Research review paper

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. . . .e engine. . . .. . . .eringts . .. . . .ctable hrices .ptides

3.2.4. Cells/stem cells and biomo

Biotechnology Advances 32 (2014) 449461

Contents lists available at ScienceDirect

Biotechnolog

j ourna l homepage: www.e lsev1. Introduction4. Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4585. Current scenario of translational research in cardiac tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4586. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Corresponding author at: Centre for Nanotechnology&Chemicals& Pharmaceuticals Chair Professor, School of ChUniversity, Tamil Nadu, India. Tel.: +91 4362 264220; fax

E-mail address: swami@sastra.edu (S. Sethuraman).

0734-9750/$ see front matter 2014 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.biotechadv.2013.12.010lecular signals based approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456ng cells in scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458tegies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583.2.5. Methods adopted for loadi3.2.6. Pharmaceutical based stra1. Introduction . . . . . . . . .2. Cardiac tissue engineering . . .

2.1. Hydrogel materials for tissu2.2. Natural polymers . . . .2.3. Synthetic polymers . . .

3. Various approaches in tissue engine3.1. In vitro engineered construc3.2. In vivo approaches . . .

3.2.1. In situ forming inje3.2.2. Decellularized mat3.2.3. Self-assembling pe. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 450ering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455ydrogels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456ContentsHydrogel based injectable scaffolds for cardiac tissue regeneration

Janani Radhakrishnan, Uma Maheswari Krishnan, Swaminathan Sethuraman Centre for Nanotechnology & Advanced Biomaterials, School of Chemical & Biotechnology, SASTRA University, Thanjavur 613401, India

a b s t r a c ta r t i c l e i n f o

Article history:Received 26 September 2013Received in revised form 14 December 2013Accepted 28 December 2013Available online 7 January 2014

Keywords:Cardiac regenerationInjectable scaffoldHydrogelStem cellsGrowth factors

Tissue engineering promises to be an effective strategy that can overcome the lacuna existing in the current phar-macological and interventional therapies and heart transplantation. Heart failure continues to be amajor contrib-utor to the morbidity and mortality across the globe. This may be attributed to the limited regeneration capacityafter the adult cardiomyocytes are terminally differentiated or injured. Various strategies involving acellular scaf-folds, stem cells, and combinations of stem cells, scaffolds and growth factors have been investigated for effectivecardiac tissue regeneration. Recently, injectable hydrogels have emerged as a potential candidate among variouscategories of biomaterials for cardiac tissue regeneration due to improved patient compliance and facile admin-istration via minimal invasive mode that treats complex infarction. This review discusses in detail on theadvances made in the eld of injectable materials for cardiac tissue engineering highlighting their merits overtheir preformed counterparts.

2014 Elsevier Inc. All rights reserved.AdvancedBiomaterials, Orchidemical & Biotechnology, SASTRA: +91 4362 264265.

ghts reserved.y Advances

i e r .com/ locate /b iotechadvCardiac diseases are the leading cause of morbidity, which accountsfor approximately 40%of all humanmortality despite the advancementsand improvements in the therapeutic approaches (Chen et al., 2008; Yeet al., 2011; Yoshida andOh, 2010). Approximately 50% of patients diag-nosed for myocardial infarction (MI) die within 5 years. Patients belong

450 J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461to both the developingworld and the industrialized nations and it is es-timated that about 25 million people suffer from heart failure (Chenet al., 2008; Dvir et al., 2011). Adverse remodeling of the left ventricle(LV), loss of non-regenerative cardiomyocytes and myocardial infarc-tion are the signicant processes involved in the initiation and progres-sion of deteriorating myocardial function that ultimately leads tocongestive heart failure (Huang et al., 2005). Therefore, remodeling ofthe left ventricle and angiogenesis at the infarcted site are the primaryfocus of all research strategies directed towards designingMI therapeu-tics (Shen et al., 2009). A diverse array of biomaterials has been investi-gated as scaffold regeneration of different tissues and also as deliverysystems. The selection of the appropriate biomaterial and the subse-quent fabrication of a scaffold that best suits the demands of the nativemicroenvironment of cardiac tissue are crucial steps in the developmentof scaffolds for tissue engineering (Mano et al., 2007).

Cardio-pathophysiologic conditions such as hypertension andvalvular heart disease cause coronary artery disease (CAD) that maybe accompanied with acute MI (Chen et al., 2008). Typically MI causesmyocyte slippage and along with CAD constitutes the single most com-mon cause for cardiac failure (Chen et al., 2008; Venugopal et al., 2012;Yoshida and Oh, 2010). The myocardial slippage weakens the collagennetwork in the extracellular matrix (ECM) resulting in ventricular wallthinning, dilation and impairs the pumping efciency of the heart(Chen et al., 2008). The enlarged ventricular volume causes progressivestructural and functional changes inducing remodeling process whichresults in substantial loss of cardiomyocytes at the infarct zone (Leoret al., 2005; Yoshida and Oh, 2010). This massive loss of cardiomyocytesimpairs the heart wall muscle permanently, as the terminally differenti-ated cardiomyocytes lack signicant intrinsic potential to repair andregenerate the lost cells (Chen et al., 2008; Ye et al., 2011). The initialcompensatory ventricular remodeling phenomenon later contributesto the inefcientmechanical pumping of the ventricularmuscle therebypredisposing the patient to congestive heart failure (CHF) (Chen et al.,2008).

Sequence of events activated after a myocardial tissue injury in-cludes inammation and granulation tissue formation that eventuallyleads to scar tissue formation (Leor et al., 2005). Cytokines and growthfactors released from the injured tissue recruit white blood cells, mainlyneutrophils, followed by migration of monocytes to the wound site,subsequently differentiates into macrophages, which plays a majorrole of clearing the infarcted zone. Furthermore, cells such as endotheli-al cells, broblasts and stem/progenitor cells are recruited at the infarctzone to aid in the granulation tissue formation. The formation of bloodvessels or angiogenesis is another essential event for the healing of in-farctedmyocardium (Nian et al., 2004; Sun et al., 2002). The granulationtissue is subsequently replaced by an ECM which is majorly depositedby broblasts and remodeled into scar tissue (Leor et al., 2005). Thisbroblastic scar tissue lacks contractile function and therefore mini-mizes the capacity of the heart to pump blood and maintain the neces-sary cardiac output (Yoshida and Oh, 2010). The expansion of cardiacbrosis elevates tissue stiffness, prevents cardiac relaxation, and there-by impairs cardiac function which eventually leads to heart failure(Marquez et al., 2009). Thus, the intrinsic healing process initiated atthe pathologic site does not restore the functional heart.

The treatment strategies adopted currently can be broadly catego-rized as pharmacological and interventional therapies apart from surgi-cal heart transplantation. Pharmacological therapy utilizes angiotensinreceptor blockers, angiotensin-converting enzyme (ACE) inhibitors, cat-echolamines (-blockers), and aldosterone (spironolactone) (Dobneret al., 2009). These strategies focus on reduction of the cardiacworkloadby utilizing diuretics and nitrates, improving systolic performance andoffering protection from the toxic humoral factors that are activatedduring heart failure (Chen et al., 2008; Nelson et al., 2011). Membraneoxygenator microporous hollow bers, pacing leads, prosthetic heartvalves, stents and stent coatings, and other such medical devices stand

as examples for advancements in biomaterials to improve heart failuretreatment (Nelson et al., 2011). Interventional therapy generally involvesimplantation of devices such as pacemakers to control electrical/mechanical asynchrony. Such techniques are being widely employedand have been reported to enhance the cardiac energy efciency in pa-tients with impaired heart functions (Chen et al., 2008; Hawkins et al.,2006; Nelson et al., 2011). Coronary artery bypass grafting and coronarystent deployment strategies are relied upon as highly effective and com-mon for treatingmyocardial infarction (MI), these interventional proce-dures aim at revascularization of the myocardium (Nelson et al., 2011).However, both drugs and interventional strategies cannot control dis-ease progression adequately and hence the gold standard for patientswith terminal heart failure is cardiac transplantation. Although thenumber of donor hearts is grossly inadequate to meet the demand(Chen et al., 2008; Dvir et al., 2011; Nelson et al., 2011). The inadequateorgan donors and the complications that are caused by immune sup-pressive treatments necessitate the development of adoptingnew strat-egies to repair and regenerate the injured heart (Akar et al., 2006; Chenet al., 2008). In this context, tissue engineering approaches haveevolved as a promisingmodality for therapy aswell as overcome sever-al pitfalls in the conventional systems.

Various tissue engineering strategies are being evaluated forrepairing the injured myocardium. These include acellular scaffoldimplantation, cell therapies, scaffolds combined with cells, growth fac-tors or genes (Davis et al., 2005a; Huang et al., 2005). The aneurismalthinning of the ventricularwall can be conferredwith structural supportusing biopolymer scaffolds which has also been used in combinationwith various cells such as cardiomyocytes, skeletal myoblasts, endothe-lial cells, bone marrow-derived stem cells and embryonic stem cells toprovide constructive matrix environment that improved properties ofcell viability, migration, proliferation and tissue progression (Huanget al., 2005). Over the past decade, investigation of injectable scaffoldshas given encouraging results independently as well as in combinationwith cells in both in vitro and in vivo (Hawkins et al., 2006; Nelson et al.,2011). Fig. 1 illustrates themerits of injectable hydrogels for cardiac tis-sue on comparison with the other tissue engineering strategies.

2. Cardiac tissue engineering

In human tissue almost all the normal cells except blood cells areresident and adherent to the solid matrix called extracellular matrix(ECM). The ECM performs a multitude of functions wherein it providesstructural support for cells, contributes to the mechanical properties oftissues, regulates cell behavior by inuencing cell proliferation, homeo-stasis, cell survival, shape, migration and differentiation, acts as a reser-voir of growth factors and potentiates their actions, allows remodelingduring development, and aids differentiation and wound healing pro-cesses (Daley et al., 2008; Yoshida and Oh, 2010; You et al., 2011).

Tissue engineering utilizes temporary scaffolds along with cells andgrowth promoting signals for successful tissue repair, regeneration andcomplete tissue progression (Davis et al., 2005a; Leor and Cohen, 2004;You et al., 2011). The scaffold should essentially be an ECM analog spe-cic to the tissue of interest (Vasanthan et al., 2012; You et al., 2011).Though, there are different types of ECM, they are all principally com-posed of a complex assembly of many proteins and polysaccharides,which varies tissue-specically (Daley et al., 2008; Frantz et al., 2010;You et al., 2011). The cardiac tissue-specic constructs shouldmeet sev-eral requirements. They should mimic native heart muscle, remain via-ble following implantation and improve systolic and diastolic functionsof diseased myocardium (Leor et al., 2005; Zimmermann et al., 2004).Scaffolds that possess appropriate elastic and electrical propertiescoupled in themmay be recommended for attaining contractile and im-pulse conducting functions of the heart (You et al., 2011). Thus an idealcardiac-specic construct should exhibit good contractility, should bemechanically robust and exible, should be extensively vascularizedor achieve vascularization quickly following implantation and totally

should be electrophysiologically stable and non-immunogenic (Leor

rog

451J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461et al., 2005). Variety of cardiac grafts based on different strategies hasbeen reported. These include cell-seeded scaffolds, cell-seeded bioma-terial gels, fabrication of cell lms from cardiac cells and porous or -brous sheets (Leor et al., 2005). The choice of the scaffold material is acritical parameter that can determine the success of the regenerationstrategy. The following sections present themajor biomaterials employedfor cardiac tissue engineering.

2.1. Hydrogel materials for tissue engineering

Though a lot of scaffold materials and scaffold geometry have beeninvestigated for cardiac regeneration, each category has certain advan-

Fig. 1. Schematic representation of merits of injectable hydtages and short-comings that limit their applicability. Hydrogels havereceived considerable attention as cardiac tissue constructs owing totheir viscoelastic nature and amenability to chemical as well as physicalmodications (Slaughter et al., 2009). Hydrogels belong to a class ofwater-insoluble polymers and may be either homopolymers or copoly-mers (Langer and Peppas, 2003). They are pre-formed by chemical orphysical cross-linking of water-soluble precursors, constituted of eithernatural or synthetic polymers. Natural polymers may be of polysaccha-rides, proteins and their derivatives. Poly(ethylene glycol) (PEG) andderivatives of PEG, poly(hydroxyethyl methacrylate) (PHEMA), poly(N-isopropyl acrylamide) (PNIPAM) and poly(vinyl alcohol) (PVA) aresome of the synthetic polymers (Jeong et al., 2002; Wang et al., 2010).Hydrogels absorb considerable amount of water or biouids leading toswelling and increase in dimensions while maintaining their shape(Jeong et al., 2002). This propertymakes themmore similar to soft tissues(Wu et al., 2009).

The swellingdeswelling characteristics of a hydrogel can be tailoredthrough surface modication to respond to a specic stimulus such aspH, temperature, ionic strength, molecules, electric or magnetic signals(Kopeek, 2007). This property has been effectively employed to devel-op stimuli-responsive smart hydrogels that have been used for con-trolled release of molecules encapsulated in their gels (Kopeek, 2007;Lutolf and Hubbell, 2005). Hydrogels with their water-lled interiorsand viscoelastic nature provide a conducive microenvironment for thecells and hence have been used for cell encapsulation, delivery andtissue formation (Slaughter et al., 2009). These hydrogel structurescan be modied further through incorporation of bioresponsivefunctionalities (Kloxin et al., 2009). In addition, the porosity and the ad-equate pore size in hydrogels allow free diffusion of metabolites/waterand exchange of oxygen, nutrients, and otherwater-solublemetabolitesapart from restricting cellmigration (Wang et al., 2010;Wu et al., 2009).

Properties of the hydrogel scaffolds for regeneration largely rely onthe biomaterial chosen, as it guides the cellular growth, differentiation,maturation and organization in tissue-engineered constructs. It alsoprovides physical support for cells, topographical, chemical and biolog-ical cues required for efcient formation of functional tissues. Essentiallythese biomaterials should crosstalk with cells at molecular level in acontrolled manner. In addition, the biomaterials and their degradationproducts must not be toxic and immunogenic and the degradation

els over the other systems for cardiac tissue regeneration.rate should appropriate the rate of new tissue formation (Leor et al.,2005). The choice of the polymer depends on the ultimate applicationand consideration of the polymer inherent physical and chemical prop-erties. The choice of naturally derived or synthetic polymers should bebased on the desired cellular interactions (Shoichet, 2010). Fig. 2shows the structures of some commonly used hydrogel biomaterialsfor cardiac tissue engineering.

2.2. Natural polymers

Hydrogels from natural polymers such as collagen, brin, alginate,and hyaluronic acid possess biological activities that include cellrecruiting,modulation of the inammatorymicroenvironment and pro-moting neovasculature formation (Ye et al., 2011). Table 1 summarizesthemajor natural biomaterials that have been employed for cardiac tis-sue engineering.

Collagen exhibits good cell and tissue compatibility as they form themajor protein component of the ECM (Vlierberghe et al., 2011;Wallaceand Rosenblatt, 2003). Collagen gels are viscoelastic i.e., they are semi-solid when at rest, but can be made owable under stress. Injectable sus-pensions of collagen bers and non-brillar viscous collagen solutions areavailable, which can be mixed with therapeutic proteins and drugs(Wallace and Rosenblatt, 2003). Collagen is an important protein of theECM, existing in different forms in various tissues. It is a natural cell-adhesive, promotes cell attachment and proliferation and presents an en-vironment conducive to cell viability (Chiu and Radisic, 2011; Shoichet,2010). Highly cross-linked porous scaffolds for tissue engineering

452 J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461applications can be fabricated using collagen that favors cell penetrationthrough them (Shoichet, 2010). Collagen has been used in combinationwith chitosan for the controlled release of thymosin 4, which inducedangiogenesis and epicardial cell migration (Chiu and Radisic, 2011). An-other study demonstrated that injection of collagen thickens an infarctscar and not only improves left ventricular stroke volume and ejection

Fig. 2. Chemical structures of natural and synthetic polym

Table 1Natural and modied natural polymer hydrogels for cardiac tissue engineering.

Material Salient features

Collagenchitosan composite Sustained release of thymosin 4 promotes epiPEGylated-brinogen Increased retention of cardiomyocytes, NRVCMAzidobenzoic acid-chitosan (Az-chitosan) Conjugated with the angiopoietin-1-derived pe

heart cell attachment and survivalHyaluronic acid (HA) Augmented the wall thickness in the apex and

ejection fraction and cardiac outputFibrin glue Delivery of hMSCs substantially increased localAlginate Sequential delivery of HGF and IGF-1 promoted

Intracoronary injection reverses left ventricularIn combination with PLGA microspheres for coTAT-HSP27 for longer duration, inhibiting the acultured under hypoxic conditions.RGD modied alginate reshaped a dilated aneuenhanced angiogenesisCardiac modeling and functionSequential dual delivery of VEGF-A165 and PDGmaturation, and improved cardiac function

(hMSCs: human mesenchymal stem cells, IGF-1: insulin-like growth factor-1, HGF: hepatocyderived growth factor-BB, MI: myocardial infarction, TAT-HSP27: transcriptional activator-hecardiomyocytes, hESC-CMs: human embryonic stem cell-derived cardiomyocytes, LV: left ventfraction but also avoids paradoxical systolic bulging following MI (Daiet al., 2005). However, the hydrophilicity and low viscosity of collagenat ambient temperature render poor processability (Ahn et al., 2011).

Hyaluronic acid (HA) is a non-sulfated, unbranched, linear glycos-aminoglycan with repeating units of the disaccharide, -1,4-D-glucuronic acid-1,3-N-acetyl-D-glucosamine (Prestwich, 2011; Tan

ers used as hydrogels for cardiac tissue engineering.

Animal models Ref.

cardial cell migration and angiogenesis Rat Chiu and Radisic (2011)s or hESC-CMs in graft Rat Habib et al. (2011)ptide, QHREDGS supports neonatal rat Rask et al. (2010)

basilar infarct regions, exhibited better Ovine Ifkovits et al. (2010)

cardiac retention Rat Martens et al. (2009)myocardial repair Rat Ruvinov et al. (2011)remodeling after MI Swine Leor et al. (2009)ntrolling the release behavior ofpoptotic pathways of cardiomyoblasts

Lee et al. (2009)

rysmal LV, improve LV function and Rat Yu et al. (2009)

Rat Landa et al. (2008)F-BB induced higher vessels density and Rat Hao et al. (2007)

te growth factor, VEGF-A165: vascular endothelial growth factor-A165, PDGF-BB: platelet-at shock protein 27, PLGA: poly(lactide-co-glycolide), NRVCMs: neonatal rat ventricularricular).

453J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461et al., 2009; Yoon et al., 2009). This ubiquitous, highly hydratedpolyanion occurs in sizes ranging from 100 kDa in serum to 8000 kDain the vitreous and is broadly spread throughout the ECM of most con-nective tissues (Prestwich, 2011; Tan et al., 2009). It mediates cellularsignaling, wound repair, morphogenesis, matrix organization, cell pro-liferation, and cell differentiation, promotes angiogenesis and sup-presses brous tissue formation (Prestwich, 2011; Yoon et al., 2009).Hyaluronic acid (HA, hyaluronan) is a promising material for tissue re-generation due to its viscous properties, ability to retain water, biocom-patibility and biodegradability (Choh et al., 2011; Tan et al., 2009; Yoonet al., 2009). Thermosensitive and photopolymerizable injectable HAhydrogels have been developed and have shown potential for tissueengineering (Tan et al., 2009). Injectable HA hydrogels with tunablegelation kinetics and mechanical properties have been assessed fortheir capability of normalizing myocardial stress using ovine model(Ifkovits et al., 2010).

Chitosan is a cationic polysaccharide, hydrophilic, biocompatible andnon-toxic extensively used for therapeutic applications such as tissueengineering, drug delivery, wound healing and surgical adhesives(Chiu and Radisic, 2011; Dhandayuthapani et al., 2010; Vlierbergheet al., 2011). Chitosan is obtained as partially deacetylated derivativeof chitin (1,4 -linked N-acetyl-D-glucosamine) from the shells ofcrabs and shrimps (Vlierberghe et al., 2011; Yeo et al., 2007).Chitosan-based pH-dependent, thermoresponsive systems obtainedby adding polyol salts have been developed (Vlierberghe et al., 2011).Chitosan can increase the compression modulus of collagen based in-jectable hydrogel that improved the ventricular wall stability andshowed ability to reduce heart dilatation upon myocardial infarction(MI) (Deng et al., 2010). These chitosancollagen hydrogels have beenfound to enhance neovascularization in human umbilical vein endothe-lial cells and epicardial explants in vitro and in rats and mouse in vivoupon subcutaneous administration (Chiu and Radisic, 2011; Denget al., 2010).

Alginate derived from brown algae is a negatively charged polysac-charide composed of -D-mannuronic acid and -L-guluronic acidunits (Madden et al., 2010; Ye et al., 2011). Based on the source and pro-cessing, itsmolecularweight ranges between 10 and 1000 kDa. Alginatehas been established as a biocompatible, mucoadhesive and non-immunogenic polymer (Vlierberghe et al., 2011). Upon addition ofmultivalent cations, alginate solution rapidly forms an ionotropic gel.An ionotropic gel has in contrast to conventional gels additional electro-static associations through the cation species. This rapid gelation prop-erty makes it suitable for cardiac tissue engineering (Hao et al., 2007;Landa et al., 2008; Lee et al., 2009; Leor et al., 2009; Ruvinov et al.,2011; Yu et al., 2009).

Fibrin forms naturally during thewound healing process, and is pro-duced from brinogen and thrombin. It has been extensively employedas a tissue sealant and for the delivery of growth factors specic for tis-sue repair. Fibrin supports adhesion of cells and growth, and allows tobe tuned to acquire desired physical properties by modulation of the -brinogen/thrombin composition during the development of the tissueengineered scaffold (Shoichet, 2010). Fibrin glue used for the deliveryof human mesenchymal stem cells (hMSCs) has led to a substantial in-crease in local cardiac cell retention (Martens et al., 2009). Fibrin gluewas employed as an injectable scaffold with/without skeletal myoblaststo prevent infarct wall thinning in MI-induced rat models (Christmanet al., 2004a). The gel displayed the potential to preserve wall thickness,decreased infarct size, improved cardiac function and increased bloodow to ischemic myocardium (Christman et al., 2004a; Christmanet al., 2004b).

Gelatin known for its biocompatibility, biodegradation and completebioresorbability is a denatured form of collagen (Christman et al.,2004b; Pok et al., 2013). Though it maintains the viability of cardiaccells, its usage solely in scaffolds for cardiac tissue suffers due to itslow tensile strength and rapid deformation. Hence, it is used in combi-

nation with other polymers such as poly(L-lactide-co--caprolactone)(PLCL), hyaluronic acid, chitosan, and poly(glycerol sebacate) to impartenhancement in biocompatibility to the scaffolds for cardiovascular ap-plications (Camci-Unal et al., 2010; Kai et al., 2013; Pok et al., 2013). PCLsandwiched between gelatinchitosan hydrogel forms a multi-layeredbiodegradable scaffold with sufcient mechanical strength, shows mi-gration and maintains neonatal rat ventricular myocyte viability (Poket al., 2013).

Matrigel is a basement membrane protein mixture secreted by amouse sarcoma (Li and Guan, 2011; Ou et al., 2011). Its chemical com-position has not been identied fully; some of the known constituentsare collagen IV, laminin, entactin, heparan sulfate proteoglycan andgrowth factors (Ou et al., 2011). Both assembling structure and compo-sition of matrigel bear resemblance to the native ECM (Li and Guan,2011). The heterogeneous bioactive composition of matrigel has a pos-itive inuence on cell adhesion, differentiation, and proliferation andpromotes angiogenesis. Thus, as one of the injectable matrices, it hasemerged as an engineered ECM that restores and improves myocardialfunctions in a rat MI model (Ou et al., 2011).

Decellularized extracellular matrices are biological scaffolds derivedfrom various tissues and organs that have received signicant attentionfor tissue regeneration. The use of decellularized tissue as scaffold hasthe advantage of being the best mimic of the structure and topographyof the native ECM (Singelyn et al., 2009). As cardiac patches they pro-mote endothelial cell and cardiomyocyte inltration (Singelyn et al.,2009). Decellularized matrices from cardiac ventricular tissue, pericar-dia,myocardium, bladder, small intestine and sub-intestinal submucosahave been used as scaffolds and have effectively delivered cells and bio-molecules for cardiac regeneration (French et al., 2012; Okada et al.,2010; Seif-Naraghi et al., 2010; Seif-Naraghi et al., 2012; Singelynet al., 2009; Singelyn et al., 2012; Zhao et al., 2010).

2.3. Synthetic polymers

Despite the several advantages of natural polymers, their poormechanical properties, variability in physical properties, complexitiesassociated with purication, risks of pathogen transmission and immu-nogenic issues hinder their widespread application. As a result, thefocus has shifted towards synthetic polymers (Lutolf and Hubbell,2005). Synthetic polymers are capable of being tailored tomeet specicapplications and they can be made biocompatible and biodegradable(Nelson et al., 2011). These polymers also have predictable, tunableand reproducible mechanical, chemical and physical properties suchas tensile strength, porosity, elastic modulus, and degradation ratethat allows to be constructed with great precision (Lavik and Langer,2004; Lutolf and Hubbell, 2005). Table 2 summarizes some synthetichydrogels recorded for potential repair or regeneration of infarctedmyocardium.

Poly(ethylene glycol) (PEG) a synthetic polymer of ethylene glycolused for the preparation of injectable hydrogels has been found to bebiocompatible and aids in the controlled release of growth factors(Dobner et al., 2009; Slaughter et al., 2009). A non-degradable syntheticPEG hydrogel was reported to retard adverse post-infarction left ven-tricular remodeling, although they were found to trigger a macrophagemediated inammatory reaction, which is undesirable (Dobner et al.,2009). PEG has also been used for surface modication of PLA or PLGA,to obtain biodegradable co-polymers (Slaughter et al., 2009). Thehydrophilic PEG chains control protein and peptide adsorption as theylack hydrogen bond donating groups. This property also enablesthem to regulate the cellular behavior on the surface of the polymer(Slaughter et al., 2009). Scaffolds incorporated with PEG chains can besubjected to further modication using bioactive peptides to stimulatecellular behaviors such as adhesion to proteins (Lutolf and Hubbell,2005; Slaughter et al., 2009).

Another interesting synthetic polymer that forms gels is polyvinylalcohol (PVA). It is a unique material as even in its atactic form

that lacks stereo-regularity, it exhibits semi-crystallinity. PVA is a

Salie

MSCintoCarddiffeDeliincrcard

valerolactone) [PVL-b-PEG-b-PVL]ConangandAmeimmPrevvencardOn ddenenaIncrblooLocaven

Promdiffe24 d

w-d

454 J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461hydrophilic polymer obtained by polymerization of vinyl alcoholformed through the partial hydrolysis of vinyl acetate (Seif-Naraghiet al., 2012). Poly(2-hydroxyethyl methacrylate) (pHEMA) is polymer-

Polyethylene glycol

Poly(N-isopropylacrylamide-co-acrylic acid-co-hydroxyethylmethacrylate)poly(trimethylene carbonate) [Poly(NIPAM-co-AAc-co-HEMAPTMC)]

Temperature

Poloxamer and di-(ethylene glycol) divinyl ether (multi blockcopolymer)

Temperature

Dextran-poly(e-caprolactone)-2-hydroxylethyl methacrylate/poly(N-isopropylacrylamide) [Dex-PCL-HEMA/PNIPAM]

Temperature

-Cyclodextrin/poly(ethylene glycol)b-polycaprolactone-(dodecanedioic acid)-polycaprolactonepoly(ethyleneglycol) (MPEGPCLMPEG)

Supramolecularself-assembly

Acryloyl-poly(ethylene glycol)-RGDS [Acr-PEG-RGD] Photocross-linking (UV)

(VEGF: vascular endothelial growth factor, MI: myocardial infarction, BMSCs: bone marrobroblast growth factor, MSCs: mesenchymal stem cells).Table 2Synthetic polymers as in situ forming hydrogels for cardiac tissue engineering.

Synthetic polymer Gelationmechanism

Hydroxyethyl methacrylatepoly(trimethylene carbonate)[HEMA-PTMC]

Temperature

Poly(N-isopropylacrylamide) Temperature

Poly(N-isopropylacrylamide-co-propylacrylic acid-co-butylacrylate) (p [NIPAM-co-PAA-co-BA])

Temperatureand pH

Poly(-valerolactone)-b-poly(ethylene glycol)-b-poly(- Temperatureized from the 2-hydroxyethyl methacrylate monomers by free radicalprecipitation polymerization. pHEMA hydrogels have been used as ma-trix material. The resultant hydrogel is relatively inert biologically,weak, shows delayed calcication and shows high resistance to proteinadsorption and thereby prevents adhesion of cells (Slaughter et al.,2009). Poly(N-isopropylacrylamide) (PNIPAM) and its copolymersbased hydrogels exhibit lower critical solution temperature (LCST) atapproximately the physiological temperature (Klouda and Mikos,2008). Various copolymers of PNIPAMwith hydroxyethyl methacrylate,poly(trimethylene carbonate), acrylic acid, polycaprolactone, dextran,and dimethyl--butyrolactone acrylate have been reported as effectivestrategy for cardiac regeneration (Fujimoto et al., 2009; Li et al., 2011;Wang et al., 2009a). Poly(glycerol sebacate) (PGS) is an elastomericpolymer with robust mechanical properties, biodegradability, andin vitro and in vivo biocompatibility (Nijst et al., 2007). Recently, an in-jectable scaffold based on PGS has improved rabbit cardiomyocyte cellretention and survival, as well as increased expression of major cardiacmarker proteins connexin 43, troponin, and actinin, and myosin heavychain was observed (Ravichandran et al., 2012).

3. Various approaches in tissue engineering

Tissue engineering strategies adopted can be broadly categorized asin vitro and in vivo approaches (Leor et al., 2005). The in vitro ap-proaches include tissue engineered constructs, with or without cells inculture dishes or bioreactors that are designed to mimic the extracellu-lar microenvironment of the affected site (Leor et al., 2005). Scaffold-free cell sheets are another major category of in vitro tissue engineeringstrategy (Vunjak-Novakovic et al., 2011). Cell transplantation, injectablescaffolds with or without cells and delivery of active molecules for selfrepair that helps to regenerate the cardiac tissue in situ followingadministration and integration with existing tissue are classied asin vivo approaches (Leor et al., 2005).

nt features Animalmodel

Ref.

s encapsulated inside hydrogel differentiatedcardiac cells

In vitro Li et al. (2012)

iosphere-derived cells proliferated andrentiated into mature cardiac lineage

In vitro Li et al. (2011)

very of bFGF augmented regional blood ow byeasing microvessel density and enhancediovascular function

Rat Garbern et al. (2011)

jugated with VEGF increased regionaliogenesis, reduced adverse cardiac remodelingenhanced function

Rat Wu et al. (2011)

liorates pathological remodeling in theediate postinfarction healing phase

Rat Dobner et al. (2009)

ented ventricular dilation, enhanced thetricular contractile action and improvediovascular function

Rat Fujimoto et al. (2009)

elivery of hVEGF plasmid, increase in capillarysity and larger vessel formation observed, thusbling effective angiogenesis

Rat Kwon et al. (2009)

eased retention of encapsulated BMSCs, elevatedd vessel density and improved cardiac functions

Rabbit Wang et al. (2009a)

l sustained delivery of rhEPO inhibits postinfarcttricular remodeling without polycythaemia.

Rat Wang et al. (2009c)

oted C2C12 myocyte adhesion, proliferation,rentiation and maintains active VEGF level overays period

Rat(ex vivo)

Yeo et al. (2007)

erived mesenchymal stem cells, rhEPO: recombined human erythropoietin, bFGF: basic3.1. In vitro engineered constructs

In vitro engineered constructs are used to replace or reconstruct de-fective regions of the heart (Leor and Cohen, 2004). Several polymersincluding poly(caprolactone) (PCL), poly(ethylene glycol) (PEG),poly(caprolactone):poly(glycerol sebacate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(L-lactide-co--caprolactone)(PLCL), and poly(L-lactide-co-glycolic acid) (PLGA) have been used forfabrication of nanobrous scaffolds for cardiac tissue regeneration(Lakshmanan et al., 2012). Electrospinning, phase separation, and self-assembly are some of the major techniques for fabrication of nanoberscaffolds (Ayres et al., 2010; Subramanian et al., 2009). Hsiao et al.(2013) have recently developed an electrically active aligned compositenanobrous mesh of poly(lactic-co-glycolic acid) (PLGA) and polyaniline(PANI), this scaffold coordinated the pulsation of the cultured cardio-myocytes synchronously. Other scaffold types fabricated for this appli-cation include hydrogels, microsphere-based scaffolds, sponges, etc.(Levenberg and Langer, 2004). Smith et al. (2012) have fabricated PEGmicrosphere based three-dimensional scaffold in the presence of dex-tran by phase separation that can support the long-term culture ofHL-1 cardiomyocytes. The cells exhibited good cell proliferation andcardiomyocyte marker expression and retained electrical activity.

Polymers that are currently being exploited for tissue engineeringhave been chemically modied to enhance the functional efciency ofscaffolds to exert precise control over quantitative adhesion, growthand migration signals (Davis et al., 2005a; Koo et al., 2002; Leor et al.,2005). Suchmodications in biomaterials can be either over the surfaceor throughout the bulk of the material. Biomimetic materials can beattained through simple surface modication with bioactive molecules(Leor et al., 2005). Incorporation of cell adhesion peptides such asRGD, DGEA, and RRETAWA enables formation of focal adhesion contactson the surface of the biomaterial leading to cell adhesion (Bakov

455J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461et al., 2004; Koo et al., 2002; Shoichet, 2010). The density and localiza-tion of the focal adhesion points also inuence cell spreading, viability,migration, proliferation and differentiation (Bakov et al., 2004).Modications are generally achieved by chemical reactions that resultsin covalent bond formation between the bioactive peptide and the poly-mer backbone. Polymeric hydrogel cross-linked using bi-functionalpeptides which consist of a signaling domain for interactions withreceptors of cell membrane has also been reported (Halstenberget al., 2002; Leor et al., 2005). Various short amino acid sequences(oligopeptides) have been recognized and have been found to mediatecell adhesion and cell-specic function. Arginineglycineaspartic acid(RGD) is the most extensively investigated cell-adhesive peptide de-rived from an ECM component bronectin, to which cells adherein vivo (Koo et al., 2002; Ratner and Bryant, 2004; Ulijn et al., 2007).This concept of peptide modied hydrogel surfaces had been effectivelyemployed in the design of an adhesion-based cell separation tech-nique for selective separation of specic cell type from a heterogeneouscell population. Here, microuidic channels were coated with RGDSpeptide-functionalized alginate gels for controlled capture and releaseof cardiac broblasts (Plouffe et al., 2009).

Angiogenesis is a vital aspect in cardiac tissue engineering as itcan promote cell survival and inhibit brotic tissue formation. Pro-angiogenic hydrogel scaffolds have been investigated for enhancingthe cardiac tissue formation. Cardiomyocyte (CM)-enriched neonatalrat heart cells were embedded into submillimeter-sized modulescomposed of bovine collagen type I supplemented with Matrigel(25% v/v), these modules were further seeded with rat aortic endothe-lial cells (RAEC). This contractile, macroporous, pro-angiogenic, sheet-like construct showed promise for the development of vascularizedheart tissue (Leung and Sefton, 2010). Cardiac implantation of acellularconstructs using bimodal poly(2-hydroxyethyl methacrylate-co-methacrylic acid) (pHEMA-co-MAA) hydrogel scaffolds were evaluatedfor their feasibility as pro-angiogenic constructs. This scaffoldwas foundto favor oriented growth of human embryonic stem cell (hESC)-derivedcardiomyocytes apart from inducing neoangiogenesis and retarding -brosis (Madden et al., 2010). Post-MI treatment with a biocompatiblegelatin hydrogel patch was found to improve LV remodeling and func-tions by acting as an anti-brotic and angiogenic construct and activat-ing prosurvival signaling (Kobayashi et al., 2008). Coupling of sheet-likeconstruct in cardiac scaffolds may be recommended for cardiovascu-lar function. A hybrid, tunable hydrogel based scaffold fabricated byhomogeneously synthesizing gold nanoparticles throughout a poly-mer templated gel has been prepared and characterized (You et al.,2011). Neonatal rat cardiomyocytes exhibited increased expressionof the gap junction protein connexin 43 on this hybrid scaffoldboth in the presence as well as absence of electrical stimulation(You et al., 2011).

Despite the diverse in vitro engineered constructs examined, thereexist substantial challenges that hinder the implantation of existing cardi-ac tissue constructs. These include poor vascularization and stimulation ofthe host inammatory response (Davis et al., 2005a; Zimmermann et al.,2004). In addition, thesematerials require suturing over the infarcted sitethat still remains a critical issue.

3.2. In vivo approaches

3.2.1. In situ forming injectable hydrogelsIn situ hydrogels are a unique class of hydrogels that exhibit sol-like

properties at ambient conditions and undergo a transition to a gel phaseunder physiological conditions. Such in situ forming gels can be injecteddirectly at a desired location and can ll any defect. Injectable scaffoldsare advanced compared to their respective preformed scaffolds as itpromises improved patient compliance, easy administration to the in-farct zone via minimally invasive technique for the treatment of largeand complex lesions (Balakrishnan and Banerjee, 2011). In contrast, ex-

ternal scaffold patches are xed onto the epicardial surface surgically(Huang et al., 2005; Singelyn and Christman, 2010). The strategies forin situ gelling of polymers include photo-cross-linking, chemicalcross-linking, ionic interaction, enzymatic cross-linking, temperature-induced gelation, pH-induced gelation, electric eld, magnetic eld,hydrophobic interactions, and presence of antigen, glucose and theircombinations (Balakrishnan and Banerjee, 2011; Huynh et al., 2011;Jeong et al., 2002; Singelyn and Christman, 2010; Wu et al., 2009; Wuet al., 2011). The hydrogels following injection in situ should deformwith the dynamic myocardial microenvironment and simultaneouslyalign with the ECM of the injured site (Singelyn and Christman, 2010).This facilitates its better integration with the host tissue, provides me-chanical support to the infarcted heart, attenuates wall stress, compen-sates the contraction function and prevents ventricular remodeling(Huang et al., 2005; Ifkovits et al., 2010; Singelyn and Christman,2010; Ye et al., 2011).

Certain polymers exhibit solgel transition above the critical gel con-centration (CGC) in response to temperature change (Jeong et al., 2002;Klouda andMikos, 2008). They remain in sol state at lower temperatureand above a temperature known as critical solution temperature (CST)as their hydrophobicity increases and they become a gel. Such polymersare known as lower critical solution temperature (LCST) polymers. Incontrast, some of them form gel on cooling below their critical solutiontemperature and such systems are referred to as upper critical solutiontemperature (UCST) polymers (Klouda andMikos, 2008). Typical exam-ple of an LCST polymer is poly(N-isopropylacrylamide) (PNIPAM) andits copolymers that are thermoresponsive and exhibit a phase transitionfrom the sol to gel phase at around 32 C (Jeong et al., 2002; Klouda andMikos, 2008). This phase transition occurs as the water moleculesbound to its isopropyl groups are released resulting in increased inter-and intra-molecular hydrophobic interactions above the LCST ofPNIPAM (Tan and Marra, 2010). Synthetic injectable poly(N-isopropylacrylamide-co-acrylic acid-co-hydroxyethyl methacrylate)poly(trimethylene carbonate) (Poly(NIPAM-co-AAc-co-HEMA-PTMC))copolymers exhibited attractive mechanical properties, enhancedcontractile function and prevented ventricular dilation in a chronicrat MI model (Fujimoto et al., 2009). Injection of thermosensitivedextran-poly(-caprolactone)-2-hydroxyethyl methacrylate/poly(N-isopropylacrylamide) (Dex-PCL-HEMA/PNIPAM) hydrogel has beenfound effective to prevent adverse cardiac remodeling and dysfunctionin MI induced rabbits (Wang et al., 2009a).

Triblock copolymers such as PEG-PCL-PEG, PCL-PEG-PCL, PLGA-PEG-PLGA, PCLA-PEG-PCLA, PEG-PLLA-PEG and PEO-PPO-PEO (Pluronic) arereversible thermogelling polymers that exhibit solgel transitions basedon bridged micelle formation (Tan and Marra, 2010). Chitosan is watersoluble at acidic pH up to 6.2 and it precipitates to appear gel-like withincreased basicity which neutralizes the amine groups. On addition ofpolyol salts such as -glycerophosphate (GP), the pH-responsive chito-san can be extended to pH-dependent, thermoresponsive system(Vlierberghe et al., 2011).

In situ formation of cross-linked hydrogels can be achieved byphotopolymerization at physiological pH and temperature, which onthe other hand allows encapsulation of cells without affecting their via-bility. This system generates radicals from the photoinitiators by utiliz-ing ultraviolet irradiation and the active end groups such as acrylate andmethacrylate enable the propagation of the reaction to form covalentcross-linking. The advantage of this system is the facile incorporationof a variety of chemistries by derivatization of macromers. A typicalexample is poly(ethylene glycol)-dimethacrylate (PEGDMA) andpoly(ethylene-glycol)-diacrylate (PEGDA) that carry unsaturatedC_C groups. Some other polymers chitosan, alginate, chitosan,chondroitin sulfate and hyaluronic acid have been methacrylatedthereby making them amenable for photocross-linking (Tan andMarra, 2010). Photopolymerized PEG-acrylate cross-linkermodied -brinogen hydrogels have supported in vitro culture of neonatalcardiomyocytes, which further promotes the development of spontane-

ously contractile tissue (Shapira et al., 2008).

456 J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461In Michael addition reaction, addition of a nucleophile (Michaeldonor) with an activated electrophilic olen (Michael acceptor) resultsto the formation of Michael adduct. For tissue engineering applications,Michael addition networks based on acrylate and thiol precursors areideal as their gelation process is controllable (Mather et al., 2006). HA-based injectable hydrogel fabricated based on the reaction betweenPEG-thiol and acrylated hyaluronic acid improved the functioning ofthe heart comparable to the normal heart (Yoon et al., 2009).

Gelation can also be induced by varying the ionic concentrationof the polymeric solution. For example, alginate, a linear blockco-polymer consisting of -D-mannuronate (M-block) and -L-guluronate (G-block) units linked by 1,4-glycosidic linkage (Cho et al.,2009) exhibits such phenomenon. On addition of divalent ions such asstrontium (Sr2+), calcium (Ca2+) and barium (Ba2+), cross-linkingof alginate chains occurs quickly through the stacking of G-blocksresulting in the formation of gel network with an egg-box structure, re-ferred to as ionotropic gels (Cho et al., 2009). Calcium cross-linked algi-nate has been shown to be an effective injectable implant for cardiacremodeling and function restoration. This absorbable hydrogel was ef-fective in both early (6 days) and aged (60 days) infarcts in rats(Landa et al., 2008). A similar scaffold has been reported to be feasible,effective and safe for intracoronary injection in swine model. In thiscase, the alginate gel crossed the injured leaky coronary vessel, deposit-ed on the infarct tissue and reversed left ventricular (LV) remodeling(Leor et al., 2009). Another group has reported an RGD modied algi-nate scaffold that reshaped dilated aneurysmal LV and improved LVfunction, and enhanced angiogenesis, thereby proving positive inu-ence of surface modication on the microenvironment of the infarctedmyocardium (Yu et al., 2009).

Some polymers in high viscous solution or in slightly cross-linkedgel form tend to deform and ow when under shear stress (Gutowskaet al., 2001). On removal of the shearing force, gelation occurs and thisphenomenon has been employed in injectable hydrogels (Gutowskaet al., 2001; Lu et al., 2012). The main criterion for cross-linking inthese gels is self-assembly and these hydrogels may be based on pro-teins, peptides, colloidal systems and polymer blends (Guvendirenet al., 2012). An alginate based shear-thinning hydrogel has been re-ported to increase the viability of human umbilical vein endothelialcells (HUVECs) (Aguado et al., 2012).

3.2.2. Decellularized matricesDecellularized ECM was developed based on the concept that the

better alternative of the complex milieu of a tissue could be the nativeECM itself. The process involving physical, chemical or enzymatic re-moval of the cellular content of an organ or tissue leaving behind theECM is given the term decellularization. Although this may modify themorphological and biochemical compositions of the ECM, it is advanta-geous as it removes cellular antigens that potentiate foreign body reac-tion via antibody activation, inammation, and potential transplantrejection (Singelyn and Christman, 2010). Xenogeneic decellularizedmaterials are well tolerated as the ECM proteins are reasonably wellconserved among species and hence some of them are FDA approvedfor clinical use (Singelyn and Christman, 2010). Epicardial cardiacpatch material from the urinary bladder matrix has proved to be bene-cial for tissue engineering applications as a reparative process sugges-tive of tissue regenerationwas observed on this scaffold (Robinson et al.,2005).

A current advancement in this eld is the modication ofdecellularizedmatrix into injectablematerial forminimally invasive de-livery. Decellularized small intestinal submucosa (SIS) was processed toobtain a powder which was injected directly into the ischemic myocar-dium of rat in the form of an emulsion (Zhao et al., 2010). These inject-able SIS gels potentially repair ischemic myocardium (Okada et al.,2010). However, decellularized matrix from cardiac tissue would bemore specic and appropriate to replace the ECM of damaged myocar-

dium, as each tissue contains a unique combination of proteoglycansand proteins (Singelyn and Christman, 2010). Decellularized ventricularand pericardial ECM has been processed to obtain a liquid which ex-hibits gelation via self-assembly at physiological temperature bothin vitro and in vivo (Seif-Naraghi et al., 2010; Singelyn et al., 2009).The biochemical composition of decellularized ventricular ECM retainedthe complexity of peptides, proteins and glycosaminoglycans whichwas revealed when characterized (Singelyn et al., 2009). Furthermore,this myocardial matrix promoted the migration of rat aortic smoothmuscle cells and human coronary artery endothelial cells in vitro, andenhanced the inltration of vascular cells and arterioles formationin vivo (Singelyn et al., 2009). Initially, the potentiality of utilizingdecellularized pericardial gel for autologous therapy has been studiedand offers benecial effects (Seif-Naraghi et al., 2010). Recently, cardiacprogenitor cells (CPCs) that were cultured on plates coated with natu-rally derived porcine cardiac extracellularmatrix (cECM) showed an el-evated expression of early cardiomyocyte markers when compared tothose cultured on collagen I coated plates (French et al., 2012).

Singelyn et al. (2012) derived an injectable hydrogel from ventricu-lar extracellular matrix (ECM) and delivered it percutaneously viatransendocardial injection to treat myocardial infarction (MI). This ma-trix exhibited retention within the myocardium, preserved cardiacfunction and showed initial translation to large animals. Seif-Naraghiet al. (2012) demonstrated that the decellularized porcine pericardialmatrix hydrogel, with its sulfated sugars, has the capacity to sequesterbasic broblast growth factor (bFGF) and exhibit enhanced acute neo-vascularization post-MI in rodents.

3.2.3. Self-assembling peptidesSelf-assembling peptides form stable nanober hydrogels when in-

troduced into the physiologicalmilieu (Segers and Lee, 2007). An inject-able self-assembling RAD16-II peptide (AcN-RARADADARARADADA-CONH2) gels in situ and provided an appropriate microenvironment inthe myocardium (Davis et al., 2005b). This microenvironment promot-ed vascular cell recruitment and repopulation of the injected area withendothelial cells, smooth muscle cells and also some nonvascular cells.Hsieh et al. (2006) have employed the same peptide for the sustaineddelivery of platelet-derived growth factor with two B chains (PDGF-BB) to the myocardium that resulted in decreased cardiomyocytedeath and preserved systolic function after MI. RAD16-II has also beenemployed as cell-carrying scaffolds (Lin et al., 2010). Lin et al. (2010)demonstrated that RAD16-II peptide injection alone prevented ventric-ular remodeling.When combinedwith autologous bonemarrowmono-nuclear cells (BMMNCs) improved cell retention and cardiac function inpigs with MI, which has the potential to be translated to clinics (Linet al., 2010).

A novel biomimetic designer self-assembling peptidewas construct-ed by attaching a cell-adhesion motif with the sequence of RGDSP tothe self-assembling peptide RAD16-I (AcN-RADARADARADARADA-CONH2). This scaffold improved the adhesion, survival of c-kit+/Nkx2.5low/GATA4low marrow-derived cardiac stem cells (MCSCs) andwas conducive for their differentiation to cardiomyocytes thereby im-proving cardiac repair and function (Guo et al., 2010). The same grouphas also designed another novel modied self-assembling peptide byattaching the heparin-binding domain sequence LRKKLGKA to the se-quence of RAD16-I self-assembling peptide. The modied peptide self-assembled under physiological conditions into nanober scaffolds,which provided sustained delivery of VEGF, improved cardiac function,markedly reduced scar size and collagen deposition and enhanced themicrovessel formation (Guo et al., 2012).

3.2.4. Cells/stem cells and biomolecular signals based approachesCardiomyocytes possess limited self-repair or regeneration capacity

once terminally differentiated or after injury, and thus pose a challengefor regenerativemedicine interventions (Giraud et al., 2008). Cell-basedtherapeutics has recently emerged as a signicant modality in regener-

ative medicine, cancer immunotherapy, and promises cures to a

457J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461multitude of diseases and disorders, via tissue repair (Klug et al., 1996;Wang et al., 2010). Cell transplantation potentially replenishes irrevers-ibly damagedmyocardiumwith intrinsic progenitor cells, including au-tologous and induced pluripotent stem cells and cells from othersources such as embryonic stemcells, adult stemcells and differentiatedcells isolated from adult tissue (Levenberg and Langer, 2004; Yoshidaand Oh, 2010).

The rst cardiac cell implantation experiments were carried outusing genetically selected embryonic stem cell-derived cardiomyocytes.These cells were administered directly to the ventricular wall or coro-nary vessels using syringe injection (Klug et al., 1996; Zammaretti andJaconi, 2004). Following this, cells of various origins including fetal, em-bryonic or adult sources have been investigated (Zammaretti andJaconi, 2004). Stem cells from adult tissue sources such as peripheralblood, bone marrow, umbilical cord and adipose tissue could give riseto the formation of autologous cardiac myocytes as well as endothelialand smooth muscle cells (Eschenhagen and Zimmermann, 2005; Orlicet al., 2001). The sources of stem cells for cardiac regeneration andtheir potential have been reviewed comprehensively in many reports(Klug et al., 1996; Lakshmanan et al., 2013; Leor and Cohen, 2004;Leor et al., 2005; Nelson et al., 2011; Nunes et al., 2011; Passier andMummery, 2003; Ye et al., 2011; Zammaretti and Jaconi, 2004).

Most studies support the hypothesis that engraftment of cells in MIanimal models can undoubtedly improve cardiac contractile function(Passier andMummery, 2003). The possibility of using human umbilicalcord cells (UCC) from autologous cell source has been studied in vitro(Kadner et al., 2002). The UCC established excellent growth properties,tissue formation and attain mechanical properties that are comparableto the native tissue. In addition, these cells are easily obtained andhence use of these cells for cardiac repair avoids the need for harvestingcells using invasive protocols from intact vascular structures (Kadneret al., 2002).

Generation of rejuvenated andhistocompatible stem cells by nucleartransfer techniques could resolve the inadequate supply of stem cells.Lanza et al. (2004) derived cloned embryos through somatic cell fusiontechnique, the nuclei of cultured LacZ-positive broblasts and enucleat-ed oocytes of different mouse strains were fused. c-kit+ fetal liver stemcells obtained from these cloned embryos were shown to induce tissuereconstitution, when injected in the MI border zone of infarcted mice.Fragments of cell sheets formed from hydrogels have been reported toserve as a cell delivery vehicle that provides favorable ECM environ-ment and retains the transplanted human amniotic uid stem cells(hAFSCs) in an infarcted myocardium of an immune-suppressed ratmodel. The transplanted hAFSCs improved the cardiac function by dif-ferentiating into the cardiomyocyte-like cells and the endothelial celllineages, as well as through regulation of growth factor mediated para-crine function (Yeh et al., 2010).

However, the clinical trial outcome to date haswell depicted that theactual benets realized seem to bemuch lesser than they assure. Almostmore than 90% of cells/stem cells in the suspension do not engraft andare lost after injection, thus hinder successful clinical translation ofthis strategy (Kurdi et al., 2010; Zammaretti and Jaconi, 2004). Cellulartherapy is applicablewhen the pathology is relatively less severe and lo-calized than in diffused conditions (Leor and Cohen, 2004). Therefore,concerted efforts are directed towards developing various tissue-engineering strategies using biomaterial scaffolds for successfullyengrafting new cells into the myocardium (Zammaretti and Jaconi,2004). Recently, hydrogels have been used as carriers for delivery ofcells and biomolecules (Huynh et al., 2011). The hydrogel-mediatedcell delivery systemprovides three dimensional (3D) environment sim-ilar to in vivo conditions and allows the maintenance of normal cellularfunction (Hunt and Grover, 2010; Wang et al., 2010). Fig. 3 representsan epicardial delivery of in situ forming hydrogel.

A family of thermosensitive and injectable hydrogels were fabricat-ed to deliver cardiosphere-derived cells (CDC), atom transfer radical po-

lymerization of N-isopropylacrylamide, 2-hydroxyethyl methacrylateand dimethyl--butyrolactone acrylate used to synthesize thesehydrogels that possess a well-dened structure and well-controlledproperties (Li et al., 2011). They possessed physical properties suitablefor MI treatment; promoted proliferation of CDC and differentiationto cardiac lineage cells (Li et al., 2011).Wang et al. (2009b) demonstrat-ed that intramyocardial injection of bone marrow stem cells with-cyclodextrin/MPEGPCLMPEG hydrogel at MI site in rabbits im-proved the impaired cardiac function and increased the longevity oftransplanted cells at the infarct zone.

Signaling molecules form the third component of the tissue engi-neering triad that also constitutes the scaffolds and cells (Kuppanet al., 2012). Biomolecular signaling by means of growth factors is criti-cal for promoting cell adhesion, proliferation, differentiation, migrationand the subsequent tissue development and repair (Whitaker et al.,2001). Suitable biomolecules other than growth factors such as antiox-idants can be co-delivered with the hydrogel to neutralize the harsh is-chemic environment, which lacks nutrients/oxygen and protect thedelivered cells. Choice of the appropriate growth factor and a robust de-livery system are the primary requirements for successful regeneration.This is because the biomolecules are highly unstable in vivo and there-fore should be chemically conjugated or physically entrapped to beprotected and gradually released from the hydrogels (Li et al., 2011).Pro-angiogenic and/or pro-survival growth factors can be delivered intheir native form or expressed using recombinant technology throughscaffold-mediated delivery. Growth factors such as vascular endothelialgrowth factor (VEGF) and basic broblast growth factor (bFGF) contrib-ute to angiogenesis, and hence delivering them in combination with asuitable scaffold would be benecial for cardiac regeneration(Yamamoto et al., 2003).

Proper spatio-temporal delivery of multiple therapeutic proteinsmajorly favors myocardial regeneration at the infarcted site. For in-stance, the dual delivery of hepatocyte growth factor (HGF) andinsulin-like growth factor-1 (IGF-1) using injectable afnity-binding al-ginate has been observed to maximize their therapeutic effects(Ruvinov et al., 2011). A synthetic, in situ forming bioactive injectablehydrogel responsive to matrix metalloproteinase (MMP) has beenused to deliver a pro-angiogenic and pro-survival factor thymosin 4(T4), in combination with hESC-derived vascular cells, to a rat ische-mic heart model. The paracrine action of factors released by thetransplanted cells was analyzed by the cytokines measurement(Kraehenbuehl et al., 2011). This strategy exhibited considerable prom-ise towards cardiac repair by elevation of blood vessel density(Kraehenbuehl et al., 2011).

Wang et al. (2009c) have reported a non-toxic -cyclodextrin/MPEGPCLMPEG hydrogel. It was employed for delivering recombi-nant human erythropoietin in an acute MI rat model and was foundto improve the cardiac function without polycythaemia. Garbernet al. (2011) showed that the temperature and pH-responsive randomcopolymer poly(N-isopropylacrylamide-co-propylacrylic acid-co-butylacrylate) (p(NIPAM-co-PAA-co-BA)) elevated microvessel density,improved regional blood ow and enhanced cardiovascular function,following injection with basic broblast growth factor (bFGF). Anew, temperature-sensitive, aliphatic polyester hydrogel (poly(-valerolactone)-b-poly(ethylene glycol)-b-poly(-valerolactone) (PVL-b-PEG-b-PVL)) polymer conjugated with vascular endothelial growthfactor (VEGF) was administered in rat after MI, stabilized the infarctand induced angiogenesis, thereby preserved the ventricular function(Wu et al., 2011). A thermo-responsive hydrogel reported increased an-giogenesis in the infarct area, upon administration along with plasmidsencoding genes for VEGF. This multi-block amphiphilic hydrogel wassynthesized by alternatively cross-linking Pluronic P-104 and di-(ethyl-ene glycol) divinyl ether via an acid-labile acetal linkage (Kwon et al.,2009). Recently, sustained delivery of an effective chemoattractantstromal cell-derived factor-1 (SDF-1) from pro-angiogenic starPEG-heparin hydrogels has been reported to effectively attract early en-

dothelial progenitor cells (EPCs), and subsequently triggered the

endogenousmechanisms of cardiac regeneration (Prokoph et al., 2012).Bone marrow mononuclear cells (BMMNCs) implanted to myocardialinfarction site differentiated to cardiomyogenic lineages in situ whenexogenous transforming growth factor-1 (TGF-1) was delivered tothe cell implantation site (Yang et al., 2012).

3.2.5. Methods adopted for loading cells in scaffoldsVarious cell loading methods are followed to achieve distribution of

cells in scaffolds such as cell dispersion in the biomaterial, cell spheroidencapsulation, cell seeding over the preformed construct or cellssandwiched between two layers (Ma et al., 2013). Previously, to achieveeven distribution of cardiomyocytes, brief centrifugation after introduc-ing the cell suspension on a 3D porous alginate scaffold has been report-ed (Dar et al., 2002). The strategy for injectable cell-bearing scaffoldinvolves use of a viable cells suspension in an aqueous solution ofhydrogellable precursors prior to gelation leading to formation of cell-loaded 3D gel matrices (Hunt and Grover, 2010; Wang et al., 2010). Insitu gelation in response to specic stimuli such as temperature, pH,electromagnetic radiation or chemical cross-linkers usually occurs rapidlyleading to uniform cell distribution (Wang et al., 2010). The optimal den-sity of cells to be suspended in hydrogel precursor solution is another keyaspect of concern,which varies according to the cell types and the scaffold

4. Limitations

It has become evident from the reports that considerable progresshas been made and injectable hydrogels may become promisingmeans to ameliorate the post-MI ventricular functioning, angiogenesisand thereby cardiac tissue regeneration (Dobner et al., 2009; Fujimotoet al., 2009; Wang et al., 2009b; Wu et al., 2011). The underlyingmech-anisms have to be unveiled for successful translation to the clinic (Touset al., 2011). In this review, the animal models on which injectablehydrogels were assessed have been mostly rats (Fujimoto et al., 2009;Garbern et al., 2011; Kwon et al., 2009; Wang et al., 2009c; Wu et al.,2011) or rabbits (Ou et al., 2011; Zhao et al., 2010). Rats are the mostcommon model employed for its ease of implementation and cost con-siderations. This model suffers from lack of clinical relevance as the leftventricular volume and structure, injection volume of the material andinjection method differs in the rat (Tous et al., 2011). Like the animalmodel, the duration of study is another factor to be considered. Moststudies have reported the injectable material on animal models for 28to 35 days (Wang et al., 2009b; Wang et al., 2009c; Wu et al., 2011) or8 weeks (Fujimoto et al., 2009). A 13 week-post-MI treatment has re-vealed that the material fails to preserve the heart dimension and theleft ventricular end-diastolic diameter was similar to sham animals

458 J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461materials. For instance, about 2.5 million cardiomyocytes in 50 L and10 million hMSCs in 1.25 mLwere suspended in photocrosslinkable pep-tide modied chitosan and thermoresponsive PNIPAM hydrogel forin vitro evaluation of the scaffold (Li et al., 2012; Rask et al., 2010).

3.2.6. Pharmaceutical based strategiesPolizzotti et al. (2012) reported that drug delivery using gel foams

intrapericardially increasedmyocardial retention, therapeutic efciencyand bioactivity of recombinant Periostin (rPN) and decreased systemicrecirculation by triggered clot formation in a clinically relevant porcinemodel of myocardial infarction. Periostin has been reported to possess amultitude of functions like improved ventricular remodeling and myo-cardial function, anti-brotic property, angiogenic nature and abilityto reduce infarct size (Polizzotti et al., 2012). Bezuidenhout et al.(2013) have incorporated dexamethasone in injectable polyethyleneglycol hydrogels by acrylation, PEGylation, and tethering to acrylatebased hydrolytically degradable and nondegradable (vinyl sulfonebased) PEG hydrogels by Michael-type nucleophilic addition. Asustained release of the active drug was achieved from the hydrogelsystem. This strategy was aimed to avoid graft rejection and any in-ammatory response.Fig. 3. Epicardial delivery of hydrogellable solution carrying cells and biomolecular signals, whi(Dobner et al., 2009). Hence, it has become evident that there still existsa lacuna in establishing the most appropriate animal MI model and theduration of study for preclinical study.

5. Current scenario of translational research in cardiactissue engineering

Clinical translation of the various tissue engineering research to treatcardiac diseases is the ultimate goal of all regenerative strategies(Ptaszek et al., 2012). Preclinical studies using animal models to evalu-ate the efcacy of cells, scaffolds, growth factors and combinationsin vivo have been discussed in previous sections. Chavakis et al.(2010) have summarized the progress in cell-based therapies for cardi-ac repair. Hematopoietic and mesenchymal stem cells from bone mar-row source, adipose tissue-derived cells, cardiac stem cells andcardiosphere-derived cells have been used in clinical trials (Chavakiset al., 2010). The past decade has evidenced a number of cellular thera-pies for treating cardiovascular diseases in clinical phase-I and phase-IItrials (Chavakis et al., 2010; Salem and Thiemermann, 2010). Recentsignicant clinical phase-I trials include autologous human cardiac-derived stem cell (ALCADIA), autologous c-kit+1 cardiac stem cellsch on administration forms 3D hydrogel over the infarct site due to cross-linked networks.

459J. Radhakrishnan et al. / Biotechnology Advances 32 (2014) 449461(SCIPIO), and cardiosphere-derived cells (CADUCEUS) that have exhib-ited promise and have emerged as prime candidates for phase-II trials(Chugh et al., 2012; Makkar et al., 2012; Ptaszek et al., 2012). Anotherphase-I clinical study, myocardial assistance by grafting bioarticialmyocardium (MAGNUM trial) has proved the safety and feasibility ofcollagen based tissue-engineered approach to improve the efciencyof cellular cardiomyoplasty (Chachques et al., 2008). Commercialproducts available include pericardial patches of xenogenic sourcessuch as Perpatch, PerGuard, SJM pericardial patch and polymericpatches Gore-Tex, Neoveil. CROSSEAL Fibrin Sealant and OmrixBiopharmaceuticals are intramyocardially injectable to treat chronic is-chemic myopathy (Lakshmanan et al., 2013). Validation of regenerativemedicine by clinical trials would bring about promising changes in thetherapeutics of cardiovascular diseases in future.

6. Conclusions

The signicant traits of scaffold for cardiac tissue engineering in-clude appropriate mechanical properties, electrical excitability or con-ductibility to transmit the pumping signals such that the scaffold andsurrounding tissue function in unison to restore normal functions of car-diac tissue. On the other hand, vascularization is an important essential-ity to enrich the blood supply at the infarcted site. The emergence of insitu forming hydrogels for tissue engineering proves to be a signicantmilestone. This semi-invasive system paves way for the next generationof personalized therapeutics as it facilitates tailoring of the scaffold tosuit the severity or defect in the cardiac tissue. However, the challengelies in meticulously choosing the best biomaterial and manipulatingthem to suit the patients' requirements. The reliability on translatingthis system to clinical applications should be preceded by the robust as-sessment of their efciency, compatibility and toxicity in pre-clinicaltrials.

Acknowledgment

This work is supported by INSPIRE fellowship (reg. no.: IF120692) ofthe Department of Science and Technology (DST), India.

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