מאמר תרביות

1. Introduction

2. ‘Injectable’ platforms

3. Patch-based platforms

4. Future platform technologies

5. Expert opinion

Review

Engineering cell platforms formyocardial regenerationUdi Sarig & Marcelle Machluf†

Technion--Israel Institute of Technology, The Laboratory of Cancer, Drug Delivery & Mammalian

Cell Technology, Faculty of Biotechnology & Food Engineering, Haifa, Israel

Introduction: Various engineered ‘cell-platforms’ have been reported in

recent years for the possible treatment of myocardial infarction (MI) and

end-stage heart failure. These engineered platforms rely on two key factors:

cells and/or biomaterial scaffolds for the regeneration of the infarcted

heart tissue.

Areas covered: Two major cell-platform approaches are described and

broadly categorized as ‘injectable cell platforms’ and ‘patch-based cell

platforms’. The recent advancements in these cell-platforms in terms of their

relative successes in-vivo as well as their clinical feasibility are summarized.

Natural as well as synthetic scaffolds, with or without the cellular compo-

nent, are compared with cell based therapy alone. This review focuses on

achievements, as well as the gaps that are presently checking any progress

towards producing clinically relevant panacea for myocardial regeneration.

Expert opinion: Cardiac and induced pluripotent stem cells will probably be

the focus of future research. The combined cell-biomaterial scaffold therapy

is superior to cell therapy alone. Nevertheless, encouraging pre-clinical

successes have limited translation into clinical practice due to limited cell

survival post transplantation, inadequate construct thicknesses for human-

sized hearts and the traditional use of ‘flat (2D) tissue culture’ techniques.

The development of complementary dynamic 3D cultivation platforms will

probably lead to improved outcomes and enable fast screening of various

therapeutic approaches.

Keywords: cardiomyocyte cell sources, cell therapy, extracellular matrix (ECM),

injection- and patch-based cell platforms, myocardial infarction,

myocardial tissue engineering, scaffolds

Expert Opin. Biol. Ther. (2011) 11(8):1055-1077

1. Introduction

In myocardial infarction (MI) the blood supply to the cardiac muscle (myocardium)is halted, leading to profound cell necrosis, downstream of the blocked artery. Ascardiomyocytes (CM, the actively contracting heart cells) have a limited regenera-tion capacity, rapid inflammatory response takes over, leading to the formation ofa fibrotic scar tissue that is unable to actively contract and imposes an extra burdenon the remaining healthy myocardium. In the USA alone, approximately 1 millionindividuals suffer from acute MI annually, making it the leading cause of heart fail-ure [1]. Current pharmacological therapies and surgical interventions can slow theprogression to end-stage disease, but they rarely prevent or reverse the progressionof the failing state [2]. At present, the only therapeutic options available to treatpatients with terminal end-stage heart failure are heart transplantations or ventricu-lar assist devices (VADs). These options have significant cost and availability limita-tions, leading to vast research in emerging fields such as cell therapy and tissueengineering [3]. Such research has resulted in the development of various engineered

10.1517/14712598.2011.578574 © 2011 Informa UK, Ltd. ISSN 1471-2598 1055All rights reserved: reproduction in whole or in part not permitted

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‘cell-platforms’ that rely on two key factors: cells and/or bio-material scaffolds for the regeneration of the infarctedheart tissue.The cellular component, the more complex part of the

engineered cell-platform, has to contract, remodel and ulti-mately regenerate the defective myocardium. The ideal cellsource should be easy to obtain and cultivate in great num-bers as the native cardiac tissue is densely populated (~ 5 �108 cells/cm3) [4]. Furthermore, the cell source and/or its dif-ferentiated products should be biocompatible, to avoid rejec-tion post transplantation. Last but not least, cell safety is amatter of concern, particularly when referring to geneticallymodified cells.The heart muscle (i.e., myocardium) consists of at least

three basic cell types (i.e., 20 -- 40% CMs; 60 -- 80% car-diac fibroblasts and endothelial cells) [4]. Thus any appliedtissue engineering technique should be able to combinethese three cell populations resulting in CM that are elec-tromechanically coupled to the host tissue while having aproper vascular supply and connective tissue to ensurefunctionality [5]. However, as only CM contribute to func-tional contraction, and these cannot be expanded ex-vivo,several other CM cell sources have been suggested for thereplacement and regeneration of the infarcted heart,including embryonic stem cells (ESCs) [6-12], induced plu-ripotent stem cells (iPSCs) [11-18], mesenchymal stem cells(MSCs) [19-27], and cardiac stem cells (CSCs) [28-32]. Eachof these cell sources has its own pros and cons, as discussedin recent reviews [1,3,33].Nonetheless, cellular therapy by itself is still limited and does

not yet lead to sufficient cardiac tissue regeneration [3], demon-strating the need for other supporting, yet crucial factors, suchas the biomaterial scaffold. Biomaterial scaffolds serve to mimicthe natural extracellular matrix (ECM) and aim to provide thephysical and biological support to the grafted cells duringin-vitro/ ex-vivo cultivation, as well as to the host cardiac tissuepost transplantation. Ideally, these scaffolds should degradeto biocompatible byproducts in parallel to the secretion of

a substituting ECM by the host tissue and the grafted cells.Further incorporation of growth and pro-survival factors withinthese scaffolds may enhance tissue regeneration by affectingboth host and transplanted cells. However, the applicationand effect of such growth factors will be discussed briefly, asit is outside the scope of this review.

Despite the importance of these two key elements (i.e., cellsand biomaterial scaffolds), they cannot solely account for theperformance of engineered myocardial cell platforms withoutmechanical stimuli in the form of the cardiac rhythm andelectrical cues, which mimic the natural cardiac environment.This area of dynamic cultivation technologies is an emergingone that plays a fundamental role at the level of cell -- celland cell -- scaffold interactions. It is also thoroughly reviewedelsewhere [34,35].

In this article, recent cell platforms that have been engi-neered for myocardial regeneration are addressed (Figure 1).We discuss, in detail, their relative successes in-vivo and theirclinical feasibility. Furthermore, the most promising emergingtechnologies, particularly those that might contribute toimproved outcomes in-vivo are also described.

2. ‘Injectable’ platforms

Injection of cells alone, biomaterial alone or combinationsthereof, is a clinically preferred delivery technique, as it is con-sidered to be minimally invasive and a controlled procedure.The injection can be performed either directly into the infarctarea or, when cell size permits, through the coronary circula-tion [36]. Injected platforms can take the form of any infarctwith relatively little effort, delaying subsequent wall thinning.Furthermore, the ability of injected cells and active biomateri-als to attract host stem and progenitor cells into the infarctarea is compelling [37]. Studies have reported the injection ofcells alone or in combination with polymers such as liquidizedECM, collagen, fibrin, alginate, chitosan, hyaluronic acid(HA) hydrogels, and synthetic derivatives of poly(N-isopropy-lacrylamide) (PNIPAAM) and PEG (Tables 1,2,3). Thesetwo forms of injections will be discussed separately in thefollowing sections.

2.1 Injectable cell-therapyIn general, the contribution of cells to cardiac regeneration isbelieved to be mitigated through three independent mecha-nisms of action: angiogenesis, myogenesis and paracrineeffects [3]. The intracoronary injection of cells has shown lim-ited success in terms of their delivery efficiency into the myo-cardium and in terms of transplanted cells’ homing andsurvival, when compared with direct intramyocardial injec-tions [36]. This review focuses on the use of the most reportedand clinically relevant cell sources for CM differentiation:ESCs [10,38-51], iPSC [11-18], MSCs [52-65] and CSC [28-32].Nevertheless, as many studies have reported the utilizationof other cell types (such as skeletal myoblasts, bone marrowmononuclear cells and endothelial progenitor cells), we would

Article highlights.

. Cell platforms for myocardial regeneration can bebroadly categorized as either injection- or patch-basedplatforms.

. Combined cell--biomaterial scaffold platforms showsuperior results compared with cell therapy alone.

. Even though many reports have shown significantimprovement in pre-clinical studies, the translation intoclinical practice is still limited.

. We review the latest achievements and drawbacks ofthese strategies and mark the technological hurdlesblocking the achievement of clinically relevant solutions.

. Evolving technologies that may enable improvedpre-clinical outcomes are discussed.

This box summarizes key points contained in the article.

Engineering cell platforms for myocardial regeneration

1056 Expert Opin. Biol. Ther. (2011) 11(8)

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like to direct the reader to recent in depth reviews discussingthe cellular aspects of myocardial regeneration [3,66-68].

2.1.1 Embryonic stem cellsESCs have the ability to self renew and also to differentiatein a robust biochemical procedure into beating and contract-ing CM-like cells, having similar (although not identical)characteristics to that of the adult CM [6,7,11,41,69]. InjectedESC-derived CM have been shown to integrate with hostmyocardium and improve the electrical conduction ininfarcted mouse and rat hearts [38,41,44,50]. Similar resultswere obtained using human-ESC-derived CM in a porcinemodel [39] and early cardiac progenitors (stage-specificembyonic antigen (SSEA)+) of primate ESCs transplanted inan infarcted primate model [12].

Nonetheless these cells are considered immunogenic [67]

and are known to form teratomas post transplantationin-vivo [9,42,43,70]. Immunogenic rejection could be overcomewith the use of autologous induced pluripotent cells (iPSCs,further discussed hereafter) and the occurrence of teratomaformation is reduced by the selection of phenotypically dif-ferentiated CM produced from ESCs [9,42,51]. Further majordifficulties include collecting unadulterated hESC-derivedCM (hESC-CM) in large quantities and assuring their longterm survival. These represent major obstacles for cell-basedtherapy [10,44].

To increase hESC-CM yield, Laflamme et al., have utilizedactivin- A and bone morphogenetic protein 4 (BMP4). Theyfurther incorporated a pro survival factors’ cocktail (PSC) tolimit hESC-CM death post transplantation into a rat acute MImodel [41]. The engrafted human myocardium attenuated ven-tricular dilation and preserved regional and global contractilefunction compared with controls receiving non-cardiac hESCderivatives or vehicle. However, the significance of these resultshas been challenged by van Laake et al. who evaluated thelong-term (12 weeks) fate of hESC-CM transplanted in immu-nocompromised acute MI mice model. Their results exhibitedrapid formation of grafts with organized CM that havingmatured over time, were further accompanied with significantimprovement in cardiac function at 4 weeks post transplanta-tion, yet this was not sustained at 12 weeks [44]. From theseresults, it was argued that there is a requirement for a minimum3 month follow-up in studies that claim to observe improvedcardiac function. We further infer that this requirement is inde-pendent of whether hESC or other (adult) cell types are used fortransplantation. In a more recent study by the former group,Intramyocardial injection of hESC-CM with the same PSCinto chronic rat MI models was evaluated. Histology performedat the 3 month time point revealed that human CM survived,developed increased sarcomere organization andwere still prolif-erating. Despite successful engraftment, both echocardiographyand MRI analyses showed no significant difference in left

Injectable platforms Patch-based platforms

C.

B.

A. D.

E.

F.

Figure 1. Schematic representation of the major strategies for myocardial tissue engineering. These strategies rely on

cardiogenic cell sources, which are administered either alone or in combination with various natural or synthetic scaffolds.

Injectable platforms (A. -- C.) could be delivered through the vena cava (intravenous, A.), through the coronary arteries

(intracoronary, B.) or into the infarcted zone or its border (intramyocardial, C.). Patch based platforms (D. -- F.) include

scaffold-free or cell-sheet transplantation (D.), tissue engineered constructs made out of various cell--biomaterial scaffold

combinations (E.) or patches made out of biomaterial scaffolds alone (F.).

Sarig & Machluf

Expert Opin. Biol. Ther. (2011) 11(8) 1057

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Table

1.Cardiomyocyte

cellso

urces.

Cell

type

Cell

source

Advantages

Disadvantages

Pre-clinical

studies

Clinical

trials

ESCs

Embryo

BiochemicaldifferentiationtowardsCM

likebeating

cells

iswellcharacterized

Giverise

tootherregenerative

celltypes(i.e.,

endothelialcells

andfibroblasts)

Integrateswithhost

tissueandim

prove

conductivity

andfunction

Requireschronicim

munosuppression

Capable

ofteratomaform

ation

(particularlyifnotfully

differentiated)

Relatively

low

yield

ofCM

Ethicalissues

Murine[38,40-46,49,51,

136] ;Porcine[39]

Nonereported

iPSCs

Derm

alfibroblasts

Autologouscellsource(noim

munosuppression

required)

Knownbiochemicaldifferentiationtowards

CM-likebeatingcells

Integrate

withhost

tissueandim

prove

conductivity

andfunction

Requiresgeneticmanipulation

Relatively

low

yield

ofCM

Murine[16];Primate

[12]

Nonereported

MSCs

Bonemarrow

Umbilicalvein

Adipose

tissue

Dentalpulp


munosuppression

required)

Relatively

easy

toisolate

andculture

inlarge

quantities

Improve

cardiacfunction

Giverise

tootherregenerative

celltypes(i.e.,

endothelialcells

andvascularmuscle)

Nolong-term

effect/survivability

(>6months)

Little

integrationwithhost

tissue

Positive

effectsare

basedonparacrine

effectsandangiogenesisinductionrather

thanCM

differentiation

Norobust

andsafe

CM

differentiation

procedure

reportedto

date

Murine[21,56,59,60,133,

139,157,158,166,167,175,178,

190-191] ;Porcine[61-63]

Phase

I[72]

CSCs

Heart


munosuppression

required)

Relatively

easy

toisolate

andculture

inlarge

quantities

Improve

cardiacfunction

Secrete

acocktailofgrowth

andprosurvival

factors

thatprobably

best

fitmyocardial

regeneration

Giverise

tootherregenerative

celltypes

(i.e.,epithelialcells

andvascular

muscle)

Norobust

CM

differentiationprocedure

inlarge-anim

almodelsreportedto

date

Murine[29,31-32]

Phase

I[80]

CM:Cardiomyocytes;

CSCs:

Cardiacstem

cells;ESCs:

Embryonic

stem

cells;iPSCs:

Inducedpluripotentstem

cells;MSCs:

Mesenchym

alstem

cells.



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Table

2.Naturalbiomaterials

formyocardialcellplatform

s.

Polymer

Advantages

Disadvantages

Pre-clinicalstudies

Clinicaltrials

ECM

Closest

mim

icto

naturalcellsurroundings

Bioactive,biodegradable

andbiocompatible

Improvesmyocardialgeometryandfunction

Promotesvasculature

form

ation

Inducible

gellingmechanism

insitu

Highbatchto

batchvariability

More

problematicin

term

sofmass

production

Inconvenientisolationprocedures

Murine[41,83,84,86,87,150,157,158,

168,175] ;Canine[146,148];Porcine

[147,152]

Nonereported

Collagen/

Gelatin

Themajorbuildingblock

ofnaturalECM


andbiocompatible


Enhancedcellretention/survivalupontransplantation

Commercially

available

Inducible

gellingmechanism

insitu

Adverseeffectsonangiogenesisand

vasculature

form

ation

Murine[40,89-94,131,165-168,170,175,

176,178,180] ;Porcine[95]

Phase

I[179]

Fibrin

Biologicalglueandcelladhesive

withcontrolled

polymerizationanddegradationkinetics


andbiocompatible

Leadsto

improvedmyocardialgeometryandfunction

uponinjection

Nosignificantlongterm

effects

observedin

humanpatients

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

[98]

Phase

II[102]

Alginate

Biodegradable

andbiocompatible

Inducible

gellingmechanism

insitu

Commercially

available

atrelatively

low

cost

FDA

approved

Improvesmyocardialgeometry

andfunctionuponinjection

Low

mechanicalpropertiesmatchwith

themyocardium

Nosignificantim

provementin

vasculature

form

ationuponinjection

Adversebioactivity

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

On-going

Phase

II[109]

Chitosan

Biodegradable

andbiocompatible

Commercially

available

atrelatively

low

cost

Variable

form

ulationsallow

forcontrolledandversatile

end-product

mechanicalproperties

Inducible

gellingmechanismsin

situ


uponinjection

Promotesvasculature

form

ation

Mechanism

ofvasculature

support

are

poorly

understood

Absence

ofdata

onchitosanbioactivityand

contributionin

large-anim

almodels

Murine[46,113]

Nonereported

HA

Improvesmyocardialgeometryandfunctionupon

injection


andnon-immunogenic

Commercially

available

Absentdata

onbioactivityin

largeanim

al

models

Low

mechanicalpropertiesmatchwith

themyocardium

Relatively

expensive

Murine[116]

Nonereported

ECM:Extracellularmatrix;HA:Hyaluronic

acid.

Sarig & Machluf


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Table

3.Syntheticbiomaterials

formyocardialcellplatform

s.

Polymer

Advantages

Disadvantages

Pre-clinicalstudies

Clinicaltrials

PNIPAAM

(andderivatives)

Inducible

gellingmechanism

insitu

Promotesvasculature

form

ation

Celladherence

istemperature-dependent

Mechanicalpropertiescould

matchthose

of

thenative

myocardium

Controlledandrobust

manufacturingprocesses

Commercially

available

atrelatively

low

cost

Lacksbiodegradability

(derivative-dependent)

Poorphysiologicalclearance


Lack

ofdata

inlargeanim

almodels

Murine[126];Rabbit

[121,125]

Nonereported

PEG

(andderivatives)


uponinjection(derivative

dependent)

Bio-inert

Lacksbiodegradability


Poorphysiologicclearance


Lack

ofdata

onPEG

andderivativescontributionin

largeanim

almodels

Nosignificantim

provementin

vasculature

form

ation

uponinjection

Murine[128,129];

Rabbit

[130]

Nonereported

Polyesters

(e.g.,poly-a-

Hydroxy-acid;PLA

;PGA;

PCL;

PLG

A;PCLA

;PLC

L)

Biodegradable

andbiocompatible

Co-polymers

could

allow

foramore

controlled

degradationkinetics

Controlledandrobust

manufacturingprocesses

Commercially

available


Mightcause

localhighacidconcentrationsleading

toaninflammatory

response

Suddenloss

offunctionalintegrity

dueto

bulk

degradationkinetics

Usedprimarily

forpatch-basedplatform

sLow

mechanicalpropertiesmatchwiththemyocardium

Lack

ofdata

onpolyesters

contributionin

large-anim

al

models

Murine[45,138,

139,175,176,178]

Nonereported

Elastomers

(PU;poly(TMC)

andco-polymers

withPLA

/PCL;

PGS

Biodegradable

Biocompatible


Mechanicalpropertiescould

bemadeto

match

those

ofthemyocardium

Short-term

improvementin

myocardialgeometry

andfunction

Localinflammationcausedbydegradationbyproducts


Usedprimarily

forpatch-basedplatform

sLack

ofdata

onelastomers

contributionin

largeanim

al

models

Murine[49,141,

142,175]

Nonereported

NIPAAM:Poly(N-isopropylacrylamide);PCL:

Poly(e-caprolactone);PCLA

:Caprolactone-co-L-lactidespongereinforcedwithknittedpoly-L-lactidefabric;

PGA:Poly(glycolic

acid);PGS:Poly(glycerol-sebacate);PLA

:Poly(lactic

acid);PLC

L:Poly(lactide-co-e-caprolactone);PLG

A:Poly

(lactic

co-glycolic

acid);poly(TMC):Poly(1,3

trim

ethylenecarbonate);PU:Polyurethane.



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ventricular structure or function compared with controls. Thissuggested that cell transplantation should be conducted in theacute or sub-acute phases of MI and is probably not effectiveonce the fibrotic scar tissue is already formed at the chronicphase of MI [71]. This finding contradicts previous resultsreported by Caspi et al., who injected hESC-CM into a similarchronic infarction rat model, revealing attenuation of left ven-tricular dilatation and improved cardiac function one monthpost tranplantation [9].

Although more preclinical studies are needed beforesuch therapies can be implemented clinically, a recent FDAapproval for ESC-based clinical trials in human patientsfor the treatment of spinal injuries [72] is a positive signthat ESC technology may one day be applied to clinicalcardiac regeneration.

2.1.2 Induced pluripotent stem cellsIn the last few years efforts have been invested in reprogram-ming autologous dermal fibroblasts, both murine and human,into a pluripotent state to avoid immunogenic rejection. Thephenotype of isolated iPSCs is very similar to that of ESCsand thus CM isolation from an autologous source is possible,as demonstrated by several groups [13,16-18]. In addition, theability to create iPSCs, without the use of genome-integratedviral vectors and oncogenes, has been demonstrated recentlyand supports the notion of future clinical use [14,15,73].Nelson et al., have recently reported the intramyocardial deliv-ery of iPSCs into immune-competent adult MI mice model,yielding progeny that properly engrafted without disruptingcytoarchitecture. In contrast to parental non-reparative fibro-blasts, iPSCs treatment restored post-ischemic contractile per-formance, ventricular wall thickness and electrical stabilitywhile achieving in situ regeneration of cardiac, smooth muscleand endothelial tissue [16]. However, as the isolation techniquefor iPSC-derived CM, still relies on the micromanipulation ofbeating areas within the embryonic body [10], the ability to pro-duce the needed amount of CM-like cells still limits its applica-tion for clinical cell-based therapy. Furthermore, the limitedunderstanding of the process of iPSCs senescence, may hindertheir potential use in the elderly [74].

2.1.3 Mesenchymal stem cellsMSCs are multipotent stem cells isolated from several adulttissues including bonemarrow, adipose, umbilical vein and den-tal pulp [53,75]. Their ability to differentiate into CM like cellswas first demonstrated a decade ago by the exposure of murinebone marrow stromal cells to 5-azacytidine [19,20]. Even thoughthese results have not yet been reproduced with humanMSCs [57,58] and are probably dependent on the MSC tissue oforigin and on yet unknown signaling factors within the myocar-dial milieu [75], they serve as a proof of concept for the applicabil-ity of MSC in cardiac regeneration. In addition, MSCswere demonstrated to express cardiac-specific markers [23],which increase when exposed to neonatal CM-conditionedmedia [26,27,52]. Intracoronary [59,64], intravenous [56,60,64] and

intramyocardial [64,65] injection of MSCs alone, yieldedimproved results compared with controls in mice and rat animalmodels. However, MSCs did not exhibit long term (6 months)survivability [55] and their positive influence is largely due toparacrine effects [24] rather than differentiation into active CMcell populations.

Schuleri et al. reported the autologous intramyocardialinjection of MSCs in a swine chronic MI model. High-dose(200 million cells) MSC therapy reduced infarct size andincreased regional contractility in both infarct and borderzones [63]. They also reported that engrafted MSCs can differen-tiate into three lineages: CM, endothelial and vascular mus-cle [62]. Poncelet et al., have shown that allogeneic porcinemesenchymal stem cell transplantation enhanced angiogenesisafter myocardial infarction, yet this effect was limited to the via-ble myocardium in the borders of the infarct area [61]. Theseencouraging results have led to a recent clinical trial with alloge-neic MSCs administered intravenously to MI patients. Inaddition to significant improvement in cardiac function, noimmunosuppression was required post transplantation and nosafety issues were detected, hence demonstrating the feasibilityof using allogeneic MSC cell sources [54]. Unfortunately, thelack of a reproducible differentiation procedure holds back thedegree of regeneration that could be expected using this cellsource as non differentiated MSC do not actively contract.

2.1.4 Cardiac stem cellsThe traditional view that myocardium lacks regenerativecapacity, has been questioned in the past decade with thediscovery of cardiac progenitor cells [28,76-78] Several groupshave since reported the successful isolation of CSC frommurine [31,78], rat [28], porcine [32] and human hearts [30-32].Oh et al., performed magnetic enrichment of stem cellantigen positive cells (Sca1+) from mice hearts and differenti-ated them in-vitro upon exposure to 5-azcytidine [78].Messina et al., developed a robust technique for the isolationof CSC from cardiac explants of murine and human origin.Using a cardiosphere intermediate step, they isolated cellsthat are capable of self-renewal and give rise to vascular andcontractile cells. While murine cells exhibited spontaneousdifferentiation upon formation of cardiospheres, human cellsexhibited CM differentiation only upon exposure to rat neo-natal CM [31]. Transplantation into infarcted murine heartsled to functional improvement which was also reported in asubsequent collaborative study with the same group usinghuman and porcine CSC [31,32]. In a more recent study, Davisand co-workers further improved the isolation procedure byskipping the cardiosphere step altogether, while exhibitingsimilar results when transplanting the directly explanted crudepopulation into a rat MI model [29]. Despite the fact thatthe efficiency of the cardiosphere methodology has yet to bereproduced sufficiently, with some reports challenging thestemness of these cardiospheres [79], these cells are regardedas holding great promise for future cardiac regeneration andare currently under evaluation in a clinical trial [80].

Sarig & Machluf


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2.2 Biomaterial-injection-based platformsTo improve cell attachment, survivability and proliferation,studies have also focused on the incorporation of liquidizedbiomaterials. These biomaterials can be injected to the myo-cardium either alone, with various homing and growth factorsor in combination with various cell sources with the aim ofsupporting the infarcted scar tissue or recruiting endogenousstem and progenitor cells. These attractive biomaterials canbe rapidly polymerized in-situ by various chemical, thermalor optical stimuli [81], thus assuring site-specific delivery in aminimally invasive technique.Different injectable biomaterials have been utilized for

myocardial tissue engineering, which include both syntheticand natural biomaterials. Natural materials feature high bio-activity, biocompatibility and biodegradability. However,there is a high variability associated with their productionand no exact control of their composition and physical char-acteristics, hence standardization and quality control in massproduction becomes overly complex. In contrast, syntheticmaterials have a more robust manufacturing capability, allow-ing for a controlled biochemical composition and characteris-tics. Unfortunately, their bioactivity and biocompatibilityqualities are less than satisfactory in comparison with thenatural scaffolds.

2.2.1 Extracellular matrixThe cardiac ECM consists of a variety of structural proteins (col-lagen, elastin), adhesive proteins (fibronectin, laminin) andother active components (MMP, tissue inhibitors of MMP(TIMP) and plasmin) within a hydrated proteoglycan andglycosaminoglycan-rich milieu [82]. To mimic this complexenvironment, three distinct strategies have been employed: I.Using commercially manufactured ECM (MatrigelTM, BDBiosciences), II. Using acellular ECM and III. Using definedsingle ECM components or mimetics.Matrigel is a secreted ECM mixture that is commercially

manufactured, exploiting mouse tumor cell ECM production.It has been proven effective in the delivery of mouse ESCs tothe left ventricle (LV) either by injection into a pouch madein the left ventricle of rats [83] or by using a direct injectioninto the infarcted myocardium of a mouse model [84]. Boththese studies have resulted in improved heart geometry andfunction with no apparent side effects as compared with eithercells-only or Matrigel-only controls. Similar results were con-firmed by Zhang et al., who mixed Matrigel, collagen and cellculture medium for the delivery of neonatal CM into a rat MImodel [85]. In another study, Laflamme et al., formulated acocktail of pro-survival factors preventing ESC-derived CMdeath. This formula contained Matrigel, which was shownto independently prevent anoikis and increase vasculatureformation after injection into the infarcted myocardium [41].In an attempt to regenerate the infarcted heart using acellular

matrices, Singelyn et al., liquidized acellular cardiac ECM usingpepsin and subsequently injected it into the myocardium viacatheter in a rat MI model [86]. Gelation occurred within the

infarcted scar, leading to a significantly increased infiltrationof endothelial and smooth muscle cells 11 days post injectionalong with an increase in the number of arterioles. The groupalso reported similar results when using pericardial matrix [66].Zhao et al. injected an emulsion made out of acellular sub-intestinal ECM directly into the rat myocardium followingan ischemia--reperfusion event [87]. They reported an improve-ment in functional cardiac parameters (i.e., ejection fraction,fractional shortening and stroke volume) compared with con-trols given injections of saline solution. Taken together, theseresults raise a question as to the difference between variousECM origins, as to that which one has the optimal characteris-tics for promoting myocardial regeneration. As no reporteddata so far has compared these different materials, it is difficultto assess which would be most beneficial for long-term effect.Future experimentation may focus on elucidating this questionas well as on the incorporation of various CM cell sourcestogether with the injected acellular ECM.

2.2.2 CollagenAs the most abundant structural protein in our body’s ECM,collagen has been widely researched for its suitability forinjectable myocardial tissue engineering. So far 28 distinct typesof collagen have been described [88], of which, types I, III and Vare the most abundant in cardiac tissue. Most studies reportedimproved LV geometry and cardiac function when dissolvedcollagen was injected. However, a question arises as to its contri-bution to angiogenesis with some reporting no involvement [89]

whereas others showing improved capillary density and myofi-broblasts infiltration [90]. Researchers also attempted to incorpo-rate cells into injectable collagen or its gelatinous derivatives.According to these studies this approach resulted in improvedcell retention, survival and angiogenesis accompanied by abetter cardiac function compared with either cell-only orcollagen-only controls. Suuronen et al., have shown a significantrestoration of the vascular supply to ischemic tissues uponinjection of a collagen-based matrix with progenitor cells [91].Kutschka et al., further indicated, through bioluminescence,that injection of cardiomyoblasts into pre-transplanted collagenbased GelfoamTM (Pfizer) [92] or together with liquidizedGelfoam [93] enhances their survival in rat hearts comparedwith cell-only injections and non treated controls. Moreoverthe incorporation of reduced Matrigel (with no growth factors)together with the pre-transplanted Gelfoam matrix, showed aneven better improvement than that of cardiomyoblast injectedGelfoam alone. This may be attributed to other ECM compo-nents within the Matrigel that contributed to cell adhesionand survival [92,93]. In another study Zhang et al., usedpositron-emission tomography in addition to histology to assessthe bio-distribution of collagen-seeded progenitor cells. Theirresults showed enhanced cell retention and survival comparedwith cells-only-seeded controls [94].

Takehara et al., used gelatin, a collagen derivative, todeliver basic fibroblast growth factor (bFGF). This growthfactor, with or without gelatin, was injected into a porcine



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MI model alone or in combination with either humancardiosphere-derived cells (CDCs) or MSCs. Their resultssuggested increased microvasculature formation in the gelat-in--bFGF group compared with bFGF directly injected withno gelatin. They also reported a synergistic effect of bFGF--gelatin when combined with human CDCs (but not withMSCs), which included improved cardiac function, LV wallmotion, CM differentiation and infarct scar size [95].

2.2.3 FibrinFibrin, the active truncated form of fibrinogen (cleaved bythrombin), is an important physiological protein that hasbeen widely used in tissue engineering [96,97]. The attractivefeatures of fibrin are its ability to serve as a biological glue,holding cells together in the desired site as well as its abilityto stimulate angiogenesis [96]. Due to these properties, fibrinhas been used both as a delivery vehicle as well as a tissue engi-neering bioactive scaffold [97]. Several groups have shownimprovement in geometry and function of the LV when itwas subjected to fibrin alone or in combinations with variouscells (i.e., endothelial cells, skeletal myoblasts and bone mar-row mononuclear cells) compared with controls that receivedinjections of saline [98-100]. In addition, under physiologicalconditions, fibrin undergoes degradation in parallel to the for-mation of a healthy replacement tissue. This process can becontrolled using inhibitory drugs such as aprotinin [97,101],therefore enabling the adjustment of the degradation kineticswith that of substitute ECM production by the host andseeded cells. Fibrin polymerization kinetics in situ has beencharacterized by Martens et al., proving its suitability forcatheter-based delivery. By maintaining fibrinogen andthrombin concentrations below 5% w/v and under 20 U/mlrespectively, polymerization rates can be controlled to allowadequate time for delivery [81].

Despite these encouraging pre-clinical results in animalmodels, and the vast use of fibrin glues in the clinical setting,little is known on the effects of fibrin on MI in humanpatients. In a clinical trial aimed to reduce reperfusion injurypost MI, Atar et al., have shown that the intravenous bolusinjection of a fibrin derivative (FX06) led to a significantshort term effect on the size of the necrotic center withinthe infarct zone compared with controls receiving placeboinjections [102]. Yet, no significant long-term effect wasobserved apart from that there were fewer patients with seri-ous cardiac events in the treated groups compared with theplacebo group patients.

2.2.4 AlginateThe natural polysaccharide alginate forms compliant hydro-gels upon exposure to divalent ions such as Ca2+ [103]. Thisinduced gelling mechanism, the low cost of high-quantity pro-duction and most importantly the FDA approval for humanuse as a wound dressing material, makes alginate an ideal celldelivery platform for myocardial tissue engineering. Severalrecent studies [104-106], have demonstrated the effectiveness

of alginate injections as it had similar and in some casesbetter effects on the infarct size and function, compared withsaline [105], neonatal CM [104] or fibrin injections [106] inrodents. Alginate has also been reported to cause reverse remod-eling in swine [107]. Its positive effect on the remodeling andfunction of the left ventricle is probably related to its chemicaland physical traits [105]. Ruvinov et al., have developed aninjectable affinity-binding alginate enabling the sequentialdelivery of IGF-1 and hepatocyte growth factor (HGF). Theaffinity-binding alginate protected the proteins from proteolysiswhile maintaining their bioactivity as demonstrated by massspectroscopy and several in-vitro assays respectively. Intramyo-cardial delivery to a rat model of acute MI, preserved scar thick-ness, attenuated infarct expansion and reduced scar fibrosisafter 4 weeks, concomitantly with increased angiogenesis andmature blood vessel formation at the infarct. Apparently thistreatment prevented cell apoptosis, induced cardiomyocytecell cycle re-entry and increased the incidence of GATA-4+

cell clusters implying that this biomaterial has potential toinduce endogenous regeneration of cardiac muscle [108]. Theseimpressive results in animal models, are currently progressingtowards a clinical trial [109].

2.2.5 ChitosanChitosan is another naturally occurring polysaccharide, whichis widely studied as a biocompatible scaffold for skin, bone,cartilage, liver and blood vessel tissue engineering [110]. Chito-san allows the formation of relatively hard porous structures,nanofibrous meshes or compliant hydrogels -- depending onthe choice of chitosan derivative [67]. Chitosan hydrogel isformed either by temperature changes (chitosan -- glycerolphosphate [67]) or optical stimuli (acrylate conjugated [111];or photoinitiator based hdrogels [112]), hence it is capable ofpolymerizing in-situ while entrapping genes, growth factorsand, most attractively for tissue engineering, cells.

Temperature responsive chitosan-glycerol phosphate (GP)hydrogels have been studied for the delivery of ESC and nucleartransferred ESCs to an infarcted rat myocardium, showingimprovement in heart function and wall thickness comparedwith the injection of chitosan-GP or cells only [46,113]. Interest-ingly, both studies showed improvement in the micro-vessel density of the infarcted scar in the chitosan-GP onlyinjected hearts. Though the exact mechanism by which thisangiogenesis process occurs is unknown, it is probably relatedto the ability of chitosan to modulate the proliferation ofvascular cells in-vitro as well as in-vivo [114].

2.3 Other biomaterial-injection based platformsDavis et al., have developed a self-assembling peptide-basedscaffold, which was injected into healthy murine hearts eitheralone or in combination with neonatal CM. In both cases,recruitment of endogenous smooth muscle cells and endothe-lial cells was reported, particularly when CM were added tothe injectable scaffold [115]. Recently, Yoon et al., reportedthe epicardial injection of a hyaluronic acid (HA)-based

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hydrogel for the treatment of MI in rats. They reported a sig-nificant improvement in wall thickness and cardiac function-ality (assessed by histology and hemodynamic measurementsrespectively) of the HA-gel-treated group compared with themyocardial infarction non-treated control group [116].Injectable platforms based on synthetic biomaterial

used for the treatment of MI, include mostly variations of poly(N-isopropylacrylamide) (PNIPAAM) and PEG. PNIPAAM isa synthetic hydrogel that is in a liquid state at room temperatureyet solidifies to a compliant gel form at 37oC. However, PNI-PAAM is non-biodegradable and is not cleared from the bodyat physiological temperature [117]. Thus, several modificationswere explored to enhance its biodegradability by incorporat-ing either natural biodegradable materials (i.e., hyaluronicacid [118], collagen [119], gelatin [120], dextran [117,121] and peptidederivatives [122]) or by copolymerization with synthetic mono-mers having hydrolytic polyester side chains [123,124]. Upon cleav-age of these side chains the polymer hydrophobicity wasincreased, leading to an increase in the lower critical solutiontemperature of the polymer above body temperature enablingpolymer dissolving and physiological clearance. Recently, severalvariations of PNIPAAM have been utilized for the treatmentof MI in rabbit [121,125] and rat models [126] showing overallimprovement in cardiac function, neovascularization andventricular remodeling. Thus the addition of a biodegradabledextran grafted to poly (e-caprolactone)-2-hydroxylethyl-methacrylate (Dex-PCL-HEMA/NIPAAM) yielded a reductionin scar size and wall thinning accompanied by improvement inthe left ventricular ejection fraction and overall cardiac func-tion [121]. Li et al., also incorporated bone marrow mononuclearcells (BMMC) with this composite [125] leading to an increase inneovascular formation. Similar modification to PNIPAAM wasemployed by Fujimoto et al., who evaluated the copolymeriza-tion of NIPAAM, acrylic acid (AAc) and hydroxyethylmethacrylate-poly(trimethylene carbonate) (HEMAPTMC).They assessed a range of monomer ratios, poly(NIPAAm-co-AAc-co-HEMAPTMC), showing that at a feed ratio of86:4:10 the polymer formed a hydrogel at 37� C and in-vitro itgradually became soluble over more than 5 months, throughhydrolytic cleavage of the PTMC residues. Injecting the hydrogelinto rats with chronic infarction has led to the preservationof the LV cavity area and contractility, compared to the controls.Furthermore, tissue ingrowth was observed in the hydrogel-injected area, together with a thicker LVwall and higher capillarydensity. These effects were found in the hydrogel-treated groupand were absent in the PBS-treated controls [126]. Another varia-tion of PNIPAAM in combination with other natural and syn-thetic polymers was recently reported by Wang et al. [127].Hydrogel composites were fabricated from Type I collagen,chondroitin sulfate (CS) and a copolymer based on NIPAAM,AAc, N-acryloxysuccinimide and the macromer poly(trimethy-lene carbonate)-hydroxyethyl methacrylate. These compositeswere capable of being injected through a 26-gauge needle andgelled within 6 sec at 37�C forming highly flexible gels withmoduli matching those of the rat and human myocardium.

Finally, PEG, well known as a commonly used bio-inertmaterial has also been studied as a non-degradable alternativefor MI injection [66]. Nevertheless, injection of PEG-vinylsulfone in a rat MI model, showed no difference in the long-term functional benefit as compared with saline injections [128].As it is believed that PEG’s non-degradability prevents along-term sustainable effect [66], other PEG derivatives thatenable proteolytic degradation have been suggested. Theseinclude, PEGylated fibrinogen [47] and PEGylated fibrinbiomatrix [129] and a hydrogel made out of a-cyclodextrin(a-CD) combined with PEG-b-polycaprolactone-(dodecane-dioic acid)-polycaprolactone-PEG (MPEG-PCL-MPEG) tri-block polymer [130]. Both these biomaterials have shownimproved heart geometry and function upon injection intomouse [129] or rabbit [130] MI models. The effect of thesematerials in larger animal models has yet to be reported.

3. Patch-based platforms

Patch-based platforms, are extensively studied for cardiacregeneration. They are composed of biomaterial either withor without cells, and can be transplanted on the epicardial sur-face of the infarct. They serve as a ‘bag pack’ that contains bio-logical therapeutics and growth factors needed for the supportof the transplanted cells and host tissue, until angiogenesisoccurs and the natural ECM is remodeled by the cells, eventu-ally replacing the artificial patch platform. It is suggested thatwith time, the recruitment of endogenous stem and progeni-tor cells may enable a mechanical and electrical integrationof such a patch with the host myocardium hopefully enablingthe regeneration of the infarcted scar tissue.

Patch deposition requires a more invasive surgical inter-vention in contrast to the relative ease of employment ofthe injection-based platforms. Nevertheless, a recent sideby side comparison between intra-myocardial injections ofskeletal myoblasts, and a patch of skeletal myoblasts in colla-gen foams showed that the latter is much more effective interms of improved cardiac functionality as determined byechocardiography and histological assessment one monthpost transplantation in rats [131].

Two major patch-based platform groups have been dis-cussed in literature; patch-based platforms that are composedof solely cells forming cell sheets and patch-based platformscomposed of synthetic or natural polymers with or withoutthe combination of cells.

3.1 ‘Cells only’-patch based platformsShimizu et al., were the first to report, in 2002, the construc-tion of a cell patch stacked out of four CM monolayers [132].They utilized poly-NIPAAM-coated dishes for the cultivationof neonatal rat cardiomyocytes which were later detached usingtemperature reduction (below 32�C). The engineered con-structs were macroscopically observed to pulse spontaneously,in-vitro, and ‘morphologically communicate’ via connexin43.In-vivo, layered cardiomyocyte sheets were transplanted into



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subcutaneous tissues of nude rats, leading to the formation ofneovasculature. Miyahara et al. transplanted stacked mono-layers of adipose tissue-derived mesenchymal stem cells ontohearts of myocardial infarcted rats [133]. One of their mostimpressive findings was that the monolayers expended,in situ, producing a 600-µm-thick tissue over the infarct scar.This finding contradicted the previously held view that myo-cardial patches could not grow beyond ~ 100 µm due to diffu-sion limitation. Nevertheless, given the thickness of human leftventricles (circa 10 -- 15 mm), this diffusion limitation stillneeds to be surpassed to achieve human cardiac restoration [134].In a more recent work published by the same group, the pro-motion of neovascularization by increasing densities ofco-cultured endothelial cells within the same cell-sheet-basedplatform was studied. In-vivo studies, performed in a MI ratmodel, revealed that the cardiac function was significantlyimproved in correlation with the increase in endothelial celldensities of the original co-cultured constructs. They alsoreported a significant increase in the number of capillaries inthe grafts which connected with the host tissue vasculatureand functionally supplied the grafted construct [135].

A different novel and scalable method, which is a ‘scaffoldfree’-based platformbut in a shape of a patchwas lately developedby Stevens et al. [48,136]. A rotating orbital shaker was used to formmacroscopic disc-shaped 300 -- 600 µm thick beating ESC-derived CM patches having diameters directly proportional toinput cell number [48]. CMs were concentrated around the patchedges and exhibited increased purity and maturation with time(determined by the abundance of b-MHC transcript using quan-titative reverstranscription (RT)-PCR and immunohistologicalstaining). Patches also exhibited automaticity and synchronouscalcium transients, indications of electromechanical coupling.A recent study published by this group, demonstrated that thesepatches, composed only of enriched CM, did not survive to formsignificant grafts after implantation in-vivo in rats [136]. Thus,‘second-generation’ prevascularized, and entirely human patchesfromCM, endothelial cells and fibroblasts were created. Implan-tation of these patches resulted in 10-fold larger cell grafts com-pared with patches composed of CM only. Despite the greatinterest these new technologies have generated, they still needto be tested in large-animal models.

3.2 Synthetic patch-based platformsTwo classes of synthetic materials have been proposed in thelast decade for the generation of patch based platforms: poly-esters and elastomers. Each of these materials has unique char-acteristics in terms of structure and composition. They cangive rise to a family of derivatives with distinct propertiesthorough structural and biochemical modifications, such asdifferent toxicity profiles of their degradation byproductsand various mechanical elasticities and strengths.

3.2.1 PolyestersThe general definition ‘Polyester’ refers to a group of polymers,which contain the ester functional group in their main chain.

Although this term is applicable to many types of materials,we will focus on the ones that are the most commonly usedfor myocardial tissue engineering such as polylactic acid(PLA), polyglycolic acid (PGA) (or their copolymer (PLGA)),poly(a-Hydroxy acid) and poly-e-caprolactone (PCL) [134,137].These polymers are of great interest because their degradationproducts are mostly biocompatible (lactic, glycolic and hydrox-ycarboxylic acids respectively). However, these polymers arenot without flaws. Their degradation products may cause alocal high acidic concentration leading to an inflammatoryresponse, which will impair the targeted tissue [2]. Furthermore,due to their bulk degradation kinetics, a sudden loss of struc-tural integrity is expected and might be less desirable. Nonethe-less, seeing as their mechanical properties, morphology anddegradation kinetics are relatively easy to control, they aresimple to manufacture and so became a most useful tool forpatch-based myocardial tissue engineering research.

Combinations of PLA and PCL (PCLA) were first producedby Ozawa et al., who employed PCLA patches, seeded withsyngeneic rat aortic smooth muscle cells to replace a surgicallycreated defect in the right ventricular outflow tract (RVOT)of rats. Compared with gelatin- and PGA-seeded patches,PCLA patches permitted better cellular penetration in-vitro,did not thin or dilate in vivo and did not produce an inflamma-tory response [138]. Jin et al., have investigated the effect oftransplanted poly(lactide-co-e-caprolactone) (PLCL) scaffoldspre seeded with MSC in a rat MI model. Ten days postimplantation, some transplanted MSCs survived and differenti-ated into cardiomyocytes in the injured region, which wasaccompanied with some 20 -- 30% improvement in cardiacfunction [139].

Notable work using PLGA-based patch platforms wasrecently published by the Levenberg group [9,45], who used atri-culture of ESC-derived CM, endothelial cells and embry-onic fibroblasts to form a synchronously contracting engi-neered human cardiac-like tissue containing endothelialvessel networks [9]. This tissue construct was further assessedin terms of its ability to engraft in-vivo into the rat heartand was found to promote functional vascularization [45].Interestingly, in common with the ‘scaffold free’ techniquediscussed above, the patch thickness did not develop tomore than 600 µm. Even though, this thickness representsalmost half the wall thickness of the rat heart, it is far fromthat required for clinical application. In addition, there isincreasing evidence that successful scaffold materials musthave elastic properties that resemble those of the native heart,allowing them to move in synchrony with every heart beat,thus maintaining their structural integrity. Unfotunately, pol-yesters, are generally less flexible than the heart tissue, hencethere is a requirement for more elastic synthetic materialssuch as ‘elastomers’.

3.2.2 ElastomersElastomers are synthetic mimics of natural rubber. Accordinglythey are defined as synthetic materials that have the ability to

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undergo elastic deformation under the influence of a force andregain their original shape once the force has been removed.A 3D cardiac construct made out of polyurethane (PU), exhib-ited, good cell adhesion in-vitro [140]. Further in-vitro andin-vivo evaluations showed no tissue inflammation and tran-sient inhibition in the progression towards heart failure. Thistransient inhibition was no surprise, as transplanted cells wereundetectable one year post treatment [141,142]. These promisingobservations are flawed by the fact that PU release a toxicbyproduct -- disocyanate that hampers its clinical use [2]. Otherelastomeric materials that were reported as suitable for myocar-dial patch engineering are 1,3 trimethylene carbonate [143] andPEG, which was suggested as a screening method for the opti-mization of tri-culture conditions [144]. A more biocompatibleand absorbable elastomer - poly(glycerol sebacate) (PGS) wasdeveloped by the renowned Langer group in 2002 [145]. Thesame material was recently reported by Chen et al., to supportthe LVs of transplanted rat hearts while serving as an efficientdelivery vehicle for ESC-derived CM [49]. The promise ofthese materials cannot yet be fully judged as to date nolarge-animal studies have been performed.

3.3 Natural patch-based platforms

3.3.1 Extracellular matrixThe use of ECM, as a patch platform for myocardial tissueengineering was introduced by the Badylak group in 2005.They reported the successful implementation of urinarybladder ECM multilaminate as an acellular myocardialpatch that significantly augmented ventricular function incanine and porcine models [146,147]. Further work on acanine RV defect model, demonstrated that the mechanicalfunction generated in the patch region correlates to thequantity of local tissue myocytes infiltration [148]. Neverthe-less, when using the natural ECM, its origin needs to beconsidered as different tissues exhibit different ECM compo-sitions and ultrastructure that may affect the formation ofthe desired tissue [66,149-151]. Therefore, a suitable ECM forcardiac regeneration may be the one that originates fromheart tissue. Such ECM has the potential of better preservingthe unique strutted, woven and coiled structures that enableefficient CM contraction. It may also allow the appropriatecell guidance and differentiation signals, while avoidingnon-favorable remodeling towards other tissue types as alsodiscussed by Badylak et al. [152].Four groups (including our own) independently reported

the successful isolation of cardiac ECM scaffold from rat [150]and from a more human relevant porcine xenogeneicorigin [153-155]. An in-vivo application was reported in a ratMI model by Ott et al., who utilized an impressive perfusiondecellularization technique to obtain an ECM template of anentire rat heart. This ECM was further re-seeded with ratneonatal CM and cultivated in a modified Langendorffapparatus to yield active beating synchronous heart. Hetero-topic transplantation of this cardiac ECM platform in rats,demonstrated that functional connectivity of the vascular

infrastructure is preserved during the decellularization pro-cedure [150]. As the scale up from rat to porcine myocardiumdecellularization is a challenging one, the isolation of por-cine acellular myocaridal ECM was achieved either fromthin (2 -- 3 mm) left ventricular slices [153,155] or by usingperfusion through the inherent vasculature [154]. Nonethe-less, the yellowish appearance of the acellular perfused car-diac tissue in the latter report indicates the need for a moreextensive decellularization technique as demonstrated byOtt et al. [150]. Indeed, large scale acellularity that is sup-ported by an immunogenic evaluation has yet to be achievedto allow large animal model studies to be performed.

Our own group has recently reported a procedure for isolatingporcine myocardial acellular ECM (1 -- 2 mm in thickness) [153].We demonstrated the effectiveness of such decellularization pro-cedure, which selectively removes all inflammation-inducing cel-lular components while preserving much of the ECM (Figure 2).In our studies the decellularization process produced yellow areason the ECM that were indicative of a non-complete decellulari-zation (Figure 2B). As remaining cellular debris is potentiallyimmunogenic [149] and may possibly affect cell attachment andviability, any decellularization process, particularly one of thicktissue, should produce complete acellular ECM and also addressthe remains of the coronary adipose tissue. Moreover when iso-lating a thick heart tissue, the left ventricular tissue can be advan-tageous, as the inherent vasculature can be used for the perfusioncleaning process and can also be proven to be essential for futureconstruction of functional inherent blood vessels by seeding withendothelial cells (Figure 2C). Once having ensured that the ECMis totally acellular, it was evident that it encourages cell growthand cellular remodeling both in-vitro as well as in-vivo (Figure 3).Current work in our lab focuses on the optimization of thisprocedure for thicker tissue specimens that could be potentiallyused as patch material and also for transmural replacementtherapies (Figure 4).

Another approach for obtaining an acellular patch materialis by the decellularization of pericardial matrix as reported byChang et al., [156]. In more recent studies performed by thesame group, mesenchymal stromal cell [157] or MSC mono-layers [158] were sandwiched together with pre cut acellularpericardia to form a multilaminate. This tissue graft (sand-wiched patch) was used not only as a patch, but also as areplacement for the infarcted wall in a syngeneic Lewis ratmodel with chronic MI. At retrieval, the area of the emptypatch serving as control, was relatively enlarged, suggestingreduced structural support while that of the sandwiched patchremained about the same. The sandwiched patches, were pop-ulated with both host and originally seeded cells [157].Whether these results could be scaled up in larger animalmodels where infarct sizes are by far bigger, remains tobe proven.

3.3.2 CollagenThe casting of liquid collagen with neonatal CM into circularmolds yielded in-vitro engineered heart like tissue (EHT) that



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was subsequently electrically [159-161] or mechanically [160,162]

stimulated to create synchronous beating hoops. This EHTwas used for both patch implantation on infarcted immune-suppressed rat hearts [163] as well as to create a novel bio-logical assist device out of a pouch like EHT [164]. Othergroups have also attempted to use collagen-patch-basedplatforms [40,165-166]. Kofidis et al., transplanted a collagen

patch, which was solidified with human ESC-derived CMon an athymic nude rat heterotopic heart transplant model.Their results indicated that the three dimensional matrix pre-vented myocardial wall thinning and improved contractil-ity [40]. In a recent study by Pozzoboen et al., a porouscollagen patch was transplanted on a cryoinjured rat model,and allowed to neo-vascularize for a period of two weeks after

A.

C.

B.

D.

C.

Figure 2. Isolation of porcine cardiac extracellular matrix (ECM). Slices of fresh porcine heart (A.), incomplete decellularized

slices showing retention of cellular debris (Yellow/Brown) marked by an arrow (B.) and clean acellular ECM preserving

structural properties such as blood vessel ECM marked by arrows (C.). Masson tri-chrome staining of representative cross

sections shows high cellular density (red -- cytoplasms) adjacent to ECM fibers (Blue) in native porcine cardiac tissue (D.). No

cellular remains are apparent in the acellular scaffold (E.). Scale bars in D. and E.: 100 µm.

A.

D. E.

B. C.

Figure 3. Cellular remodeling of porcine cardiac extracellular matrix (ECM). Rat neonatal cardiomyocytes (CM) seeded on

porcine cardiac ECM. Immunological staining for a-cardiac actinin (A.) and connexin-43 (B.) at day 7 shows CM organization.

Seeded CM secretes ECM fibers and remodels the acellular scaffold visualized by scanning electron microscopy (C.).

Subcutaneous transplantation in mice shows the transplanted ECM at one-week post transplantation (D.) and 8 weeks post

transplantation (E.). Arrows mark cell penetration and remodeling. Scale bars: A. -- C. 20 µm; D. -- E. 200 µm.

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which, human CD133+ cells were injected directly into thepatch. Their results indicated that this strategy, though suit-able for angiogenesis and arteriogenesis, does not produce bet-ter results, in terms of selective homing and differentiationinto CMs, than the ‘traditional’ method of cell injectioninto the myocardium [166].Simpson et al., have also attempted to assess whether an

epicardially applied, tissue-engineered cardiac patch contain-ing progenitor cells would result in enhanced exogenouscell engraftment [167]. They have embedded human MSC(hMSC) into patches made of rat tail type I collagen andglued this patch to rat MI model hearts using fibrin glue.Although hMSC engraftment, one week post implantationwas about 23 ± 4% of the original seeded quantity,long-term cell survival was not detected at 4 weeks.A patch composed of collagen and Matrigel, was used by

Giraud et al., to deliver skeletal myoblasts into the infarctedmyocardium of rats [168]. The patch was glued onto the myo-cardium using fibrin glue leading to some functional benefits,namely improved systolic heart function, however, skeletalmyoblasts survival rate was low and the arrhythmogenic effectwas not properly monitored [169].

3.3.3 GelatinSeveral studies have reported the use of gelatin for the produc-tion of cardiac patch platforms [137]. Li et al. seeded fetal ven-tricular muscle cells onto a gelatin mesh in-vitro andcultivated it for 7 days prior to implantation on a cryoinjured

rat heart. Although cells survived the transplantation andinterconnected with the native tissue, no analysis of cardiacfunction was made [170]. In a follow-up study, they reportedon increased cellular proliferation and better performancewhen mechanical stress regimes were applied on the gelatinfoam [171].

3.3.4 AlginateAlginate patches were first proposed by Leor et al., who iso-lated and grew fetal rat cardiac cells within 3D porous alginatescaffolds. Scaffolds were manufactured using a freeze dryingtechnique and had 90% porosity with an average pore sizeof 100 µm. Intensive neovascularization was revealed posttransplantation of the alginate scaffold in a rat MI model [172].In more recent studies performed by the same group, similaralginate scaffolds were implanted for 7 days on the omentumto achieve pre-vascularization and than transplanted on a MIrat model. The vascularized cardiac patch showed structuraland electrical integration into host myocardium and inducedthicker scars that prevented further dilatation of the chamberand ventricular dysfunction [173]. In a heterotopic heart trans-plant model similar alginate patches also exhibited improvedleft ventricular function [174].

3.4 Synthetic-and-natural-combined patch platformsDifferent studies exploited the advantages of both the syntheticas well as the natural biomaterials producing bio-syntheticchimeras for the construction of cardiac patches. The in-vitro

A. B.

C.

Figure 4. Perfusion -- decellularization of thick porcine cardiac extracellular matrix ECM. Porcine left ventricle is perfused

through the coronary artery while immersed in the same decellularization solutions for a week. Note the difference in cellular

remains in the perfused area (black arrows) compared with the non-perfused parts that were exposed to the decellularization

solutions only from submerging the tissue in solution (A., white arrows). Hematoxylin and eosin staining of cryosectioned

slices obtained from the dashed square in A. (effective decellularization, B.) and from the continuous square in A. (ineffective

decellularization, C.).



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fate of such scaffolds has been reviewed by [2,33,137]. The currentreview addresses the combined-patch platforms which demon-strated in-vivo success. Krupnick et al., seeded a mixture ofbone marrow-derived MSC, collagen types I and IV andMatri-gel onto a nonwoven polymermeshmade out of PLA reinforcedwith poly(tetrafluoroethylene) (PTFE). This construct was thenheterotopically implanted into infarcted syngenic rat hearts.Their results suggested that this construct is non-immunogenicand biocompatible as minimal intracardiac inflammation wasobserved [175]. Similarly, Fukuhara et al., seeded crude bonemar-row cells (cBMC) onto a bioengineered polyglycolic acid cloth(PGAC) impregnatedwith collagen type I and bFGF. The patchwas subsequently sutured on top of a MI scar tissue of rat heartsdemonstrating improved cardiac function, and a distinct cellularpopulation within the patch material including infiltratinginflammatory cells at 4 weeks post implantation [176].

Siepe et al., produced another interesting combination of syn-thetic and natural polymers, which is composed of polyurethane(PU) patches coatedwith laminin, a natural adhesion and signal-ing glycoprotein of the ECM [141]. When implanted with myo-blasts, this type of patch led to significantly improved heartfunction in a MI rat model as assessed by echocardiography. Itwas concluded that myoblast-seeded PU scaffolds preventedpost--myocardial infarction progression toward heart failurewith the same efficiency as direct intramyocardial injection. Ina follow-up study by the same group, a progression toward heartfailure was significantly prevented for up to 6months after injec-tion of myoblasts and for up to 9 months following biograftimplantation [177]. This effect vanished after 12 months, withimmunohistological examinations revealing the absence oftransplanted myoblasts within the scaffold, thus, suggestingonce more that polymer-based platforms in combination withcells are superior to cell therapy alone. However, the beneficialeffects were transient and are probably dependent on the survivaland viability of the seeded cells [177].

Finally, Miyagi et al., developed a new, biodegradable patchmade of a sponge like inner core (modified Gelfoam, MGF)that encourages cell engraftment and an outer coating of PCLproviding sufficient strength to permit ventricular repair. Usingrat MI model, they demonstrated that animals whose heartswere repaired with untreated Gelfoam died of ventricular rup-ture, while the MGF-treated group had significantly improvedmyocardial systolic function. Further incorporation of cyto-kines (stem cell factor, stromal cell-derived factor-1a) and/orbone-marrow-derived MSC within the patched platform, pro-moted a-smooth muscle actin-positive cells, greater capillaryformation, increased wall thickness and better preserved systolicelastance than MGF alone [178].

4. Future platform technologies

Several emerging technologies in tissue regeneration havebeen recently reported to improve cell survival, differentia-tion, spatial organization and/or biomechanical integrationwith host myocardial tissue upon transplantation. These

include combinations of injection- and patch-basedplatforms [179,180] and the use of either physical (mechanicalstretching [162,171], perfusion [181,182] and electrical [183-189])or biochemical (hypoxic pre-conditioning [190,191])stimulation procedures.

Combinations of patch and injectable platforms exploitboth themechanical support of the patch with the ease of deliveryaccompanying intramyocardial injections. Cortes-Morichettiet al., tested in a mouse MI model, the effect of intrainfarctinjection of human umbilical cord blood mononuclear cells(HUCBS) followed by fixation onto the epicardium of a collagenmatrix seeded with the same cell type. This treatment proved tobe advantageous in terms of post-ischemic ventricular dilationand remodeling, over cell-only or non-seeded-patch-only con-trols [180]. Further proof of concept was later provided using asimilar approach in a Phase I controlled clinical feasibility studyperformed in 20 patients, half of whom received the combinedtreatment (using bone marrow cells) while the rest receivedonly the intra-infarct cell injection. The results indicated thatthe combined strategy appears to improve the efficiency of cellu-lar cardiomyoplasty as the collagen scaffold increased the wallthickness with viable tissue while limiting remodeling andnormalizing cardiac wall stress in the injured regions [179].

Effective bio-mechanical pre-conditioning for collagen (andgelatin) stretching has already been discussed with respect topatch-based platforms above [160,162,171]. Perfusion offersanother approach to exert mechanical stimuli on cells and con-structs. This methodology confers increased mass transfer effi-ciency for nutrient supply and waste products removal [181,182].Radisic et al., employed a custom built bioreactor system todemonstrate that the interstitial flow of culturemedium throughrat neonatal CM-seeded porous collagen scaffold, yielded thick,compact and contractile cardiac constructs. These constructsexhibited physiological densities of metabolically active and via-ble cells containing cardiac markers [182]. Similar observationwere also reported by Dvir et al., who also developed a perfusionbioreactor prototype for alginate based constructs seeded withthe same cell type [181]. The in-vivo evaluation of these perfusionend-products compared with non-perfused controls is still tobe reported.

Another physical condition associated with cardiac celldifferentiation and maturation is that of electro-stimulation.Electrical stimuli have shown increased pre-commitmenttowards cardiac lineages of neonatal cadiomyocytes [185-188],fibroblasts [186], hMSCs [184], skeletal myoblasts [183] andhESC [188,189]. Genovese and colleagues have recentlyshown that electrostimulation induces cardiomyocyte pre-commitment of hMSCs in-vitro and is an effective alternativeto cytokine-induced differentiation. ElectrostimulatedhMSCs have exhibited both morphological and biochemicalchanges, resulting in a shift toward a striated muscle cell phe-notype expressing cardiac specific markers. This partiallydifferentiated phenotype may allow a gradual, ongoing differ-entiation within the cardiac environment thus conveyingpotential advantages in clinical applications. The same group

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further evaluated the effects of in-situ electrostimulation(using biventricular pacemakers) on skeletal myoblasts trans-planted in sheep MI model. Stimulation resulted in a signifi-cant improvement in the ejection fraction and limitation ofleft ventricular dilatation compared with controls. Eventhough the arrhythmogenic nature of skeletal myoblasts is stilla matter of controversy [192], no evident arrhythmias wereobserved in this study, rendering the strategy of skeletal myo-blast electrical stimulation prior to transplantation clinicallyrelevant. Tandon et al., have recently reported the develop-ment of a set of electrical stimulation procedures enablingthe use of modified culture dishes [188]. Such reports wouldhopefully lead to the establishment of standard proceduresthat in turn would enable a better understanding of theelectrical stimulation mechanism of action.Finally, hypoxia (i.e., oxygen starvation) had long been

known to have a considerable effect on the proliferation, dif-ferentiation and maintenance of the cardiovascular systemduring development [193]. As such, the use of hypoxia to pre-condition transplanted cells lead to improved results in termsof increased cell survival and angiogenesis [190,191]. Hu et al.,reported that mouse MSCs grown under hypoxic conditions(0.5% oxygen for 24 h) displayed increased expression ofpro-survival and pro-angiogenic factors (hypoxia induciblefacor 1, angiopoietin-1, vascular endothelial growth factorand its receptor fetal liver kinase 1 (Flk-1), erythropoietin,B cell lymphoma 2 (Bcl-2) and Bcl-2-like X (Bcl-xL)).When transplanted to the peri-infarct region of a rat MImodel, hypoxia preconditioned MSC exhibited higher sur-vival rates leading to an increase in angiogenesis and to othermorphologic and functional benefits, most probably associ-ated with MSC paracrine effects [190]. Similar results werealso recently reported by Wang et al., who demonstratedthat anoxia preconditioning enhanced the capacity of MSCsto repair the infarcted myocardium. This was attributed toincreased cell survival and angiogenesis [191]. This studytogether with other in-vitro evidence concerning the effective-ness of sub-physiological oxygen concentrations on ex-vivocell proliferation, gene expression and commitment [193-198]

may call into question the traditional and standard laboratorycell conditions (i.e., 5% CO2, 95% air) used in most of thestudies mentioned in this report.Taken together, these evolving technologies, offer genuine

opportunity to new discoveries as boundaries of ‘traditional’cell therapy and tissue engineering techniques are beingchallenged to create platforms having actual bio-mimeticproperties in an ex-vivo environment.

5. Expert opinion

The future choice of the cell or cell combinations for heartregenerative cell-based platforms are probably autologous iPSCsand CSCs. These cells offer a genuine clinical alternative mainly

due to their autologous origin and their overall tendency tospontaneously give rise to several supporting cell types includingbeating CM and endothelial cells. Nonetheless, major researchstill needs to be performed towards achieving a robust andtime effective differentiation procedure(s) that enhances theyield of CMproduction with as little genetic interference as pos-sible being introduced. It is expected that clinical trials of in-situ-injectable biomaterial platforms would produce similar results tothose already obtained in large-animal models, showingimproved cardiac function, with the hope of total cardiac regen-eration. Nonetheless, one cannot ignore the promising resultswith cardiac patches. This potential should motivate furtherbasic in-vitro research aiming to deal with several major limita-tions still hampering the production of relevant thick andorganized tissue constructs for patch or infarct replacement ther-apies. These limitations include the lack of cell survival posttransplantation, as it is evident from the data presented hereinthat without cells populating these tissue engineered constructs,no long-term effect can be achieved. Most importantly, the oxy-gen diffusion limitation in culture media, which dictates thatin-vitro produced constructs exhibit inadequate thicknesses forhuman sized hearts. Finally, the static ‘flat tissue culture’ isinappropriate for 3D tissue constructs. Hence a ‘Biomimeticapproach’ to cardiac tissue engineering would probably directthe ‘assembly of functional tissues using biologically deriveddesign requirements’ [34]. Several pivotal reports, reviewedelsewhere [34], have already been published in recent years byleading researchers in this field, such prestigious groups includethose of Robert Langer, Smadar Cohen, Gordana Vunjak-Novakovic, Milica Radisic, Thomas Eschenhagen, WolframHubertus Zimmerman, Juan Carlos Chachques and Alain FCarpentier [35,159,162,163,182-185,187,199-200]. In summary, ourgrowing understanding and ability to supply pulsed-flowfeeding as well as timely mechanical, electrical and biochemicalstimuli would enable the establishment of complementarydynamic cultivation platforms. These will in turn producemore densely populated, thicker and aligned constructsprior to implantation. Furthermore, in-vitro-developed tissueconstructs, serving as cardiac mimetics, would also enablerapid screening of novel therapeutics and their effects oncardiac regeneration.

Acknowledgments

The authors would like to sincerely thank S Irvineand Y Eitan for their help in the preparation of thismanuscript. We would like to acknowledge D Machlouffor contributing Figure 1.

Declaration of interest

The authors state no conflict of interest and have received nopayment in preparation of this manuscript.



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AffiliationUdi Sarig & Marcelle Machluf†

†Author for correspondence

Technion--Israel Institute of Technology,

The Laboratory of Cancer,

Drug Delivery & Mammalian Cell Technology,

Faculty of Biotechnology & Food Engineering,

Haifa 32000, Israel

Tel: +972 4 8294916/3079;

Fax: +972 4 8293399;

E-mail: [email protected]

Sarig & Machluf


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