values of 4 mNare similar to the force of intact muscle preparations such asrat papillary muscles, but experiments in the latter underes-timate maximal forces developed by adult, mature cardiacmyocytes due to core ischemia. Very thin adult rat heartmuscle strips develop forces of 56 mN/mm2.57 The diameterof mature rat EHTs is 0.8 mm, therefore the diameter of thetwo sides of the rings together is 1 mm2. Thus, the normalizedoptimal force of EHT is 4 mN/mm2. The difference of EHTforce generation as compared with mature heart muscles mostlikely reflects both a quantitative and a qualitative aspect, asfor example a lower fractional occupancy of the EHT tissueby cardiac myocytes and the lower degree of sarcomeredevelopment. Both are likely to be important for tissueformation in the absence of perfusion in vitro, but are, at leastpartly, overcome after implanting onto the heart.56

Critical SizeOne of the central unresolved problems of all tissue engineer-ing approaches is the limitation of maximum size which isdefined by maximum diffusion distances for nutrients andoxygen. Indeed, none of the various tissue engineeringapproaches developed today generate cardiac tissue-like,contracting constructs in which compact muscle strandsexceed 50 to 100 �m. Early tumor angiogenesis studies haveshown that, in the absence of capillarization and perfusion,tumors implanted into nude mice did not reach a size of morethan 2 to 3 mm.58 These values, however, depend both on themetabolic demand and the cellular density of the respectivetissue. Beating cardiac myocytes obviously have a very highmetabolic activity. Accordingly, the density of capillaries inthe adult heart is very high, amounting to approximately 2400to 3300/mm2 in rat and human at different postnatal stages(intercapillary distance �19 to 20 �m).59,60 On the otherhand, the early embryonic rat heart (until ED 16) as well asthe adult frog heart are avascular and nourished exclusivelyfrom the lumen by blood circulating within the trabecularsystem.61,62 Thus, large heart muscles either develop physio-

logically through vascularization or through intense trabecu-larization with single strands smaller than 50 to 75 �m. Thelatter pattern is recapitulated in EHTs, in which the networkof cardiac myocytes strands is loose in most parts andconsists of muscle bundles with a diameter of 30 to 50 �m(see Figure 2 in reference 35). Only in some parts compactmuscle strands form that reach up to 100 �m. Similarly, 3 to4 cardiac myocyte monolayers can be successfully stacked,more did not improve the thickness of tissue constructs.18

Some groups increase oxygen and nutrient delivery bycultivating constructs in bioreactors, increasing ambient ox-ygen concentrations and/or by adding oxygen carriers such asperfluorocarbon.63,64 Indeed, such optimization of cultureconditions has had beneficial effects on cell density andmetabolic activity.63 and has been modeled to allow construc-tion of cardiac tissues with a clinically meaningful thicknessof up to 500 �m.64 Another strategy is to incorporate aperfused natural vessel into engineered heart muscle con-structs65 or to stimulate angiogenesis in cardiac tissue con-structs. In EHTs, primitive capillaries and larger vessel-likestructures lined with CD31 positive endothelial cells developspontaneously.35 It seems unlikely that these vessel structuresplay a significant role in oxygen and nutrient exchange underin vitro conditions, but they may facilitate perfusion afterimplanation. Levenberg and colleagues have recently mixedendothelial cells and fibroblasts to a culture of skeletalmyoblasts on a synthetic polymer and observed generation ofskeletal muscle constructs with increased density of vascularstructures.66 A third strategy to overcome size limitations invitro is to weave several EHTs or comparable constructstogether, thus forming a network in which each individualconstruct remains accessible for unlimited diffusion andexchange of nutrients. Large networks could be useful as animprovement of the “ACORN” technology (CorCap CardiacSupport Device), a therapeutic device currently tested inpatients with terminal heart failure with predominant ventric-ular dilatation.67 Such a “chain mail” biological network(Figure 2) could have the advantage over the syntheticCorCap Cardiac Support Device in that it could support bothdiastolic and systolic function of a failing heart. Takentogether, several potential strategies have been developed toovercome size limitations, both in vitro and after implantationin vivo. Yet, critical size remains an unresolved problem andits solution is one of the most important tasks in the field.

Implantation StudiesThe early studies by Li and Leor indicated that cardiacmyocyte-populated gelatin and alginate-grafts, respectively,were visible for extended time after implantation as a patchonto the heart and contained cells, despite the absence ofimmunosuppression.38,39 No definite proof for the develop-ment of new cardiac tissue was provided. Shimizu et aldemonstrated survival and vascularization of their sandwichconstructs and recorded beating activity after implantationinto nude rats.18 Survival, vascularization and terminal car-diac differentiation of cardiac myocytes was observed inEHTs after implantation onto rat hearts in the presence ofimmunosuppression.56 Even though these data collectively




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suggest that constructs are rapidly vascularized after implan-tation and that hypoxia during and after implantation is not amajor problem, several important questions remain unan-swered: Is vascularization sufficient? How many of thecardiac myocytes initially implanted survive over time? Dothe constructs couple electrically and mechanically to the hostmyocardium? Does the implantation of cardiac constructsbeneficially affect cardiac performance? The early studiesreported improved function as evidenced by enhanced devel-oped pressure in isolated Langendorff-perfused hearts38 andimproved left ventricular dimensions as well as fractionalshortening.39 However, the effects were generally small andsimilar effects have been reported in numerous cell injectionstudies, apparently independent of the cell type injected.Thus, the specific functional contribution of grafting 3Dconstructs as compared with cell injection remains unclear atpresent. Thorough evaluation of this issue is indispensable forthe further development of the field.

Future DevelopmentsMatrix MaterialsAs outlined above, the optimal scaffold for engineeringcardiac muscle tissue has not yet been found. Alginate,gelatin, polyglycolic or polylactic acid, as presently used forengineering cartilage, bone, ear, skin, blood vessels, or heartvalves, will not likely be the materials of choice. The reasonlies in the physiological properties of cardiac muscle tissue,particularly the need for high mechanical stability coupledwith great compliance. Moreover, muscle fibers and bundlesin the heart form layers with different orientation to provideoptimal pump function. At present it is difficult to imaginethat any technically fabricated material will ever mimic suchsophisticated structure and even if it would do so, the cardiaccells, stem cells or stem cell-derived cardiac myocytes stillhave to grow into the free spaces, interconnect and therebyform an electrical and mechanical syncytium. On the otherhand, material sciences and particularly nanotechnology are

Figure 2. Perspectives of cardiac tissue engineering exemplified with the EHT technology. Constructs will be made from ES cells thatare ideally autologous, derived from a nuclear transfer strategy, or from adult cardiac progenitors that may be obtained from a heartmuscle biopsy. By miniaturizing the setup and combining casting, culturing, and force-measurement in one 96-well plate, engineeredcardiac tissues can be used for drug screening and target validation. For cardiac regeneration, several circular constructs can bewoven together to form a multi-loop construct. Several multi-loop constructs together can form large “chain-mail”–like networks for thetreatment of dilated cardiomyopathy or a patch for the treatment of myocardial infarction.




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progressing rapidly and it may be that in a collaborationbetween engineers, physicists, biotechnologists, biologistsand morphologists the ideal scaffold material for creatingtruly “artificial” heart tissues will be designed.68,69 Recentexamples are tubular scaffolds for the engineering of bloodvessels that were generated by electrospinning of collagenand elastin70 and collagen tubes for cardiac tissue engineer-ing.71 Another innovative approach is to use MRI-derived 3Dimages of an organ as a blueprint for the fully automatedfabrication of a synthetic copy.72 It will be interesting to seewhether these sophisticated methods in nanotechnology andimaging will prove useful in creating optimized cardiacconstructs.

Cell Sources for Cardiac Tissue EngineeringThe greatest conceptional problem of cardiac tissue engineer-ing (as for all cell-based therapies) relates to a suitable cellsource. It has been estimated that the adult human leftventricle contains 5�109 cardiac myocytes,73 ie, 40 millioncardiac myocytes per gram of native myocardium. Thus, eventhe creation of a small tissue patch would require tens ofmillions of cardiac myocytes, a number impossible to obtainfrom a primary human cardiac cell source, not to speak of theethical aspect. Thus, the primary cells used today for cardiactissue engineering can only serve as a proof-of-principle.Great hope has been created by recent findings indicating thatstem cells exist in the adult organism that could give rise tothe formation of autologous cardiac myocytes as well asendothelial and smooth muscle cells. Such cells have beenfound in various tissues, including the bone marrow,74,75

peripheral blood,76,77 umbilical cord,78 and adipose tissue.79

Initial studies in mice showed that lin-, c-kit� cells can beisolated from bone marrow and regenerate new myocardiumwhen injected into the heart of recipient mice after myocar-dial infarction.75 These data prompted the initiation of clinicalstudies in patients with acute myocardial infarction indicatingthat such treatment is safe and may provide functional benefit(review in reference 5). The first results of larger, randomizedand placebo-controlled studies are eagerly awaited in late2005. Yet, recent studies in mice using similar cells as theoriginal study75 reported no cardiac regeneration at all.80–82

Thus, the capacity of bone marrow stem cells to regeneratethe heart is currently under intensive debate and a detaileddiscussion is beyond the scope of this article (for review seereferences 5 and 7).

New hope was raised by the demonstration of progenitor cellsresident in the heart that have the capacity to differentiate intofunctional cardiac myocytes and repair the heart.47–49,75,83 Thefirst report by Beltrami and colleagues described small, roundc-kit� cells organized in niches in the adult rat heart that could beclonally expanded and, when injected into infarcted hearts,generated new viable myocardium.47 This study has been sub-sequently supported by data showing that a c-kit� cell populationcan be propagated from murine and human heart specimens and,in case of murine cells, form spontaneously contracting cardio-spheres even in the absence of coculture with primary heartcells.84 Moreover, c-kit� cells could be mobilized to regeneratenew myocardium in infarcted dog hearts by injecting IGF-1 andHGF-1.83 Another study isolated sca-1� cells from a total adult

murine heart cell population and found that these cells canacquire a cardiac phenotype after treatment with 5�-azacytidine,a compound known to induce muscle cell differentiation.48 Byusing an extensive genetic labeling strategy it was also shownthat sca-1� cells home to the injured myocardium when injectedintravenously and have the capacity to differentiate into cardiacmyocytes as well as to fuse with host cells. Subsequent studiesextended these findings showing that sca-1� expressing cells canbe slowly expanded in vitro and, in the absence of primary heartcells, differentiate to beating cardiac myocytes when treated withoxytocine.85 Moreover, data were presented showing that car-diac differentiation of sca-1� cardiac stem cells depends onFGF-286 and that only a CD31--, isl-1--subset of sca-1� cells mayactually represent cardiac precursors.87 Finally, the LIM domaintranscription factor isl-1 has been identified by genetic means tomark a cardiac progenitor population of which few cells appearto remain in the postnatal heart.49 Isl-1� cells have also beenexpanded and differentiated into contracting cardiac myocytesunder coculture with neonatal rat cardiac myocytes. Fusion wasmade unlikely by pretreatment of myocytes with formaldehyde.It is not clear at present whether c-kit, sca-1 and isl-1 indeedlabel three different cardiac progenitor populations as indicatedby some of the above studies49,87 or whether expression of thesemarkers reflects the dynamic phenotype of the same one. In anycase, these exciting findings open the door not only to anautologous cell therapy approach but also to the engineering ofautologous cardiac tissues. Such tissues would be ideal forcardiac repair as being likely devoid of tumorigenic and immu-nological problems. However, tissue engineering from autolo-gous stem cells has yet to be shown.

The alternative to adult stem cells or cardiac progenitorsare embryonic stem cells which are able to form virtually anycell type of the body in vitro under appropriate condi-tions.88–90 Genetically selected ES cell-derived cardiac myo-cyte form electrical connections with the host myocardiumwhen injected into the heart.91 The pluripotency of ES cellsmay be of particular importance for cardiac tissue engineer-ing when considering the role of noncardiomyocytes in EHTformation (see above). Indeed, recent experiments showedthat spontaneously contracting EHTs can be generated frommouse ES cells and produce forces similar to EHTs fromprimary cardiac cells (own unpublished data). Human EScells possess principally the same features as mouse ES cellsincluding the propensity to spontaneously differentiate intocardiac myocytes92–94 and a recent study showed that trans-planted human ES cell-derived cardiomyocytes survived inthe swine hearts under immunosuppression and acted as arate-responsive biological pacemaker.95 The potential of car-diac differentiation apparently strongly depends on the cellline, but also on the quantification algorithm. Some linesseem to have a very limited or basically no potential forcardiac differentiation at all. This raises questions with regardto the present federal restrictions to a limited number ofalready existing human ES cell lines (listed by the NIH athttp://stemcells.nih.gov/research/registry/). Moreover, it callsfor studies deciphering the mechanisms of cardiac differen-tiation from ES cells. On this basis pharmacological strategiescould be developed to direct cardiomyogenic differentiation.Because ES cells can be easily and quickly propagated in




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unlimited quantities, even the immense cell number theoret-ically needed to produce cardiac muscle patches to curehuman heart diseases appears a realistic option.

Major problems of ES cells are their immunogenicity96 andtheir potential to form tumors.7,97 Data exist that ES cells atleast partly escape the normal immune responses98 and somegroups have even injected mouse ES cells into rat hearts andfound large numbers of surviving cardiac myocytes up to 32weeks thereafter without evidence for tumor formation.99,100

In contrast, significant immune responses were seen afterinjecting GFP-labeled mouse ES cells into allogenic mousehearts.96,101 The reasons for these discrepancies are not clearat present, but it is evident that immune responses have to beanticipated. Options to deal with this problem could be tocreate ES cell banks containing immunologically diversephenotypes from which the best fit for the respective patientcould be chosen. And finally the option exists to derivepotentially autologous ES cells by nuclear transfer.102–104

The most significant problem of ES cells in regenerativeapproaches is the problem of tumor formation as noted incareful studies.7,101,105,106 Even if the risk can be minimizedby various means, it is likely that even 1 tumorigenic cell in1 million can create problems in a cardiac repair approach inwhich 100 million cells are needed. Thus, injection ofundifferentiated ES cells for cardiac regeneration must beviewed with scepticism. Two principal strategies are cur-rently used to reduce this risk. One is to positively select cellsthat acquire a cardiac phenoytpe, for example by introducinginto the genome of an ES cell line a neomycin resistance geneunder control of a cardiac specific promotor.91 With thisstrategy noncardiac myocytes can be eliminated before im-plantation of the cells/tissue by adding neomycin-derivativesinto the culture medium. Alternatively or in addition, it maybe necessary to introduce a suicide gene into the ES cell clonesuch as herpes simplex virus thymidine kinase which makesall undifferentiated potentially tumorigenic ES cell-derivedcells sensitive to drugs such as ganciclovir. This strategycould be used as an emergency treatment if tumor formationoccurs. However, none of these genetic approaches is 100%efficient or safe. In fact, any approach to alter a stem cells’genome may by affected by genetic and epigenetic instabil-ity.107 Moreover, the genetic manipulation itself could createnew tumorigenic problems by deleterious integration of thevector into the genome.108 It will therefore be essential toapply rigorous tests to exclude such problems before anyapplication, because safety will be crucial for the success ofthe entire ES cell concept. Other unresolved issues relate toinsufficient vascularization, one of the likely reasons for graftsize reduction after ES cell injection.101

Immune ResponseAll allogenic cell-based therapies face the problem of immu-norejection and it is questionable whether patients and doc-tors would accept lifelong immunosuppression with poten-tially fatal consequences such as tumor development,cyclosporine-induced renal failure, Cushing syndrome, andosteoporosis. This issue is in fact a strong argument forautologous stem cell therapies, most likely with residentcardiac stem cells. Alternatively, ES cell-based approaches,

will require either a way to circumvent immunorejection orthe opportunity to do therapeutic cloning, either with oocytesderived from volunteers104,109 or from ES cells.110

Another somewhat neglected problem is that most of thecommonly used media supplements are xenogenic (eg, fetalcalf serum, horse serum, chick embryo extract, matrigel,collagen). This results in several problems: Infectious risks,nonadherence with standards of good laboratory and manu-facturers practice (GLP, GMP), and the generation of im-mune responses even with autologous or syngenic cells. Forexample, complete immune rejection was seen when EHTsmade from neonatal Fischer rats and Fischer rat tail collagenI were implanted into adult syngenic Fischer rats.56 The mostlikely explanation is that, despite extensive washing beforeimplantation, remnants of the chick embryo extract and horseserum, or Matrigel had impregnated the genetically autolo-gous cells or the extracellular matrix and provoked animmune response. Another explanation could be induction ofself-antigens during prolonged culture. It will be necessarytherefore to identify suitable culture conditions for engi-neered heart tissues that are devoid of xenogenic growthsupplements. Some critical factors for cardiac myocyte cul-tures including epidermal growth factor, hydrocortisone,L-thyroxine, albumin, selenium, and transferrin have alreadybeen identified.111

Realistic Perspectives of Cardiac TissueEngineering in the Next 10 Years

Statements on the perspectives of science share the limi-tations of stock market predictions and remain essentiallyspeculations, founded on facts and spiced with personalconvictions. With this in mind, some predictions can bemade. The total artificial heart will likely remain fictionfor a while, but some important milestones toward thisambitious goal have already been achieved as outlinedabove. The following summarizes realistic accomplishableachievements:

Drug Screening Assay Based on EngineeredCardiac ConstructsEngineered cardiac tissues will be useful for drug develop-ment and target validation in a format that allows formedium-to-high throughput assays (Figure 2). Experimentsare on the way to construct a 96-well plate ES-cell EHTformat to eventually generate a humanized experimentalmodel for drug research.

Serum-Free Culture Conditions for CardiacTissue GraftsExperiments to define such culture conditions are currentlybeing pursued in several laboratories. High throughputscreening systems as mentioned above will be helpful toidentify cardiogenic or cardiac growth supportive factors inthe right concentration and combination as well as the righttiming. Given the availability of serum-free media for manyother cell types there is little reason to assume that a similarcondition cannot be established for cardiac cells and tissues.




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Prevention/Slowing of Remodeling AfterMyocardial Infarction by Tissue Grafts in LargeAnimal ModelsA realistic perspective is that large networks composed ofseveral engineered cardiac grafts can be generated andsutured around the heart. Such “biological chain-mail” asexemplified with the EHT-technology in Figure 2 could bedesigned both from primary cardiac and ES cells and tested inrats, mice and pigs after myocardial infarction.

Correction of Heart Defects by a ContractingPatch in a Large Animal ModelCongenital heart defects such as complex Tetralogy of Fallot,and aplastic right and left ventricles would profit from theimplantation of actively contracting engineered tissuepatches. This approach will likely be tested first in the lowpressure system of the right heart with patches made from EScell-derived myocytes.

Autologous Cardiac Graft From an Autologous ESCell Line Generated by Nuclear TransferTechniques to generate autologous blastocysts by nucleartransfer and to produce autologous ES cell lines (ntES cell)from these early embryos have been successfully establishedover the past 5 years, in various animal species and recentlyalso in human.104 Thus, despite the present formal hurdles andthe ongoing ethical debate, it seems realistic to create autol-ogous artificial heart muscle from ntES cell lines and testtheir applicability in an animal model in the next years.

Autologous Cardiac Graft From ResidentCardiac PrecursorsGiven the discovery of cardiac progenitors it should also bepossible to isolate such cells from a cardiac biopsy, amplifyand differentiate them into functional cardiac myocytes andcreate a 3D tissue construct.

ConclusionsCardiac tissue engineering is a relatively young, activelydeveloping field. Its great attraction, both for researchers andthe public, lies, first of all, in the fascinating natural capacityof heart cells to form spontaneously beating, well organizedheart-like tissues in the culture dish—a finding that was mademore than 50 years ago. Apart from the naive enthusiasm thisphenomenon excites, there are good reasons to assume thatengineered cardiac tissues will be of practical use in the nearfuture, as an improved experimental model, as a modelsystem for cardiac development and, much later, as a thera-peutic option for cardiac repair. The latter optimism is mainlybased on the enormous progress in stem cell research. Whathas been pure fantasy 10 years ago now becomes a realisticprospect—to create contracting heart muscles from the pa-tients own stem cells and to use them to alleviate heartdisease. Many critical caveats remain, however, and intenseand stringent scientific work is mandatory, before suchstrategies should head into the clinic. And finally, futuredevelopments in mechanic devices, xenotransplantation, celltherapy and not at last pharmacology may decide whether or

not cardiac tissue engineering will indeed find its place in thepractical therapy of heart disease.

AcknowledgmentsThe work of the authors has been supported by Bundesministeriumfur Forschung und Technologie (BMBF 01GNO124 to T.E.), Deut-sche Forschungsgemeinschaft (DFG Es 88/8-2 to T.E.), DeutscheStiftung fur Herzforschung (W.H.Z.), and Novartis Foundation(W.H.Z. and T.E.). We thank L.J. Field, Indianapolis, and L. Carrier,Hamburg, for critical reading of the manuscript.

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Thomas Eschenhagen and Wolfram H. ZimmermannEngineering Myocardial Tissue

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2005 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.0000196562.73231.7d2005;97:1220-1231Circ Res.

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In an article by Eschenhagen et al (Circ Res. 2005;97:1220–1231), the authors incorrectly citedthe source of Figure 1E. The top of Figure 1E is reproduced with permission from Bursac et al(Am J Physiol Heart Circ Physiol. 1999;277:433–444). The bottom of Figure 1E is reproducedwith permission from Papadaki et al (Am J Physiol Heart Circ Physiol. 2001;280:168–178). Theauthors apologize for this error.

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Reviews - Circulation Researchcircres.ahajournals.org/content/circresaha/97/12/1220.full.pdf · create 3-dimensional heart muscle equivalents that may not only serve as experimental