engineering the heart piece by piece: state of the art in cardiac tissue engineering

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Engineering the heart piece by piece: state of the art in cardiac tissue engineering
Louise Hecker1 & Ravi K Birla2†
†Author for correspondence1Cell and Developmental Biology, The University of Michigan, MA, USA2Section of Cardiac Surgery, University of Michigan, B560 Medical Science Research Building II, 1150 West Medical Center Drive, Ann Arbor, MI 48109, USATel.: +1 734 615 5203;Fax: +1 734 763 0323;E-mail: [email protected]

part of

Keywords: active force, artificial heart, biomaterials, bioreactors, fibrin gel, heart muscle, perfusion, scaffolds, self-organization, tissue engineering

10.2217/17460751.2.2.125 © 2

According to the National Transplant Society, more than 7000 Americans in need of organs die every year owing to a lack of lifesaving organs. Bioengineering 3D organs in vitro for subsequent implantation may provide a solution to this problem. The field of tissue engineering in its most rudimentary form is focused on the developed of transplantable organ substitutes in the laboratory. The objective of this article is to introduce important technological hurdles in the field of cardiac tissue engineering. This review starts with an overview of tissue engineering, followed by an introduction to the field of cardiovascular tissue engineering and finally summarizes some of the key advances in cardiac tissue engineering; specific topics discussed in this article include cell sourcing and biomaterials, in vitro models of cardiac muscle and bioreactors. The article concludes with thoughts on the utility of tissue-engineering models in basic research as well as critical technological hurdles that need to be addressed in the future.

Overview of tissue engineeringTissue engineering is a rapidly evolving fieldinvolving collaborative expertise from diverse dis-ciplines including engineering, medicine and thelife sciences [1–5]. In its simplest form, the aim oftissue engineering is to promote functional regen-eration of damaged tissue utilizing cells culturedin vitro within 3D scaffolds to repair and/orreplace damaged tissue in patients. Tissue-engi-neering strategies are focused on four main areasof research: cell sourcing, scaffold design, func-tional tissue development and finally, develop-ment of viable commercialization models(Figure 1). The identification of a suitable cellsource remains a formidable challenge, especiallyfor cardiac applications, since adult-derived cardi-omyocytes are difficult to obtain and nonprolifer-ative in vitro, thereby limiting their applicability.The main areas of opportunities for cell sourcinginclude human embryonic stem cells (ESCs),adult-derived stem cells and autologous cellsderived from patients. The choice of cell sourcewould vary significantly depending on the appli-cation; autologous-derived skeletal muscle cellscan be utilized for cardiac regeneration, whileautologous-derived cardiac cells may not be themost feasible choice. Selection of suitable scaf-folding material depends on the ability of thematerial to simulate properties of the extracellularmatrix (ECM), promote cell viability and prolif-eration, possess easily controllable degradationkinetics and have a high degree of immune toler-ance when implanted in vivo. There are severalmatrices currently available that meet many of

these requirements, while new and improved bio-materials with improved functionality are contin-uously being developed. The next stage typicallyrequires successful colonization of the scaffold bythe cells; the viability of the cells during culturewithin the scaffold, the ability of the cells tomaintain differentiated phenotype and the abilityof the cells to functionally interact with the bio-material become important considerations. Oncecellularization of the scaffold has occurred,guided phenotypic maturation of the cellsbecomes important to promote tissue formationthat closely resembles the physiological make-upof normal mammalian tissue. It becomes neces-sary to provide mechanical, electrical and chemi-cal/hormonal cues to support the functionaldevelopment of the tissue. Bioreactors need to beimplemented to induce electro–mechanical stim-ulation of the bioengineered tissue, leading togene expression that closely resembles the geneexpression of in vivo tissue. The development ofmicroperfusion systems becomes increasinglyimportant to replicate the physiological flow con-ditions observed in vivo. As tissue growth andmaturation occurs, vascularization and/or func-tional innervation of the bioengineered tissueconstruct become important.

The task of engineering functional 3D con-structs is often confounded by the degree ofcharacterization required to evaluate the physi-ological performance of the construct. Func-tional tests often require in vitro assessment ofmechanical (force, pressure and compression)and histological/biological (gene/protein

007 Future Medicine Ltd ISSN 1746-0751 Regenerative Med. (2007) 2(2), 125–144 125

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126 Regenerative Med. (2007) 2(2) future science groupfuture science group

Engineering the heart piece by piece: state of the art in cardiac tissue engineering – REVIEW

future science groupfuture science group

Figure 1. Pathway f

The complex roadmap todonor human tissue, derexpanded and characteriutilized to colonize prefabof electrical and mechaniinduction of angiogenesidevelopment and implem

expression and distribution) properties. Thein vitro testing phase is followed by in vivoevaluation of biocompatibility and immuneacceptance/tolerance. During the next stage oftesting, it becomes important to demonstratethe ability of the bioengineered construct tofunctionally integrate with the host tissue topromote the regeneration of damaged tissueusing suitable animal models. This is followedby clinical evaluation of the bioengineeredconstructs to demonstrate the ability to trans-late the research to the clinical setting. Thebioengineered constructs are then evaluated byguidelines established by regulatory process,such as the US FDA, which serve to evaluatethe safety and efficacy of the bioengineeredconstructs. For definitive success of bio-engineered functional constructs, they mustmeet the challenges of commercialization,including the development of business modelsbacked by strong financial resources.

Integration of core technologiesDevelopment of tissue-engineering technolo-gies requires collaborative efforts from diversescientific disciplines. This model of scientificcollaboration is fairly well established in manyscientific endeavors and the novelty of tissue-engineering places additional challenges inimplementing successful collaborations. Devel-opment of core technologies for tissue engi-neering research requires expertise fromengineering, medical and life sciences disci-plines. Evaluating the phases of research for oneparticular example – development of a func-tional 3D cardiac patch – will illustrate the flowof information and technology between thethree key players (Figure 2).

Identification of the need for an alternativetherapy to treat cases of myocardial injury wouldneed to begin within the medical profession.Although there have been several therapeuticoptions available to treat acute myocardial infarc-tion, one can see the advantage of using a func-tional tissue-engineered 3D patch. The first and

foremost problem would be the identification,isolation, purification and characterization of asuitable cell source, typically carried out by thecell biologists. The next step would be the devel-opment of bioactive biomaterials by the engineer-ing team and would require expertise inbiomaterial synthesis, characterization and induc-tion of bioactivity, thereby allowing functionalinteraction with cells. The ability of the cells tofunctionally interact with the biomaterial andpromote the formation of 3D cardiac musclewould depend on many factors; attachment ofthe cells to the fibers of the biomaterials via inte-grin-mediated mechanisms and the ability of thecells to maintain differentiated phenotype duringcolonization of the scaffold. Understanding andmanipulating cell–material interactions necessi-tates scientific input from engineering as well asthe life sciences experts.

During the early stages of research, there needsto be an effort directed towards the developmentof small animal models, bioreactor technology tosimulate physiological parameters and modulatethe fluid environment of the constructs by devel-oping continuous perfusion systems as well asthe utilization of an array of biochemical mark-ers. Every group of experts has to contribute totheir full potential to head these initiatives.Later stages of research involve the develop-ment of large animal models as well as the abil-ity to develop feedback control for bioreactorsand real-time monitoring of material propertiesand biochemical markers. This again demon-strates the need for true genius by scientistsfrom each discipline.

A true collaborative effort between variousdisciplines is imperative to the success of eachphase and it is crucial to promote the exchangeof technology between each phase, revisiting theproblem definition at every stage of the process.This simple example serves to demonstrate thedegree of complex interactions and exchange ofinformation required at the very early stages ofscientific development between scientists fromthe medical, engineering and life sciences.

or the formation of bioengineered constructs.

bioengineering constructs begins with the identification of a suitable cell source; cells can be obtained from ived from an embryonic origin, obtained from animal tissue and/or derived from cell lines. The cells are isolated, zed under controlled in vitro conditions, manipulated to modulate the expression of specific genes and then ricated biomaterials. The bioengineered constructs are then subjected to various environmental cues in the form

cal stimulation as well as microperfusion to promote phenotypic maturation of the constructs. This is followed by s, in vitro testing of the constructs followed by in vivo testing using animal models, clinical testing and entation of commercialization strategies.

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Figure 2. Core techn

Implementation of a succexpertise from several scipatches, the interplay beas bioengineered construearly stages of the researtowards cell isolation, pubioengineered construct.sophisticated biological mfabrication of novel biorereal-time monitoring of fperformance of bioenginphase of technological d

Development of a successful model to accom-plish this degree of scientific and technologicalcollaboration will be a significant challenge forthe field of tissue engineering.

Tissue engineering has traditionally lacked amodel for cohesive research. The general ten-dency has been for the establishment of laborato-ries by single investigators in an academicsetting. This has led to the development of excel-lent technological development in a single areaof tissue engineering, without emphasis on thefunctional interaction across disciplines.Although single laboratories claim to collaboratewith disciplines outside their area of expertise oreven recruit research personnel across theseboundaries, we do not believe this is often ade-quate. Rather, multidisciplinary centers need tobe established, often through the collaborativeefforts of professionals from academic, govern-ment and industrial backgrounds. Leadershipneeds to be jointly established by all participat-ing members. Funding will need to be secured bydiverse sources, such as federal agencies, philan-thropic donations and support from industry.Intellectual property will need to be managed ina manner that promotes a significant degree ofopenness to allow technology development whileprotecting ownership of the inventors. In ouropinion, this will be the only successful way todevelop tissue-engineering models.

Potential clinical applications of bioengineered cardiovascular constructsThe field of cardiac tissue engineering includesnot only heart muscle, but also tri-leafletvalves, cell-based cardiac pumps/ventricles andvascular grafts. Although the focus of this arti-cle is on cardiac muscle, other areas of cardiactissue engineering will be briefly discussed forcompletion. Most of these fields are at earlystages of technological development and there

lies an opportunity to have a significantimpact on the future of treatment modalitiesfor cardiovascular disorders (Figure 3).

The most successful strategy for the treatmentof end-stage congestive heart failure has been sur-gical transplantation. However, widespread appli-cability of heart transplantation is often limitedby a chronic shortage of donor organs [6–8]. Car-diac tissue engineering may provide a potentialsolution to the donor heart crisis in the future.The clinical utility of cardiac patches could beparticularly advantageous in cases of acute myo-cardial infarction, where integration of bioengi-neered patches with host myocardium couldtheoretically improve the contractile function ofthe failing left ventricle (Figure 3A) [9–15].

The current treatment options for vascularsubstitution require the use of the mammaryartery or saphenous vein for by-pass grafting.The major disadvantages of this strategy is theneed for invasive patient surgery and the lim-ited quantity of autologous grafting materialavailable. Synthetic vascular substitutes, such asDacron and expanded polytetrafluoroethylene(ePTFE), lack the cellular components, therebylimiting functional performance and have lim-ited utility in small-diameter applications.Patients often suffer from thrombus formationand intimal hyperplasia. Tissue-engineeringalternatives could potentially eliminate some ofthese complications and may be particularlysuitable for small-diameter applications(Figure 3B) [16–20]. The ability to fabricate hollowtubular structures surrounded by smooth mus-cle cells with a lining of endothelial cells on theluminal surface would constitute a viabletissue-engineered alternative.

Heart valve substitution is the only effectivetreatment for end stage heart valve diseases whenrepair is not feasible [21]. Current options arelimited to mechanical valves [22] and biological [23]

ologies required for tissue engineering.

essful pathway for the formation of bioengineered constructs requires functional integration of technical entific disciplines: medicine, engineering and the life sciences. Using an example of the development of cardiac tween the various cores can easily be illustrated. Identification of the need for novel treatment modalities, such cts, begins within the medical discipline necessitating the identification of multidisciplinary teams. During the ch, the engineering core works towards the development of novel biomaterials, while the life science core works rification and expansion. Novel cellularization strategies lead to the formation of a premature first-generation During the second stage of research, small animal models are developed by the medical experts, while arkers are developed by experts in the life sciences. Engineering folks are generally involved in the design and

actors and microperfusion systems. Later stages of research necessitate the development of large animal models, unctional performance with embedded feedback control and noninvasive biological markers for in vivo eered constructs. This model of functional integration of core technologies progresses intimately through every evelopment resulting in the development of clinically usable bioengineered constructs.

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Figure 3. Functional

(A) Bioengineered cardiabiomaterials results in thmyocardial infarction to afabricate tubular structurwalls of the tubular graftformation of bioenginee(C) Bioengineered tri-leaftissue-engineered equiva(D) Cell-based cardiac pucell-based device capablesinoatrial node receive elsynchrony with the host ventricular assist devices

substitutes. Mechanical heart valves oftenrequire long-term anticoagulation, while bio-logical substitutes lack the mechanical durabil-ity for long-term function. Valve replacementfor pediatric patients is further limited owingto the inability of the prosthetic valve to growwith the pediatric patient, necessitating severalre-operations during the childhood of apatient. Tissue engineering offers the potentialof generating heart valves of virtually any size,which would grow with pediatric patients [24].In addition to the many challenges of tissueengineering already discussed, the ability toengineer tri-leaflet valves is confounded by acomplex 3D geometry necessitating complexmold design (Figure 3C) [25–29].

The ability to engineer cell-based cardiacpumps may provide an alternative to mechani-cal left ventricular assist devices (LVADs) [30].LVADs are used as a bridge to transplantation,transporting oxygenated blood from the apex ofthe heart directly to the aorta, completelybypassing the failing left ventricle. A cell-basedalternative would be composed of a hollowchamber surrounded by contracting cardiaccells with one-way valves for unidirectionalflow and embedded electronics for feedbackcontrol (Figure 3D). Sensors would receive elec-trical input from the sinoatrial (SA) node, per-mitting contractions that are synchronized withthe heart. The cell-based alternatives tomechanical LVADs would offer a higher degreeof immune tolerance, reducing the need forlong-term anticoagulation.

Cell sourcingA critical issue in cardiac tissue engineering is thechoice of cells for construct development. Anideal cell source for cardiac tissue engineeringshould possess the following characteristics:

• Proliferative

• Easy to harvest

• Nonimmunogenic

• Ability to differentiate

Depending on the specific application, poten-tial cell types for cardiac tissue engineering mayinclude (but are not limited to): vascularsmooth muscle, skeletal myoblasts, fetal cardi-omyocytes, mesenchymal stem cells, endothe-lial progenitors, bone marrow cells, umbilicalcord cells, fibroblasts and ESCs. The composi-tion of cells is also an important considerationin cardiac tissue engineering. Cardiac tissuein vivo is complex, being composed of multiplecell types. Cardiac myocytes are the main com-ponent of the heart; however, a combination ofendothelial cells, fibroblasts, smooth musclecells, neural cells and leukocytes make upapproximately 70% of the totally cells in theworking myocardium [31]. Noncardiomyocytesplay an important role in normal cardiac devel-opment and function. Using a triple-cell-basedculture of cardiomyocytes, endothelial cellsand embryonic fibroblasts, a 3D model of vas-cularized cardiac tissue has recently been devel-oped [32]. The presence of endothelial cells ledto increased cardiomyocyte proliferation, whilethe presense of embryonic fibroblastsdecreased endothelial cell death and increasedtheir proliferation; thereby demonstrating theimportance and complexity of functionalinteractions between the various cell types.Future development of in vitro cardiac modelsthat incorporate multiple cell types should leadto improved graft/transplant survival withincreased functionality.

Utilization of adult-derived stem cells wouldprovide a cell source that can be derived frompatients and is therefore autologous. Potential

cardiovascular tissue engineering at a glance.

c muscle: the ability to engineer cardiac muscle by functionally integrating primary contractile cells with e formation of tissue-engineered cardiac patches. These cardiac patches can be utilized to treat cases of acute ugment the contractile function of the failing left ventricle. (B) Bioengineered vascular grafts: the ability to

es utilizing various scaffolds/hydrogels is dictated by the design of suitable molds, which is often fairly trivial. The are cellularized with smooth muscle cells, while the lumen is cellularized with endothelial cells resulting in the red vascular grafts. The utility of these vascular grafts would be in cases of coronary bypass surgery. let valves: the complex 3D architecture of tri-leaflet valves necessitates complex mold prototyping to fabricate lent. Once a prototype is fabricated, cellularization with myofibroblasts results in functional heart valves. mps: the ability to engineer hollow structures with cardiac cells wrapped around the periphery can serve as a of generating intramural pressure upon electrical stimulation. Embedded detectors on the device coupled to the ectrical impulses that are transmitted to electrical stimulators. This allows the cell-based pump to contract in heart. Cell-based cardiac pumps can be utilized to treat cases of chronic heart failure, with mechanical left being used as a bridge to transplantation.



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sources of adult stem cells for cardiac tissue engi-neering include bone marrow, peripheral bloodand skeletal muscle. There have been promisingresults using adult stem cells, demonstrating via-bility of transplanted cells, increased angiogene-sis in the infarct zone and improved leftventricular function. Studies evaluating the fea-sibility of cell transplantation provide a goodmodel to understand the utility of various celltypes for myocardial regeneration [33–35]. Celltransplantation therapies have utilized adult-derived stem cells skeletal myoblasts [36–41], bonemarrow-derived cells [42–46] and endothelialprogenitor cells [47,48] or ESCs [49–53].

Autologous skeletal myoblasts show expressionof contractile proteins and form well-differenti-ated myotubes in vitro and after transplantationin the heart. However, the nascent myotubes donot form electrical junctions with host myocar-dium and thus are unlikely to participate in afunctional syncytium and deliver respective work-load. Furthermore, myoblasts do not differentiatetowards a more cardiomyocyte-like phenotypeupon in vitro or in vivo physiological stimulationand have not demonstrated the fatigue resistancerequired for cardiac application. In addition, thetime needed to process and expand autologousmyoblast cultures would make it difficult todeliver them in a timely fashion to a patient inneed. Although adult stem cells are a promisingoption for tissue engineering, at the present timetheir true potential remains unclear. An impor-tant question that remains is whether it is possi-ble to induce transdifferentiation of adult stemcells at the necessary efficiency. Recent researcheffort is focused on growth factors and cultureconditions that promote cardiac lineage commit-ment and genes that may be introduced toinduce cardiac differentiation [15].

Although the utilization of human ESCs iscompounded with ethical, political and scientificdifferences of opinion, there is great potential fortheir use in cardiac regeneration. The motivationfor the utilization of ESCs are the unlimited pro-liferative capacity of these cells and ability to dif-ferentiate into multiple cells types, including allcells found in the myocardium. Various studieshave demonstrated that mouse and human ESCscan differentiate into functional cardiomyocytes[54–56]. Genetically engineered ESCs can be cul-tured to yield large-scale viable cardiomyocytesfor tissue engineering and/or implantation [57–59],thereby demonstrating the feasibility of utilizingESCs for cardiac tissue-engineering applications.Recent studies demonstrating the presence of

stem/progenitor cells in the heart may providean alternative cell source for cardiac regenerationand repair [60].

A significant amount of research is directedtowards evaluating the feasibility of utilizing dif-ferent cell types for cardiac regeneration.Although a clear direction has not evolved as yet,several promising options are being evaluated. Inconcluding this section, it may be valuable tocompare cell therapy approaches with tissue-engineering methods for cardiac regeneration.The main advantage that tissue engineering hasto offer is the fabrication of a functional 3D tis-sue construct that may be layered onto the dam-aged myocardium. This presents the opportunityfor functional interaction between the trans-planted construct and the host, thereby permit-ting functional augmentation due to thecontractions of the construct.

BiomaterialsBiomaterials guide the phenotypic maturation ofthe cardiac cells during tissue formation by pro-moting functional cell attachment and subse-quent remodeling to form functional 3D tissueconstructs [61–64]. There are several requirementsfor an ideal biomaterial for cardiac tissue-engi-neering applications. The material should sup-port the attachment, viability and proliferation ofcardiac cells in culture. The porosity (pore sizeand distribution) should accommodate the colo-nization of different cell types found in cardiactissue. The size, orientation and distribution ofbiomaterial fibers should mimic the properties ofthe cardiac ECM. The degradation kinetics of thebiomaterial should match the rate of tissue for-mation by the cardiac cells while producing non-toxic degradation products. The mechanicalproperties of the material (tensile strength andelasticity) need to be comparable to the ECM ofthe heart. In addition, for in vivo applications,the biomaterial should be immune tolerant, func-tionally integrate with host myocardium and sup-port the formation of electro–mechanicalcoupling as well as construct neovascularization.

Several biomaterials have been utilized toengineer functional 3D cardiac tissue in vitro[65]. Two classes of biomaterials currently utilizedto engineer functional 3D cardiac tissues arepolymeric scaffolds (polyglycolic acid [PGA][66–69]) and hydrogels (collagen [70–73], fibrin[Huang YC, Khait L, Birla RK: Unpublished Data] and alginate[74]). The main advantage of using polymericscaffolds is the ability to provide stable mechani-cal support to promote cardiac tissue formation.




Polymeric scaffolds can sustain higher mechani-cal loads and therefore have been preferred formany cardiovascular tissue-engineering applica-tions. Hydrogels, on the other hand, have beenshown to provide an environment that moreclosely stimulates the ECM of cardiac muscle.Several medications, including the utilization ofcross-linking agents, have been implemented toimprove the mechanical strength of the gels.

The currently utilized biomaterials have beendemonstrated to adequately support cardiac cellremodeling. Bioengineered constructs generatedby both polymeric scaffolds and hydrogels havebeen shown to exhibit contractile, histologicaland biochemical characteristics similar to normalmammalian cardiac muscle. However, as newmodels of cardiac muscle are engineered in thelaboratory with the desire to functionally matchthe performance of normal heart muscle, a newgeneration of novel biomaterials would also needto be developed.

There has been an emphasis towards thedevelopment of ‘smart’ biomaterials that arereceptive to changes in the physiological envi-ronment and are adaptive to changes in thedegree of tissue maturation [75–78]. The overallgoal is to program a feedback loop that changesthe properties of the materials as changes in thephysiology of the cells take place. Consider ahypothetical case in which cardiac cells areseeded into the 3D architecture of a ‘smart’material. As tissue remodeling takes place, thecardiac cells generate ECM components, mainlycollagen. The rate of synthesis of collagen woulddepend on several variables: the viability of thecardiac fibroblasts, mechanical conditioningand stimulation with soluble factors, and therate of synthesis of collagen would change overtime depending on the balance of these factors.One goal of a smart material would be to ‘sense’the change in collagen production and ‘pro-gram’ material degradation to match the rate ofcollagen synthesis. Conceptually, one unit ofcollagen production will result in one unit ofmaterial degradation.

Although conceptual at this stage, there havebeen several interesting examples of the devel-opment of smart materials (Figure 4). The abilityof matrix metalloproteinases (MMPs) to recog-nize and cleave specific amino acid sequencesmakes it a suitable mechanism for the function-ality of smart biomaterials [79]. A similar con-cept has been developed by utilization ofchanges in pH upon cellular endocytosis of bio-materials [80]. The pH within the endosome is

acidic, which can be used to cleave acid-sensitivecarriers of biomaterials, thereby promoting thecontrolled release of bioactive factors. Changesin the oxidative state of tissue have also beenproposed as a potential mechanism for thedevelopment of smart materials [81,82].

The goal in the development of smart mate-rials is to engineer biomaterials with specifi-cally targeted cell attachment sites, targets forcleavage by changes in the physiological envi-ronment all linked to a polymer backbone con-taining internalized growth factors (Figure 4).The objective would be to utilize the cellattachment site to deliver the biomaterial tospecific targets, while changes in the physiolog-ical state of the cells would promote the releaseof growth factors in the local environment. Forexample, matrix metalloproteins cleavage atspecific sites results in controlled release of thebone morphogenetic proteins into the localenvironment.

In vitro models of heart muscleWhen considering the functional requirementsthat tissue-engineered heart muscle needs tosatisfy, one must aim to recapitulate the anat-omy of mammalian cardiac muscle. Although itmay not be feasible to develop a perfect replicaof mammalian cardiac tissue in vitro, certainkey characteristics define conditions that wouldbe pre-requisite for the development of tissuemodels of heart muscle. Identification of a suit-able cell source, preferably adult-derived autolo-gous stem cells, with physiologicalcompositions of cardiac myocytes, fibroblastsand endothelial cells would be necessary. Onewould need novel biomaterials with propertiesthat replicate the ECM of the heart. The func-tional integration of cardiac cells with biomate-rials would need to result in contractile,histological, biochemical and electro–physicalproperties comparable with mammalian heartmuscle. Immune tolerance and neovasculariza-tion would also be additional constraints placedon tissue-engineered cardiac constructs.

Our discussion on in vitro models of heartmuscle is divided into two sections. In the firstsection, we discuss models published by investi-gators across the globe and in the second part, wediscuss three models that have been developed inour laboratory.

Carrier and colleagues utilized fibrousmeshes made from PGA as a scaffolding mate-rial for culturing neonatal cardiac myocytes[67]. PGA was processed into fibrous meshes to



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Figure 4. Smart biom

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form disk-shaped constructs with a very highdegree of porosity and seeded with primary car-diac myocytes [66,68]. These studies were thefirst to demonstrate the formation of functionalcardiac muscle utilizing scaffolding-based strat-egies. Eschenhagen developed a model of car-diac muscle by casting a mixture of neonatalcardiac myocytes and collagen into plastic

molds [70,72]. The resulting tissue constructs,termed engineered heart tissue (EHT), werecapable of generating fairly high twitch forces[72]. Eschenhagen’s model was the first 3Dmodel of cardiac muscle in vitro. Okano utilizedtemperature-sensitive polymer surfaces (TSS) toengineer 2D sheets of cardiac cells, which werestacked to form 3D tissue constructs [83–86].

aterials for tissue engineering.

mart biomaterials necessitates engineering cell-specific adhesion sites and cross-linking agents to trap soluble hitecture of the biomaterial. In vivo, the biomaterial is guided to specific target receptors on the surface of ing cell–material interaction. MMP cleavage at the cross-linking site promotes the release of the soluble factors t. This mechanism ensures the delivery of soluble factors to specifically targeted sites using the biomaterial as a he mediator. MMP: Matrix metalloproteinases.

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Regenerative Medicine




The most attractive feature of this model is theutilization of the TSS, which eliminates theneed for synthetic scaffolding materials in thecontractile region of the tissue-engineering car-diac muscle. Akins used a rotating bioreactorsystem to promote the organization of isolatedcardiac cells on the surface of microcarrierbeads [87]. Li utilized a gelatin mesh [88–92],while Leor used alginate sponges to supportcardiac tissue formation [74,93].

Our laboratory has spent the past severalyears evaluating the feasibility of using differentplatforms to engineer functional 3D cardiacmuscle in vitro [94–100]. We have currentlydeveloped three models of contractile heartmuscle (Figure 5):

• Cardioids are formed by the self organizationof primary cardiac cells in the absence of syn-thetic scaffolding materials (Figure 5A);

• Bioengineered heart muscles (BEHMs) areformed in the presence of a rapidly degradingfibrin gel (Figure 5B);

• Smart material integrated heart muscle(SMIHM) utilizes polymeric scaffolding tosupport cardiac tissue formation (Figure 5C).

Cardioids are formed by the spontaneousdelamination of a cohesive monolayer of pri-mary cardiac cells. The tissue culture surfaceutilized for cardioid formation is specificallyengineered to control surface protein concen-tration. The surface protein promotes cellattachment during the initial stages of culture,while initiating delamination during the laterstages. In addition, soluble factors introducedvia media changes promote the cardiac cells togenerate ECM components during the entireculture period. The cardiac cells and ECM selforganize by controlled guidance of the protein-adhesion molecules. The main advantage ofthis model is the ability to promote self organi-zation of a 3D tissue construct without the useof synthetic scaffolding material.

Bioengineered heart muscles are formed bythe remodeling of primary cardiac cells in thepresence of a biodegradable fibrin gel. DuringBEHM formation, the cardiac cells are layeredon the surface of the fibrin gel and allowed toremodel, promoting the formation of functional3D heart muscle. Cardiac cells degrade thefibrin gel and produce ECM components dur-ing the initial stages of construct formation. Asthis process continues, the fibrin gel is graduallydegraded and eventually replaced by endog-enous ECM within 2 weeks of culture. The

scaffolding material promotes ECM productionwhile providing temporary support for the cells.The most attractive feature of this model is theformation of heart muscle in a relatively shorttime period, within 4–5 days.

Smart material integrated heart muscles utilizea smart material to control the formation ofheart muscle. The material is considered to besmart because it closely resembles cardiac ECMin vivo and the material can undergo controlleddegradation upon formation of viable heartmuscle. Using this technology, we have engi-neered SMIHMs by cellularization of thesesmart materials using primary cardiac cells andshown close resemblance to normal heart muscletissue. Our current efforts are focused on con-trolling the rate of material degradation tomatch the rate of tissue formation. We are alsoin the process of programming a feedback loopin the material to respond to changes in the rateof collagen synthesis.

Bioreactors & microperfusionBioreactors are an integral aspect of all tis-

sue-engineering research and are particularlyimportant in the cardiovascular field owing tothe hemodynamic requirements of bioengi-neered constructs. Several classes of bioreactorshave been utilized in functional tissue engi-neering [101]. Bioreactors have been utilizedduring scaffold cellularization to obtain a uni-form cell distribution within the 3D architec-ture of biomaterial. Bioreactors have also beenused to provide physical stimulation to pre-formed 3D constructs during long-termin vitro culture; for example, electro–mechani-cal stimulation to promote maturation of car-diac constructs. In addition to bioreactors,microperfusion systems play an important rolein functional tissue engineering. The role ofmicroperfusion systems has been primarily todeliver a continuous supply of enriched mediato the bioengineered construct during in vitroculture. The goal is to closely replicate thein vivo condition, whereby a continuous bloodsupply promotes nutrient exchange as well asremoval of waste products. Bioreactor/microp-erfusion systems have been described for sev-eral cardiovascular systems, including tissue-engineered blood vessels [102–106], valves[107–112] and cardiac muscle [113–115]. Althoughthe configuration of bioreactor systems havevaried, the functional requirements haveremained similar to simulate physiologicalconditions (temperature, pH, oxygen tension,



136

Figure 5. Tissue-eng

(A) Self organization: primcoated with polydimethylsfibroblasts generate pro-cprocess. During the early sDuring later stages of cell monolayer forms a 3D contermed ‘cardioids’. The ke(B) Biodegradable hydrogremodel around. In our cacells are mixed with collagheart muscle. During the cin the environment. Theseperformance of the constrpolymeric scaffolds is to ppolymeric scaffolds often phase separation methodfunctional heart muscle. Instrategies to match the rahigh functional performan

ineering platforms for heart muscle.

ary cardiac myocytes and cardiac fibroblasts are co-cultured on the surface of a tissue culture plate that has been iloxane (PDMS), natural mouse laminin and engineered with anchor points at the center. During co-culture, the cardiac ollagen, which polymerize to form collagen fibers. The collagen provides structural support during the remodeling tages of cell culture, the primary cardiac cells form a cohesive monolayer using the laminin as an adhesion protein. culture, the spontaneous contractions of the cardiac myocytes promote delamination of the cell monolayer. The cell struct at the center of the plate and forms attachment points with the anchor points. These tissue constructs are

y element of this model is that the cells form a 3D tissue construct in the absence of any scaffolding material. el: when hydrogels are used as a tissue-engineering platform, the cells are provided with structural components to se, we utilized collagen type I as the structural element, while fibrin is used as a carrier for biofactors. Primary cardiac en and biofactors that have been embedded in a fibrin gel. The cardiac cells utilize the collagen to form functional 3D ourse of construct formation, controlled degradation of the fibrin by the cardiac cells results in the release of biofactors biofactors act on the cardiac cells during the remodeling process resulting in significantly increased functional uct. These constructs have been termed bioengineered heart muscle (BEHM). (C) Polymeric scaffolds: the use of rovide a prefabricated and predefined area to guide remodeling of cardiac cells to form 3D tissue constructs. The offer superior mechanical properties when compared with hydrogel. We utilized chitosan in our laboratory and used s to form porous 3D scaffolds. Cellularization of the scaffold using cardiac cells has resulted in the formation of addition, the degradation of the scaffold is easily controlled by the addition of lysozyme. We have developed

te of new tissue formation with the rate of scaffold degradation. This has led to the formation of heart muscle with ce, termed ‘smart material integrated heart muscle’ (SMIHM).




glucose levels, radial and circumferential stress)under controlled aseptic conditions to promoteremodeling of cells and ECM.

We have developed a bioreactor system toallow the application of coordinated mechani-cal signals to bioengineered constructs duringlong-term in vitro culture. The bioreactor sys-tem was designed and fabricated by RobertDennis, Associate Professor of BiomedicalEngineering at the University of North Caro-lina, Chapel Hill (NC, USA). We have utilizedthis system in our laboratory with our BEHMmodel. Each bioreactor allows up to 11 indi-vidual tissue specimens to be subjected as agroup to the same mechanical stimulation pro-tocol. The tissue culture plates are positionedon a movable platform that is controlled by astepper motor driver circuit. The stepper con-troller circuit allows for direct user interfaceand control of each bioreactor, both for systemset-up and to initiate the experimental proto-col. The actual mechanical strain protocol isprogrammed directly onto the embeddedmicrocontroller prior to each experiment. Thestimulation protocols can be carried out forlong periods of time (weeks or months) whilepermitting the culture media to be changedmanually, as would occur in traditional cell cul-ture using petri dishes. The design of the biore-actor is suitable for transforming the static-engineered muscle culture for dynamicmechanical loading studies without physicaltransfer of the construct. Using this system, wehave shown that stretching the BEHMs at a fre-quency of 1 Hz for 7 days results in a twofoldincrease in specific force.

We have developed a microperfusion systemin our laboratory, in collaboration with Des-mond Radnoti, CEO, Radnoti Glass Inc, tosupport the formation of cardiac muscle [Rad-

noti D, Birla RK: Unpublished Data]. Our system con-sists of a custom biochamber designed toaccommodate 11 tissue culture plates, eachplate stacked vertically on an independentstage (Figure 6). Each plate receives an indepen-dent media flow with independent outflowmanifolds for media aspiration. The internalenvironment of the biochamber is carefullyregulated to maintain temperature and carbondioxide. The biochamber is surrounded by awater jacket to permit flow of heated fluid fortemperature control. Carbon dioxide isdirectly injected into the biochamber andmaintained at a predefined level by feedbackcontrol using a solenoid valve. Cell culture

media is maintained at a constant temperatureusing a water-jacketed reservoir and is oxygen-ated prior to entering the biochamber. Mediaoxygenation is achieved using one of two pos-sible methods: direct bubbling of oxygen inthe media or perfusing the media through ahollow chamber oxygenator. The temperature,pH, oxygen saturation and carbon dioxide val-ues are measured and recorded in real time atmultiple points within the system. The mainadvantage of our system is that is does notrequire the use of a cell-culture incubator,thereby permitting complete user control ofthe cell culture environment.

Utility of bioengineered in vitro models for basic researchAs new layers of complexity are continuallyadded to our understanding of basic biologicalprocesses, interdisciplinary approaches will beincreasingly necessary for successful researchadvances. This is demonstrated by the upwardtendency of funding agencies to provide sub-stantial support for program projects involv-ing many contributors from a variety oflaboratories and the growing number of majorpublications with multiple authors fromdiverse scientific backgrounds. Tissue engi-neering combines principles of engineeringand biology, leading to seemingly endlessapplications. However, the tremendous poten-tial of bioengineered tissues for in vitro basicresearch applications has only recently beenrecognized.

Cells interpret cues from their immediatemicroenvironment and the growth and differ-entiation of many cell types is regulated by theinterplay of four major signaling sources(Figure 7):

• Soluble growth factors

• Insoluble ECM and growth substrates

• Environmental stress and physical cues

• Cell–cell interactions [116]

Given the complex mechanical and biochemi-cal interplay, researchers will miss biologicalsubtleties by only considering cells grown in a2D manner [117]. On the other hand, 3D tissueexplants are complex, being composed of sev-eral different cell types that are difficult toexperimentally manipulate individually. Forthese reasons, tissue engineering has emergedas a powerful technology where isolated cellscan be bioengineered into 3D, homogeneoustissues with characteristics similar to those



138

Figure 6. Microperfu

The biochamber consistsindependently from eachbiochamber is controlledtemperature and is oxygespecified flow rate, aspirapromote uniform distribuembedded at several poi

Thermocirculator

observed in vivo. In addition, bioengineeredtissues can be maintained for weeks or monthsin culture under physiological conditions.

Incorporation of tissue-engineering compo-nents with cellular/molecular-based projects hasbecome a powerful approach for basic research.For example, researchers have used an integratedapproach to examine the direct effects ofmicroenvironment on stem cell specification[118]. By varying matrix elasticity and measuringcell morphology, transcript profiles, marker pro-teins and the stability of responses, they demon-strated that naive mesenchymal stem cellsspecify lineage and commit to phenotypes withextreme sensitivity to tissue-level elasticity.

Bioengineered tissues can be more easilymanipulated for experiments, including transfec-tion of cells with expression vectors to control

expression of an endogenous protein or to giveexpression of mutated proteins. The use of tetra-cycline-regulated gene expression in bioengi-neered skeletal muscle tissues has beendemonstrated. Although macroscale animal test-ing remains the primary method used for evalu-ation of toxicological and pharmacologicalprofiles of a therapeutic agent, it is possible toreduce the number of experimental animalsused in the future with the use of in vitrobioengineered models. Researchers have devel-oped a microfluidic device consisting of an arrayof channels and 3D tissues embedded in cham-bers, which can be used to screen drug efficacyand toxicity in multiple tissues simultaneously.Convergence of new technologies may providefeasibility for the development and use of amicroscale bioengineered animal-on-a-chip.

sion for heart muscle.

of multiple independent stages to accommodate tissue culture plates. Media delivery and aspiration takes place plate via manifolds that are positioned directly above the tissue culture plates. The internal environment of the to regulate temperature and CO2 level. Media is maintained in a water-jacketed reservoir to maintain nated prior to reaching the plates. The system is run on several pumps that are used to deliver media at a te spent media from the plates, promote mixing of the gases inside the biochamber to equalize CO2 levels and tion of moist air. All variables (temperature, CO2, O2, pH and flow-rate) are monitored in real time via sensors

nts throughout the system.

Vacuumflask

Oxygenbubbler

Water-jacketedmedia resevoir

Peristalticpump

Fluid outFluid in

BiochamberInflowmanifold

Outflowmanifold

Standard 35-mm cell culture plates

Oxygen/temperature/pH sensors

Oxygen/temperature/pH sensors

Regenerative Medicine




Figure 7. Tissue eng

Cellular growth/prolifera(2) insoluble ECM and grmanner lack the complexin vitro, we can more adetissues allows for increasCAM: Calmodulin; ECM:

Researchers can manipulate their materialsto control the biology of the cells growingwithin. The use of biological feedback mecha-nisms in growth factor delivery has also beenexplored [119]. For example, a growth factorbound to the matrix and released upon cellular

demand through cell-mediated localized pro-teolytic cleavage from the matrix, which sub-stantially mimics the mechanism by whichthese factors are released in vivo from stores inthe natural ECM by invading cells in tissuerepair [120,121].

ineering for basic research.

tion and differentiation is regulated by the interplay of four major signaling sources: (1) soluble growth factors, owth substrates, (3) environmental stress and physical cues and (4) cell–cell interactions. Cells grown in a 2D mechanical and biochemical interplay, which occurs naturally in vivo. By developing 3D bioengineered models quately replicate tissues with characteristics similar to those observed in vivo. The 3D shape of bioengineered

ed cell–cell interplay, leading to increased ECM production. Extracellular matrix; GAG: Glycosaminoglycan.



140

The main motivation for the developmentof bioengineered constructs seems to be theirin vivo utility. Although this is likely to be onevery significant application of the constructs,this realization may not occur within the short-term as cardiac tissue engineering is still in itsearly stages of development. However, otherpotential applications of bioengineered con-structs exist and utility as a tool for basicresearch is one example.

Future perspectiveThere have been several important technologi-cal breakthroughs in the field of functional car-diac tissue engineering during the past decade.Using current technology, primary isolated car-diac cells can be programmed to form func-tional 3D constructs in vitro. Severalbiomaterials have been demonstrated to sup-port heart muscle development in vitro andvarious cell sources have been utilized to studycardiac regeneration in vivo. However, severalcritical technological hurdles need to beaddressed in order for the field of cardiac tissueengineering to advance.

Identification of a suitable cell source forcardiac tissue engineering still remains a chal-lenging task. Contractile cells derived fromcardiac origin are difficult to source, while cellsfrom a skeletal origin have not demonstratedthe necessary fatigue resistance required forcardiac applications. Human ESCs are barredby technological advancements demonstratingfate programming in addition to the politicalbattles that need to be overcome. Adult-derived stem cells are yet to prove useful for car-diac applications. Although several approachesshow promise, a clear vision has not been estab-lished for the identification of a suitable cellsource with the potential to be utilized for basicresearch, several leading to translational andfinally clinical applicability.

There needs to be a new generation of bio-materials, developed specifically for cardiactissue-engineering applications. Although thecurrent materials have adequately supportedthe formation of 3D cardiac tissues in vitro,the mechanical performance and biologicalproperties of these bioengineered tissues donot resemble the properties of native mamma-lian cardiac tissue. Novel biomaterials need bedeveloped to more closely match the proper-ties of cardiac ECM in terms of content andorientation of structural proteins. In addition,

new biomaterials need to be programmed withfeedback control mechanisms to respond tochanges in the local environment. As an exam-ple of feedback control of biomaterials, gener-ation of ECM by cardiac cells would result inprogrammed degradation of the material inproportion to the rate of synthesis ofthe ECM.

Development of a new generation of biore-actors and microperfusion systems presentsanother technological hurdle for the field offunctional cardiac tissue engineering. The cur-rent body of literature clearly supports the needto utilize bioreactors for the formation of func-tional tissue-engineered constructs. Currentsystems are fairly complex and involve somecombination of electro–mechanical stimula-tion in the presence of microperfusion. Onechallenge would be to develop the ability toevaluate the changes in the functional perfor-mance outcome of the tissue construct in realtime. As a simple example of real-time func-tional assessment, it would be valuable to mon-itor the contractile performance of 3D cardiacconstructs during long-term culture. Anotherchallenge for bioreactor/microperfusion devel-opment would be the implementation of feed-back control. As an example, as maturation oftissue engineered constructs takes place,changes in the cellular environment wouldneed to be matched by changes in the rate ofmicroperfusion to adequately support theincreased metabolic demands.

In conclusion, although several technologi-cal milestones have been accomplished withinthe field of cardiac tissue engineering, someimportant challenges remain. Future studiesdesigned to address these issues will helpdefine the field of functional cardiac tissueengineering.

Acknowledgements

The authors would like to thank Elisa Manzotti, Editorof Regenerative Medicine, for giving us the opportunity toprepare this article. We would also like to thank KarenRowland, Head of the graphics design team at Regenera-tive Medicine, for preparing the images used in this arti-cle. RB would like to thank the Section of CardiacSurgery at the University of Michigan for financial sup-port that allowed timely completion of this article. Finan-cial support for LH was provided by the American HeartAssociation (predoctoral fellowship grant # 0615557Z).We would like to thank everyone at the Artificial HeartLaboratory for supporting the preparation of this article.



Bibliography1. Fuchs JR, Nasseri BA, Vacanti JP: Tissue

engineering: a 21st century solution to surgical reconstruction. Ann. Thorac. Surg. 72(2), 577–591 (2001).

2. Langer R, Vacanti JP: Tissue engineering. Science 260(5110), 920–926 (1993).

3. Lysaght MJ, Reyes J: The growth of tissue engineering. Tissue Eng. 7(5), 485–493 (2001).

4. Nerem RM: Tissue engineering: the hope, the hype, and the future. Tissue Eng. 12(5), 1143–1150 (2006).

5. Vacanti CA: History of tissue engineering and a glimpse into its future. Tissue Eng. 12(5), 1137–1142 (2006).

6. Cleland JG, Khand A, Clark A: The heart failure epidemic: exactly how big is it? Eur. Heart J. 22(8), 623–626 (2001).

7. Goldstein S: Heart failure therapy at the turn of the century. Heart Fail. Rev. 6(1), 7–14 (2001).

8. Miniati DN, Robbins RC: Heart transplantation: a thirty-year perspective. Ann. Rev. Med. 53, 189–205 (2002).

9. Akins RE: Can tissue engineering mend broken hearts? Circ. Res. 90(2), 120–122 (2002).

10. Eschenhagen T, Didie M, Heubach J, Ravens U, Zimmermann WH: Cardiac tissue engineering. Transplant Immunol. 9(2–4), 315–321 (2002).

11. Fedak PW, Weisel RD, Verma S, Mickle DA, Li RK: Restoration and regeneration of failing myocardium with cell transplantation and tissue engineering. Semin. Thorac. Cardiovasc. Surg. 15(3), 277–286 (2003).

12. Mann BK, West JL: Tissue engineering in the cardiovascular system: progress toward a tissue engineered heart. Anatomical Record 263(4), 367–371 (2001).

13. Papadaki M: Cardiac muscle tissue engineering. IEEE Eng. Med. Biol. Mag. 22(3), 153–154 (2003).

14. Shimizu T, Yamato M, Kikuchi A, Okano T: Cell sheet engineering for myocardial tissue reconstruction. Biomaterials 24(13), 2309–2316 (2003).

15. Zimmermann WH, Eschenhagen T: Cardiac tissue engineering for replacement therapy. Heart Fail. Rev. 8(3), 259–269 (2003).

16. Weinberg CB, Bell E: A blood vessel model constructed from collagen and cultured vascular cells. Science 231(4736), 397–400 (1986).

17. L'Heureux N, Paquet S, Labbe R, Germain L, Auger FA: A completely biological tissue-engineered human blood vessel. FASEB J. 12(1), 47–56 (1998).

18. Thomas AC, Campbell GR, Campbell JH: Advances in vascular tissue engineering. Cardiovas. Pathol. 12(5), 271–276 (2003).

19. Nerem RM, Seliktar D: Vascular tissue engineering. Annu. Rev. Biomed. Eng. 3, 225–243 (2001).

20. Grassl ED, Oegema TR, Tranquillo RT: A fibrin-based arterial media equivalent. J. Biomed. Mater. Res. A 66(3), 550–561 (2003).

21. Stock UA, Vacanti JP, Mayer JE Jr, Wahlers T: Tissue engineering of heart valves – current aspects. Thorac. Cardiovasc. Surg. 50(3), 184–193 (2002).

22. Butany J, Ahluwalia MS, Munroe C et al.: Mechanical heart valve prostheses: identification and evaluation. Cardiovas. Pathol. 12(1), 1–22 (2003).

23. Butany J, Fayet C, Ahluwalia MS et al.: Biological replacement heart valves. Identification and evaluation. Cardiovas. Pathol. 12(3), 119–139 (2003).

24. Sapirstein JS, Smith PK: The ‘ideal’ replacement heart valve. Am. Heart J. 141(5), 856–860 (2001).

25. Bader A, Schilling T, Teebken OE et al.: Tissue engineering of heart valves – human endothelial cell seeding of detergent acellularized porcine valves. Eur. J. Cardiothorac. Surg. 14(3), 279–284 (1998).

26. Shinoka T, Breuer CK, Tanel RE et al.: Tissue engineering heart valves: valve leaflet replacement study in a lamb model. Ann. Thorac. Surg. 60(6 Suppl.), S513–S516 (1995).

27. Sodian R, Sperling JS, Martin DP, Stock U, Mayer JE Jr, Vacanti JP: Tissue engineering of a trileaflet heart valve-early

Executive summary

• The aim of cardiovascular tissue engineering is to develop functional models of cardiovascular structures to augment, repair and/or replace failing myocardium.

• Models have been developed for heart muscle, trileaflet valves, vascular substitutes and cell-based cardiac pumps.

• Integration of core technologies and disciplines is critical for the development of cardiovascular structures and requires a collaborative effort between experts in the life sciences, medical profession and engineering.

• Current models of 3D heart muscle utilize animal-derived cells, although alternative cell sources are being evaluated.

• Novel biomaterials are being developed with specifically engineered bioactivity to provide a mechanism for targeted growth factor delivery.

• Self-organization, biodegradable hydrogels and polymeric scaffolds have been evaluated as potential platforms for the development of heart muscle.

• Bioreactors have been developed to deliver controlled mechanical stretch to bioengineered heart muscle resulting in significant functional improvement of the constructs.

• Microperfusion systems have been developed to promote the functional remodeling of isolated cardiac cells without the need for cell culture incubators, thereby permitting greater control over the culture conditions.

• Tissue engineering models provide an excellent tool for basic research as the constructs can be maintained in culture for extended time periods, often in the order of months, thereby permitting multiple opportunities for interventions.

• Future developments in the field will need to identify a clinically viable cell source, promote the development of novel bioactive biomaterials and engineer a new generation of bioreactors/microperfusion systems with embedded feedback control.



in vitro experiences with a combined polymer. Tissue Eng. 5(5), 489–494 (1999).

28. Stock UA, Nagashima M, Khalil PN et al.: Tissue-engineered valved conduits in the pulmonary circulation. Ann. Thorac. Cardiovasc. Surg. 119(4 Pt 1), 732–740 (2000).

29. Zeltinger J, Landeen LK, Alexander HG, Kidd ID, Sibanda B: Development and characterization of tissue-engineered aortic valves. Tissue Eng. 7(1), 9–22 (2001).

30. Stevenson LW, Kormos RL: Mechanical cardiac support 2000: current applications and future trial design. J. Heart Lung Transplant. 20(1), 1–38 (2001).

31. Nag AC, Zak R: Dissociation of adult mammalian heart into single cell suspension: an ultrastructural study. J. Anatomy 129(Pt 3), 541–559 (1979).

32. Caspi O, Lesman A, Basevitch Y et al.: Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circ. Res. 100(2), 263–272 (2007).

33. Li RK, Yau TM, Sakai T, Mickle DA, Weisel RD: Cell therapy to repair broken hearts. Can. J. Cardiol. 14(5), 735–744 (1998).

34. Laflamme MA, Murry CE: Regenerating the heart. Nat. Biotechnol. 23(7), 845–856 (2005).

35. Murry CE, Field LJ, Menasche P: Cell-based cardiac repair: reflections at the 10-year point. Circulation 112(20), 3174–3183 (2005).

36. Taylor DA, Silvestry SC, Bishop SP et al.: Delivery of primary autologous skeletal myoblasts into rabbit heart by coronary infusion: a potential approach to myocardial repair. Proc. Assoc. Am. Physicians 109(3), 245–253 (1997).

37. Chiu RC, Zibaitis A, Kao RL: Cellular cardiomyoplasty: myocardial regeneration with satellite cell implantation. Ann. Thorac. Surg. 60(1), 12–18 (1995).

38. Zibaitis A, Greentree D, Ma F, Marelli D, Duong M, Chiu RC: Myocardial regeneration with satellite cell implantation. Transplant. Proc. 26(6), 3294 (1994).

39. Murry CE, Wiseman RW, Schwartz SM, Hauschka SD: Skeletal myoblast transplantation for repair of myocardial necrosis. J. Clin. Invest. 98(11), 2512–2523 (1996).

40. Robinson SW, Cho PW, Levitsky HI et al.: Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: long-term survival and phenotypic modification of implanted myoblasts. Cell Transplant. 5(1), 77–91 (1996).

41. Atkins BZ, Lewis CW, Kraus WE, Hutcheson KA, Glower DD, Taylor DA: Intracardiac transplantation of skeletal myoblasts yields two populations of striated cells in situ. Ann. Thorac. Surg. 67(1), 124–129 (1999).

42. Dawn B, Bolli R: Bone marrow cells for cardiac regeneration: the quest for the protagonist continues. Cardiovasc. Res. 65(2), 293–295 (2005).

43. Vanelli P, Beltrami S, Cesana E et al.: Cardiac precursors in human bone marrow and cord blood: in vitro cell cardiogenesis. Ital. Heart J. 5(5), 384–388 (2004).

44. Shim WS, Jiang S, Wong P et al.: Ex vivo differentiation of human adult bone marrow stem cells into cardiomyocyte-like cells. Biochem. Biophys. Res. Commun. 324(2), 481–488 (2004).

45. Hattan N, Kawaguchi H, Ando K et al.: Purified cardiomyocytes from bone marrow mesenchymal stem cells produce stable intracardiac grafts in mice. Cardiovasc. Res. 65(2), 334–344 (2005).

46. Fernandez-Aviles F, San Roman JA, Garcia-Frade J et al.: Experimental and clinical regenerative capability of human bone marrow cells after myocardial infarction. Circ. Res. 95(7), 742–748 (2004).

47. Rehman J, Li J, Orschell CM, March KL: Peripheral blood ‘endothelial progenitor cells’ are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 107(8), 1164–1169 (2003).

48. Kocher AA, Schuster MD, Szabolcs MJ et al.: Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7(4), 430–436 (2001).

49. Laflamme MA, Gold J, Xu C et al.: Formation of human myocardium in the rat heart from human embryonic stem cells. Am. J. Pathol. 167(3), 663–671 (2005).

50. Kehat I, Khimovich L, Caspi O et al.: Electromechanical integration of cardiomyocytes derived from human embryonic stem cells. Nat. Biotechnol. 22(10), 1282–1289 (2004).

51. Xue T, Cho HC, Akar FG et al.: Functional integration of electrically active cardiac derivatives from genetically engineered human embryonic stem cells with quiescent recipient ventricular

cardiomyocytes: insights into the development of cell-based pacemakers. Circulation 111(1), 11–20 (2005).

52. Mummery C, Ward-van Oostwaard D, Doevendans P et al.: Differentiation of human embryonic stem cells to cardiomyocytes: role of coculture with visceral endoderm-like cells. Circulation 107(21), 2733–2740 (2003).

53. Xu C, Police S, Rao N, Carpenter MK: Characterization and enrichment of cardiomyocytes derived from human embryonic stem cells. Circ. Res. 91(6), 501–508 (2002).

54. Amit M, Itskovitz-Eldor J: Derivation and spontaneous differentiation of human embryonic stem cells. J. Anatomy 200(Pt 3), 225–232 (2002).

55. Kehat I, Kenyagin-Karsenti D, Snir M et al.: Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes. J. Clin. Invest. 108(3), 407–414 (2001).

56. Sachinidis A, Fleischmann BK, Kolossov E, Wartenberg M, Sauer H, Hescheler J: Cardiac specific differentiation of mouse embryonic stem cells. Cardiovasc. Res. 58(2), 278–291 (2003).

57. Zandstra PW, Bauwens C, Yin T et al.: Scalable production of embryonic stem cell-derived cardiomyocytes. Tissue Eng. 9(4), 767–778 (2003). Erratum appears in Tissue Eng. 9(6), 1331 (2003).

58. Zweigerdt R, Burg M, Willbold E, Abts H, Ruediger M: Generation of confluent cardiomyocyte monolayers derived from embryonic stem cells in suspension: a cell source for new therapies and screening strategies. Cytotherapy 5(5), 399–413 (2003).

59. Schroeder M, Niebruegge S, Werner A et al.: Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control. Biotechnol. Bioeng. 92(7), 920–933 (2005).

60. van Vliet P, Sluijter JP, Doevendans PA, Goumans MJ: Isolation and expansion of resident cardiac progenitor cells. Expert Rev. Cardiovasc. Ther. 5(1), 33–43 (2007).

61. Davis ME, Hsieh PC, Grodzinsky AJ, Lee RT: Custom design of the cardiac microenvironment with biomaterials. Circ. Res. 97(1), 8–15 (2005).

62. Hollister SJ: Porous scaffold design for tissue engineering. Nat. Mater. 4(7), 518–524 (2005).

63. Lutolf MP, Hubbell JA: Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23(1), 47–55 (2005).



64. Ratner BD, Bryant SJ: Biomaterials: where we have been and where we are going. Annu. Rev. Biomed. Eng. 6, 41–75 (2004).

65. Christman KL, Lee RJ: Biomaterials for the treatment of myocardial infarction. J. Am. Coll. Cardiol. 48(5), 907–913 (2006).

66. Bursac N, Papadaki M, Cohen RJ et al.: Cardiac muscle tissue engineering: toward an in vitro model for electrophysiological studies. Am. J. Physiol. 277(2 Pt 2), H433–H444 (1999).

67. Carrier RL, Papadaki M, Rupnick M et al.: Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization. Biotechnol. Bioeng. 64(5), 580–589 (1999).

68. Papadaki M, Bursac N, Langer R, Merok J, Vunjak-Novakovic G, Freed LE: Tissue engineering of functional cardiac muscle: molecular, structural, and electrophysiological studies. Am. J. Physiol. Heart Circ. Physiol. 280(1), H168–H178 (2001).

69. Radisic M, Euloth M, Yang L, Langer R, Freed LE, Vunjak-Novakovic G: High-density seeding of myocyte cells for cardiac tissue engineering. Biotechnol. Bioeng. 82(4), 403–414 (2003).

70. Eschenhagen T, Fink C, Remmers U et al.: Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11(8), 683–694 (1997).

71. Eschenhagen T, Didie M, Munzel F, Schubert P, Schneiderbanger K, Zimmermann WH: 3D engineered heart tissue for replacement therapy. Basic Res. Cardiol. 97(Suppl. 1), I146–I152 (2002).

72. Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen T: Three-dimensional engineered heart tissue from neonatal rat cardiac myocytes. Biotechnol. Bioeng. 68(1), 106–114 (2000).

73. Zimmermann WH, Schneiderbanger K, Schubert P et al.: Tissue engineering of a differentiated cardiac muscle construct. Circ. Res. 90(2), 223–230 (2002).

74. Dar A, Shachar M, Leor J, Cohen S: Optimization of cardiac cell seeding and distribution in 3D porous alginate scaffolds. Biotechnol. Bioeng. 80(3), 305–312 (2002).

75. Davis ME, Hsieh PC, Grodzinsky AJ, Lee RT: Custom design of the cardiac microenvironment with biomaterials. Circ. Res. 97(1), 8–15 (2005).

76. Rosso F, Marino G, Giordano A, Barbarisi M, Parmeggiani D, Barbarisi A: Smart materials as scaffolds for tissue engineering. J. Cell. Physiol. 203(3), 465–470 (2005).

77. Zisch AH, Lutolf MP, Ehrbar M et al.: Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth. FASEB J. 17(15), 2260–2262 (2003).

78. Ehrbar M, Djonov VG, Schnell C et al.: Cell-demanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circ. Res. 94(8), 1124–1132 (2004).

79. Seliktar D, Zisch AH, Lutolf MP, Wrana JL, Hubbell JA: MMP-2 sensitive, VEGF-bearing bioactive hydrogels for promotion of vascular healing. J. Biomed. Mater. Res. A 68(4), 704–16 (2004).

80. Kusonwiriyawong C, van de WP, Hubbell JA, Merkle HP, Walter E: Evaluation of pH-dependent membrane-disruptive properties of poly(acrylic acid) derived polymers. Eur. J. Pharm. Biopharm. 56(2), 237–246 (2003).

81. Napoli A, Valentini M, Tirelli N, Muller M, Hubbell JA: Oxidation-responsive polymeric vesicles. Nature Materials 3(3), 183–189 (2004).

82. Napoli A, Boerakker MJ, Tirelli N, Nolte RJ, Sommerdijk NA, Hubbell JA: Glucose-oxidase based self-destructing polymeric vesicles. Langmuir 9, 3487–3491 (1920).

83. Shimizu T, Yamato M, Akutsu T et al.: Electrically communicating three-dimensional cardiac tissue mimic fabricated by layered cultured cardiomyocyte sheets. J. Biomed. Mater. Res. 60(1), 110–117 (2002).

84. Okano T, Yamada N, Sakai H, Sakurai Y: A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 27(10), 1243–1251 (1993).

85. Shimizu T, Yamato M, Kikuchi A, Okano T: Two-dimensional manipulation of cardiac myocyte sheets utilizing temperature-responsive culture dishes augments the pulsatile amplitude. Tissue Eng. 7(2), 141–151 (2001).

86. Okano T, Yamada N, Okuhara M, Sakai H, Sakurai Y: Mechanism of cell detachment from temperature-modulated, hydrophilic–hydrophobic polymer surfaces. Biomaterials 16(4), 297–303 (1995).

87. Akins RE, Boyce RA, Madonna ML et al.: Cardiac organogenesis in vitro: reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells. Tissue Eng. 5(2), 103–118 (1999).

88. Li RK, Yau TM, Weisel RD et al.: Construction of a bioengineered cardiac graft. Ann. Thorac. Cardiovasc. Surg. 119(2), 368–375 (2000).

89. Ozawa T, Mickle DA, Weisel RD et al.: Histologic changes of nonbiodegradable and biodegradable biomaterials used to repair right ventricular heart defects in rats. Ann. Thorac. Cardiovasc. Surg. 124(6), 1157–1164 (2002).

90. Ozawa T, Mickle DA, Weisel RD, Koyama N, Ozawa S, Li RK: Optimal biomaterial for creation of autologous cardiac grafts. Circulation 106(12 Suppl. 1), I176–I182 (2002).

91. Li RK, Jia ZQ, Weisel RD, Mickle DA, Choi A, Yau TM: Survival and function of bioengineered cardiac grafts. Circulation 100(19 Suppl.), II63–I169 (1999).

92. Sakai T, Li RK, Weisel RD et al.: The fate of a tissue-engineered cardiac graft in the right ventricular outflow tract of the rat. Ann. Thorac. Cardiovasc. Surg. 121(5), 932–942 (2001).

93. Leor J, Aboulafia-Etzion S, Dar A et al.: Bioengineered cardiac grafts: a new approach to repair the infarcted myocardium? Circulation 102(19 Suppl. 3), III56–III61 (2000).

94. Dennis RG, Kosnik PE: Excitability and isometric contractile properties of mammalian skeletal muscle constructs engineered in vitro. In vitro Cell. Dev. Biol. 36(5), 327–335 (2000).

95. Hecker L, Baar K, Dennis RG, Bitar KN: Development of a three-dimensional physiological model of the internal anal sphincter bioengineered in vitro from isolated smooth muscle cells. Am. J. Physiol. Gastrointest. Liver Physiol. 289(2), G188–G196 (2005).

96. Huang YC, Dennis RG, Larkin L, Baar K: Rapid formation of functional muscle in vitro using fibrin gels. J. Appl. Physiol. 98(2), 706–713 (2005).

97. Kosnik PE, Faulkner JA, Dennis RG: Functional development of engineered skeletal muscle from adult and neonatal rats. Tissue Eng. 7(5), 573–584 (2001).

98. Baar K, Birla R, Boluyt MO, Borschel GH, Arruda EM, Dennis RG: Self-organization of rat cardiac cells into contractile 3-D cardiac tissue. FASEB J. 2, 275–277 (2005).

99. Birla RK, Borschel GH, Dennis RG, Brown DL: Myocardial engineering in vivo: formation and characterization of contractile, vascularized three-dimensional cardiac tissue. Tissue Eng. 11(5–6), 803–813 (2005).



100. Birla RK, Borschel GH, Dennis RG: In vivo conditioning of tissue-engineered heart muscle improves contractile performance. Artif. Organs 29(11), 866–875 (2005).

101. Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM: Bioreactor design for tissue engineering. J. Biosci. Bioeng. 100(3), 235–245 (2005).

102. Narita Y, Hata K, Kagami H, Usui A, Ueda M, Ueda Y: Novel pulse duplicating bioreactor system for tissue-engineered vascular construct. Tissue Eng. 10(7–8), 1224–1233 (2004).

103. Niklason LE, Gao J, Abbott WM et al.: Functional arteries grown in vitro. Science 284(5413), 489–493 (1999).

104. Sodian R, Lemke T, Fritsche C et al.: Tissue-engineering bioreactors: a new combined cell-seeding and perfusion system for vascular tissue engineering. Tissue Eng. 8(5), 863–870 (2002).

105. Solan A, Mitchell S, Moses M, Niklason L: Effect of pulse rate on collagen deposition in the tissue-engineered blood vessel. Tissue Eng. 9(4), 579–586 (2003).

106. Williams C, Wick TM: Perfusion bioreactor for small diameter tissue-engineered arteries. Tissue Eng. 10(5–6), 930–941 (2004).

107. Engelmayr GC Jr, Hildebrand DK, Sutherland FW, Mayer JE Jr, Sacks MS: A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials. Biomaterials 24(14), 2523–2532 (2003).

108. Hoerstrup SP, Sodian R, Daebritz S et al.: Functional living trileaflet heart valves grown in vitro. Circulation 102(19 Suppl. 3), III44–III49 (2000).

109. Hoerstrup SP, Sodian R, Sperling JS, Vacanti JP, Mayer JE Jr: New pulsatile bioreactor for in vitro formation of tissue engineered heart valves. Tissue Eng. 6(1), 75–79 (2000).

110. Dumont K, Yperman J, Verbeken E et al.: Design of a new pulsatile bioreactor for tissue engineered aortic heart valve formation. Artif. Organs 26(8), 710–714 (2002).

111. Engelmayr GC Jr, Hildebrand DK, Sutherland FW, Mayer JE Jr, Sacks MS: A novel bioreactor for the dynamic flexural stimulation of tissue engineered heart valve biomaterials. Biomaterials 24(14), 2523–2532 (2003).

112. Mol A, Driessen NJ, Rutten MC, Hoerstrup SP, Bouten CV, Baaijens FP: Tissue engineering of human heart valve leaflets: a novel bioreactor for a strain-based conditioning approach. Ann. Biomed. Eng. 33(12), 1778–1788 (2005).

113. Akhyari P, Fedak PW, Weisel RD et al.: Mechanical stretch regimen enhances the formation of bioengineered autologous cardiac muscle grafts. Circulation 106(12 Suppl. 1), I137–I142 (2002).

114. Carrier RL, Rupnick M, Langer R, Schoen FJ, Freed LE, Vunjak-Novakovic G: Perfusion improves tissue architecture of engineered cardiac muscle. Tissue Eng. 8(2), 175–188 (2002).

115. Fink C, Ergun S, Kralisch D, Remmers U, Weil J, Eschenhagen T: Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement. FASEB J. 14(5), 669–679 (2000).

116. Bottaro DP, Liebmann-Vinson A, Heidaran MA: Molecular signaling in bioengineered tissue microenvironments. Ann. N. Y. Acad. Sci. 961, 143–153 (2002).

117. Abbott A: Cell culture: biology’s new dimension. Nature 424(6951), 870–872 (2003).

118. Engler AJ, Sen S, Sweeney HL, Discher DE: Matrix elasticity directs stem cell lineage specification. Cell 126(4), 677–689 (2006).

119. Zisch AH, Lutolf MP, Hubbell JA: Biopolymeric delivery matrices for angiogenic growth factors. Cardiovas. Pathol. 12(6), 295–310 (2003).

120. Sakiyama-Elbert SE, Panitch A, Hubbell JA: Development of growth factor fusion proteins for cell-triggered drug delivery. FASEB J. 15(7), 1300–1302 (2001).

121. Zisch AH, Schenk U, Schense JC, Sakiyama-Elbert SE, Hubbell JA: Covalently conjugated VEGF – fibrin matrices for endothelialization. J. Control. Release 72(1–3), 101–113 (2001).


engineering the heart piece by piece: state of the art in cardiac tissue engineering

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