Research Article Development of Bioactive Patch for...

Research ArticleDevelopment of Bioactive Patch for Maintenance ofImplanted Cells at the Myocardial Infarcted Site

C Castells-Sala1 A Valleacutes-Lluch2 C Soler-Botija3

M Arnal-Pastor2 C Martiacutenez-Ramos2 T Fernandez-Muintildeos1

N Mariacute-Buyeacute1 A Lluciagrave-Valldeperas3 B Sanchez4 J C Chachques5

A Bayes-Genis3 M Monleoacuten Pradas2 and C E Semino1

1Department of Bioengineering IQS-School of Engineering Ramon Llull University 08017 Barcelona Spain2Center for Biomaterials and Tissue Engineering Universitat Politecnica de Valencia 46022 Valencia Spain3Heart Failure and Cardiac Regeneration (ICREC) Research Program Health Research Institute Germans Trias i Pujol (IGTP)Cardiology Service Hospital Universitari Germans Trias i Pujol 08916 Badalona Spain4Division of Neuromuscular Diseases Department of Neurology Beth Israel Deaconess Medical CenterHarvard Medical School Boston MA 02215-5491 USA5Laboratory of Biosurgical Research University of Paris Descartes Pompidou Hospital 75015 Paris France

Correspondence should be addressed to C E Semino carlosseminoiqsurledu

Received 9 November 2014 Accepted 16 February 2015

Academic Editor Zhenhui Kang

Copyright copy 2015 C Castells-Sala et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Ischemia produced as a result of myocardial infarction might cause moderate or severe tissue death Studies under developmentpropose grafting stem cells into the affected area and we hypothesize that this mechanism could be enhanced by the applicationof a ldquobioactive implantrdquo The implant herein proposed consists of a thin porous elastomeric membrane filled with self-assemblingnanofibers and human subcutaneous adipose tissue derived progenitor cells We describe the development and characterization oftwo elastomericmembranes poly(ethyl acrylate) (PEA) and poly(caprolactone 2-(methacryloyloxy)ethyl ester) (PCLMA) Both area goodmaterial support to deliver cells within a soft self-assembling peptide and are elastic enough to withstand the stresses arisingfrom the heartbeat Both developed composites (PEA and PCLMA combined with self-assembling peptide) equally facilitate thepropagation of electrical pulses andmaintain their genetic profile of the seeded cells Preliminary studies with small animal modelssuggest that at short times the bioimplant shows good adhesion with the myocardium After three days cells loaded in the patchremain alive at the implanted site We propose that the bioactive patch (elastomeric membranes with self-assembling peptide andcells) could increase the efficacy of future cardiac cell therapy by improving cell immobilization and survival at the affected site

1 Introduction

Heart failure (HF) is the end stage of most cardiovasculardiseases such as myocardial infarction (MI) which remainsthe leading cause of premature death in the world MIis caused by a coronary artery occlusion that provokes areduction of blood supply to a portion of the myocardiumdeveloping a necrotic tissue with subsequent cell death [1]Current therapies to reestablish blood supply to the affectedtissue include percutaneous coronary intervention (PCI)stent placement coronary artery bypass surgery and drugs

administration These treatments are capable of increasingpatientsrsquo life expectancy but do not address the malfunctiondue to muscular tissue death In parallel growing evidencesindicate that heart muscle is capable of regenerating throughthe activation of cardiac stem cells or recruitment of stem cellsfrom other tissues [2] but it is not enough to compensatethe large-scale tissue loss after MI The implantation of cellswith the hope that they will contribute to the generationof new cardiac tissue appears as a good approach [3ndash5]Several cell types are being tested as a potential source forcell therapy including cardiac myocytes skeletal myoblasts

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2015 Article ID 804017 14 pageshttpdxdoiorg1011552015804017

2 Journal of Nanomaterials

embryonic stem cells (ESCs) and adult stem cells (ASCs)[2 6ndash12] ASCs are present in various organs and tissuesincluding bone marrow [13 14] adipose tissue [14ndash19] andumbilical cord [20 21] Their principal advantages are basedon the fact that they can be isolated from the own patient(autologous) and present a low risk of generating an immuneresponse and tumour development The benefits reportedfor cardiomyoplasty lead cells-based therapies to turn intonew hope in regenerative medicine and are undergoingexperimental and clinical trials Unfortunately until nowcell transplantation has not achieved clear hemodynamicbenefits for myocardial diseases [22 23] The main obstacleis that MI is an ischemic event followed by inflammatoryreaction cytokines and growth factors secretion Thereforethe transplantation of unprotected cells into this environmentresults in significant cell death and low cell bioretentionand engraftment [24] In this context it seems suitable toprovide a safe environment (niche) for cell proliferation anddifferentiation [23] On the other hand extracellular matrixthat gives structural strength to the LV is pathologicallymodified (collagen type I decreased from 80 to 40) and afibrous scar is formed [22] Therefore cardiomyocytes deathand scar formation modulate cardiac remodeling whichrefers to the changes in size shape structure and physiologyof the heart The regional structural changes lead to globalLV geometric change (dilated cardiomyopathy) altering fiberdirection and diminishing function This contributes to anincrease in LV wall stress and mitral valve regurgitation [23]

Nowadays a definitive statement about TE and bioma-terials fields cannot be done but there are some reliableremarks referred to as materials-based platforms they pro-vide mechanical support to the damaged tissue while itregenerates can be modified to supply the bioactive agentsnecessary for regeneration and are able to restrict the cells tothe defect site guiding cell growth through surface chemistryand topography [25] RECATABI consortium proposes anapproach to address both the death of cardiomyocytes and theremodelling of the heart muscle with the aim to avoid heartfailureThe strategy consists in the development of a bioengi-neering platform that contains three main components (1)elastomeric microporous membrane which provides biome-chanical support (2) self-assembling peptide nanofiber gelwhich assures an adequatemicroenvironment and (3) subcu-taneous adipose tissue derived progenitor cells (subATDPCs)[26] Each of the components used to develop the patch is notnovelty by themselves but the right combination of them is

Since the activity of heart muscle involves both con-traction and dilation the use of elastomeric membranesmay provide restoration forces In addition it is conceivablethat the transmitted stresses to the engrafted cells allowdeep structural and functional biointegration With the hopeto introduce a new insight materials used for the firsttime in this field were examined Complete biodegradationof grafted scaffolds may compromise long-term limitationof postischemic ventricular remodeling For this reasonnondegradable poly(ethyl acrylate) (PEA) with cylindricalorthogonal pores and semidegradable poly(caprolactone 2-(methacryloyloxy) ethyl ester) (PCLMA) with intercon-nected spherical pores)membranes were tested [23] PEA has

mechanical properties in the range of those of soft biologicaltissues and it has shown excellent biological performancein vitro with different cell types [27ndash33] On the otherhand PCLMA also elastomeric at body temperature hasa biologically stable skeleton with a biodegradable lateralchain of caprolactone It has been chosen to carry out thesepreliminary in vivo experiments for its outstanding biologicalbehaviour tested with several architectures and cell typesboth in vivo and in vitro [34ndash36] The elasticity of thesepolymers at physiological temperature is expected to permittheir adaptation to the curved ventricle over their entiresurface and follow its mechanical deformation This elasticproperty is crucial to not disturb the synchronous beatingand at the same time to avoid further dilatation It is alsoexpected to allow the transfer of cells from the patch to themyocardium due to its good tissue-material biointegration

The RAD16-I self-assembling peptide nanofibers (provedto be nontoxic in vivo [37]) were chosen to provide a suitableenvironment to the implanted cells It has been proved tobe a good candidate to culture different cell types in a 3Denvironment [38ndash43] but its soft nature is a drawback forits direct implantation as a cell vehicle Nonetheless it hasalso been described to improve cell attachment in polymericscaffolds with respect to bare ones [44ndash46] Here we proposethat the combination of RAD16-I and elastomeric scaffoldsshould overcome the shortcomings of the gel combining theadvantages of both materials

In the present work we study the mechanical and bio-logical properties of the developed bioactive patch as wellas a preliminary assessment of their early response 3 daysafter implantation in small animal model (mice) of acuteMI As stated the elastomeric scaffolds employed possessdifferent architectures and compositions and thus slightlydifferent mechanical properties degradability and biologicalperformance It is not intended to compare both structuresand chemistries nor to study their effects in terms of cardiactissue regeneration but to develop ldquoproof of conceptrdquo anddemonstrate their suitability for the proposed application interms of manipulability and applicability onto the infarctedarea in the affected ventricle

2 Materials and Methods

21 Preparation of the Elastomeric Membranes (MacroporousScaffolds) Poly(ethyl acrylate) (PEA) scaffolds with inter-connected cylindrical orthogonal pores were obtained bythe template leaching method described before [47ndash49] Onthe other hand poly(caprolactone 2-(methacryloyloxy)ethylester) (PCLMA) scaffolds with spherical interconnectedpores were obtained by a procedure analogous to that fol-lowed by Escobar Ivirico et al [34] For detailed methodsplease refer to the Supplementary Materials and Methodssection in the Supplementary Material available online athttpdxdoiorg1011552015804017 Finally both materialswere washed with water and dried PEA and PCLMA scaf-folds were cut into samples having 5mm in diameter and1mm thickness Bulk films of both materials obtained bypolymerization in flat glass moulds were prepared to be

Journal of Nanomaterials 3

used as controls Materials to be used for cell culture werepreviously sterilized with gamma radiation

22 Preparation of the Elastomeric Membrane Prefilled withRAD16-I Self-Assembling Peptide Nanofiber Scaffolds (Com-posite) The self-assembling peptide RAD16-I (PuraMatrix1 (wv) BD Biosciences) was used to fill the PEA andPCLMA scaffoldsrsquo pores Prior to its use RAD16-I was placedin a bath sonicator (Bandelin) for 30min at 25∘C applying30W to decrease its viscosity and diluted to 015 (wv)Each elastomeric membrane was submerged in a RAD16-I nanofiber peptide 015 solution and with the help ofvacuum it was forced to penetrate in the hydrophobicscaffolds For the induction of the self-assembly of RAD16-Iit is imperative to increase the ionic strength of themilieu Forexperiments without cells the self-assembling was inducedwith phosphate buffer saline (PBS) but for the preparationof bioimplant containing RAD16-I and cells the gelationof RAD16-I was performed simultaneously to the seedingprocess [45] The process is explained in Section 24 Theefficient filling of membranes and gelling of RAD16-I wereassessed macroscopically by 120573-sheet structure staining withCongo Red 01 (wv) aqueous solution (Sigma Aldrich) for20min followed by 30min of rinsing with PBS Scaffolds withonly PBS in their pores were stained to be used as controlsNext cryoSEM images of the composites were obtained bycryoSEM in a JSM5410 (JEOL Ltd Tokyo Japan) deviceequipped with a cryounit (Oxford CT 1500) to confirmthe uniform filling of the scaffolds with the peptide gelCross sections were previously obtained by immersion ofthe samples in liquid nitrogen Water within the peptide gelwas sublimated at minus70∘C in the cryogenic unit for 15min invacuum Samples were gold-sputtered and observed at 15 kVand 15mm of working distance

23 Structural Mechanical and Electrical Characterization ofthe Elastomeric Membranes and Its Composites

231 Morphology Bare PEA and PCLMA scaffolds werefixed in 5 glutaraldehyde (Sigma Aldrich) dehydrated insuccessive ethanol washes dried using a CO

2critical point

and subsequently coated with gold Samples were observedin a JSM 6300 (JEOL Ltd Tokyo Japan) scanning electronmicroscope (SEM) at 15 kV of acceleration voltage and 15mmof working distance The porosity of the bare scaffolds 120587(pore volume fraction) was obtained through the specificvolume of PEA (reciprocal of 113 gsdotcmminus3) [50] or PCLMArespectively V the weight 119898 and apparent (geometric)volume 119881 of the scaffolds as 120587 = 119881pores119881 = 1 minus 119898V119881The specific volume of PCLMA was previously obtainedas follows dry pieces of PCLMA films were weighed inair 119898 and immersed in n-octane (95 Fluka 120588n-octane =0703 g cmminus3)1198981015840 at room temperature Specific volume wascalculated as the volume of n-octane displaced obtained as119881displ = (119898 minus119898

1015840)120588n-octane divided by the mass of the sampleV = 119881displ119898 Mettler AX 205 balance (Mettler-Toledo IncColumbus OH USA) with a sensitivity of 001mg equippedwith a Mettler ME 33360 density accessory kit was used

232 Surface Wettability The wettability of the elastomericmembranes was assessed using aDrop ShapeAnalysis SystemDSA100 (KRUSS Deutschland) Briefly water drops weredeposited on the membranes and the contact angle betweeneach drop and the surface of the membrane was measured

233 Swelling andTensile Properties Toquantify the swellingof the scaffolds 5mm diameter pieces of PEA and PCLMAfilmswere dryweighed and after different times of immersionin phosphate-buffered saline (PBS) at 37∘C until no furtherweight change was confirmedThe equilibriumwater content(EWC) was obtained as the ratio of the mass of waterabsorbed at equilibrium to the mass of dry polymer EWC =(119898wet minus 119898)119898 Pieces of PEA and PCLMA scaffolds andtheir films were cut into 05 times 3 cm2 pieces and tensile testswere performed in a Microtest SCM3000 95 (Microtest SAMadrid Spain) device at a deformation rate of 02mmminuntil fracture The tensile modulus was obtained in each caseas the average of the slope in the strain-stress plots

234 Study of the Impedance Theelectrical impedance of themembranes filled with hydrogel and submerged in 120572-MEMmedium (Sigma Aldrich) was measured at 10 kHz usingan impedance meter (Autolab PGSTAT128N) Membranesfilled with physiologic serum (120588 = 0625Ωsdotm) were alsomeasured to normalize impedance values (Ω) to resistivityunits (Ωsdotm) Platinum electrodes (05mm diameter) wereused for measurements performed at room temperature(25∘C) The electrodes were introduced in parallel within theplane membrane and separated by a distance of 5mm

24 Cell Culture

241 Subcutaneous Adipose Tissue-Derived Progenitor Cells(SubATDPCs) Isolation and Culture SubATDPCs were iso-lated from fat pads between skin and sternum from patientsundergoing cardiac surgery Informed consent was obtainedfrom all subjects and the study protocol conformed to theprinciples outlined in the Declaration of Helsinki Adiposetissue biopsy samples were processed as previously described[53 54] Refer to Supplementary Materials and Methodssection for detailed information Adhered cells were grownunder standard conditions (37∘C and 5 CO

2) in 120572-MEM

(Sigma Aldrich) supplemented with 10 foetal bovine serum(Lonza) 1 penicillin-streptomycin (Labclinics) 1 L-glutamine (Labclinics) and 5 120583gmL Plasmocin (InvivoGen)

242 In Vitro Characterization of SubATDPCs Cultured inMonolayer SubATDPCs were characterized using clono-genic and immunosuppression assays and immunopheno-typical analysis Additionally assays for adipogenic andosteogenic differentiation evaluation were performed Pleaserefer to the online Supplementary Materials and Methodssection for details of the methods

243 Cell Seeding in Elastomeric MembraneSelf-AssemblingPeptide Composites SubATDPCs were seeded inside thecomposites at passage 8 With this aim the elastomeric


membranes were filled with the peptide solution as explainedand placed in a 96-well plate Then subATDPCs weretrypsinized and resuspended in a final concentration of625sdot106 cellsmL in culture media 40120583L of this suspensionwas loaded on the top of each composite and incubated withsoft shake during 30min This time allows the cells to diffuseinside the membrane while the ionic strength of the mediainduces the gel of the RAD16-I Finally 160 120583L of fresh mediawas added in each well and samples were cultured understandard conditions (37∘C and 5 CO

2)

25 In Vitro Characterization of SubATDPCs Loaded inComposites (Bioimplant)

251 Study of Gene Expression by RT-PCR Reverse tran-scription polymerase chain reaction (RT-PCR) was per-formed to analyse gene expression in 2D and bioimplantcultures The samples were lysed and RNA was extractedwith PeqGold Total RNA kit (Peqlab) and followed bycDNA synthesis using Quantitect Reverse Transcription Kit(Qiagen) according to manufacturer protocol PCR reactionwas carried out using 30 ng of cDNA in a final volume of25 120583L containing 1XThermoPol Reaction Buffer (stock 10X)042 units of TAQDNApolymerase (SigmaAldrich) 200 120583Mof dNTPs (Sigma Aldrich) and 03 120583M primers The PCRtook place under the following conditions 3min at 95∘C(activation) followed by 35 cycles of 20 s at 94∘C 30 s ofannealing (119879

119898dependent on primer pair see supplementary

Table 1) and 30 s at 72∘C Final extension step was performedat 72∘C during 15min PCR products were size fractionatedby 2 or 4 agarose gel electrophoresis depending on theexpected fragment size

26 In Vivo Characterization of the Bioactive Implant

261 Genetic Labelling of SubATDPCs Cells were transduced(2 times 106 transducing unitsmL MOI = 21 48 h) with CMVp-RLuc-mRFP1 lentiviral vector which contains a chimericconstruct of the Renilla reniformis luciferase (RLuc) reportergene and monomeric red fluorescent protein (mRFP1) undertranscriptional control of the cytomegalovirus (CMV) pro-moter [55]

262 Myocardial InfarctionModel The study was performedon 8 female SCID mice (20 to 25 g Charles River Laborato-ries Inc) Myocardial infarction was created as previouslydescribed [18] occluding the LAD coronary artery whichprovokes and acute infarct The animals were randomlyassigned to one of the following groups (1) PEA-bioimplantimplantation and no MI induction (the PEA sham group119899 = 2) (2) MI induction and PEA-bioimplant implantation(119899 = 2) (3) PCLMA-bioimplant implantation and no MIinduction (the PCLMA sham group 119899 = 2) and (4) MIinduction and PEA-bioimplant implantation (119899 = 2) Thebioimplants were implanted immediately after occlusion

263 Assembly and Transplantation of the Bioimplant Thebioimplants were cultured in standard conditions for 24 h

before implantation onto the healthy or the infarctedmyocardium of mice making use of synthetic surgical glue(Glubran 2) to fix their edges to the tissue and avoid slidingThree-day postimplantation hearts were arrested in diastolewith arrest solution (684mM NaCl 59mM KCl 111mMGlucose 19mM NaHCO

3 297mM BDM (23-butanedione

monoxime) 1000UHeparin)Then the sampleswere excisedfixed cryopreserved in 30 sucrose in PBS embedded inOCT (Sakura) and snap-frozen in liquid nitrogen-cooledisopentane Tissue blocks were stored at minus80∘C until section-ing

264 Noninvasive Bioluminescence Imaging (BLI) of Lucifer-ase Activity from Bioimplant Anesthetized mice bearing abioimplant seeded with PLuc expressing cells were intraperi-toneally injected with 150 120583L of luciferin for in vivo BLI(167mgmL in physiological serum) (Caliper HopkintonMA) Mice were monitored right after implantation andbefore sacrifice Quantification and analysis of photonsrecorded in images were done using the Caliper imageanalysis software (Caliper Hopkinton MA)

265 Histology and Immunohistochemistry Mouse heartcryosections were stained withMassonrsquos trichrome (collagenblue myocardium red) and photographed using a TL RCIstereoscope (Leica) for morphology evaluation Addition-ally sections were incubated with the primary antibodiesagainst cTnI (2 120583gmL) (Abcam) andRFP (2 120583gmL) (Abcam)to enhance cells detection Actin fibres were stained withPhalloidin Alexa568 (1 40 Invitrogen) Nuclei were counter-stained with Hoechst 33342 and results were analysed withLeica TCS SP2

27 Statistical Analysis All values were expressed as mean plusmnstandard deviation Samples were prepared in triplicate foreach condition analysed

3 Results

31 Composite Development (Elastomeric Membrane +RAD16-I Self-Assembling Peptide) The architecture of devel-oped scaffolds was analysed by SEM in a frontal view andcross section (Figure 2)

Cylindrical interconnected orthogonal pores of PEAscaffold can be clearly observed in a cross section image(Figure 2(b)) and different layers of cylindrical crossed poresin parallel and perpendicular planes can be visualized(Figure 2(a)) On the other hand interconnected sphericalpores of PCLMAscaffolds can be easily appreciated providinga trabecular regular aspect (Figures 2(e) and 2(f))

The measured porosities of both types of scaffoldsswelling in PBS water contact angle and their apparenttensile moduli (together with the tensile moduli of the bulkrespective samples) are listed in Table 1 Although the valuesobtained for PCLMA scaffolds are slightly higher than thosefor PEA for both it can be stated that they are highly porousand have a hydrophobic character and elasticity typical ofelastomeric polymers Since they differ in their chemical


Table 1 Structural and mechanical parameters of the elastomericmembrane Morphological parameters (porosity 120587 equilibriumwater content in PBS EWC water contact angle and Youngmodulus 119864) of PEA scaffolds with cylindrical orthogonal pores andPCLMA scaffolds with spherical pores Young modulus betweenbrackets corresponds to the bulk PEA and PCLMA polymers

120587 () EWC () Contactangle (∘) 119864 (MPa)

PEA 764 plusmn 61 114 plusmn 016 115 plusmn 095 004 plusmn 002(084 plusmn 008)

PCLMA 890 plusmn 10 1000 plusmn 073 12346 plusmn 150 040 plusmn 008(073 plusmn 007)

composition and architecture (sizes and shape of pores andinterconnectivity) their biological performances will likelybe different

The Young moduli herein obtained may differ slightlywhen the scaffolds are filled with the weak peptide hydrogelgel and loaded with cells Nonetheless it can be safely statedthat the stiffness of any of the proposed cardiac patches willcloselymatch the values reported for the heartmuscle ofmiceafter dissection (006MPa) and the ones of rat or human (014or 02ndash05 MPa resp at the end of diastole) [12 56 57]

The loading of the self-assembling peptide RAD16-Iinto the elastomeric membranes was carried out prior tothe loading of the cells Both elastomeric membranes arehydrophobic (none of them swells more than 10 in PBSTable 1) For this reason vacuumwas used to force the viscousself-assembling peptide solution to get into the membraneand a subsequent in situ gelling was carried out successfullywith the diffusion of an ionic solutionThepositiveCongo redstaining (Figures 2(c) and 2(g)) indicates that the formationof the gel leads to a120573-sheet structure and the cryoSEM images(Figures 2(d) and 2(h)) confirm that the scaffoldsrsquo pores areuniformly filled with the peptide gel The hydrogel is shownas a honeycomb-like structure after sublimation of waterAlthough the quantity of RAD16-I cannot be quantified thecomplete filling of the pores has been assessed

32 Electrical Resistivity of the Composites The electricalresistivity of the composites was analysed and data were nor-malized Electrical resistivity of 530 plusmn 85Ωsdotm was obtainedfor PEA membrane while 528 plusmn 55Ωsdotm was obtainedfor PCLMA (Table 2) By comparing PEA and PCLMAmembranes it can be observed that both membranes studiedpresent similar electrical resistivity

33 In Vitro Characterization of SubATDPCs Cultured inMonolayer SubATDPCs were spindle-shaped and clono-genic and had a duplication time of 31 plusmn 003 days atpassage 2 when seeded at a density of 1000 cellscm2 (Supple-mentary Figure 1A) Immunophenotypical characterizationof the cells revealed a mesenchymal stem-cell- (MSC-) likepattern with a high percentage of subATDPCs stainingstrongly positive for CD105 CD44 CD166 CD29 and CD90and negative for CD106 CD45 and CD14 (SupplementaryFigure 1B) Comparable to the case of bone marrow-derived

Table 2 Electrical parameters of the composite Electric impedancevalues at 10 KHz for PEAandPCLMAscaffolds previously filledwithRAD16-I 015 self-assembling peptide and healed transmural andnontransmural myocardium

120588 (Ωm)PEA 530 plusmn 85a

PCLMA 528 plusmn 55a

Infarcted nontransmural myocardium 122 plusmn 27b

Infarcted transmural myocardium 104 plusmn 31caIn workb[51 52]c[52]

MSCs [58] subATDPCs were able to inhibit peripheral bloodlymphocyte proliferation (an 82 proliferation reduction)indicating an immunosuppressive capacity of subATDPCsMoreover culture of subATDPCs in adipogenic or osteogenicdifferentiation media resulted in intracellular accumulationof lipid droplets and calcium deposition respectively indi-cating adipogenic and osteogenic differentiation potentials(Supplementary Figure 1C) [59] In addition these cells havebeen analyzed for the expression of cardiac markers andtheir cardiac differentiation under electric stimulus by Llucia-Valldeperas et al [60]

34 Bioimplant Preparation (Elastomeric Membrane +RAD16-I Self-Assembling Peptide + SubATDPCs) Once as-sessed the correct introduction of RAD16-I nanofiber peptideinside the elastomeric membrane subATDPCs suspended inmedium were loaded on the constructs

During shaking the cells were allowed to diffuse whilethe high ionic strength of the media induced the self-assembly of RAD16-I After 1 and 4 days of in vitro culturethe bioimplants were removed from the well plate and weanalysed gene expression The morphology of the cells after7 days of in vitro culture can be observed in Figure 3 Cellsgrew mostly at the surface of the construct of both typesof elastomeric membranes indicating that in vitro theywere unable to invade the scaffolds completely The surfaceswere though totally covered by cells after this culture timeDAPIPhalloidin staining in Figures 3(g) and 3(h) shows howsome cells have been able to migrate inside the constructduring the first days of culture but most remain on thesurface

35 Study of Gene Expression by RT-PCR RT-PCR analysiswas performed to assess the possible cardiac profile of thecells after 1 and 4 days (Figure 4) of culture Day 1 samplesshow gene profile before implantation of the bioimplantsin mice and day 4 presents gene profile of the bioimplantcultured in vitro for 4 days which coincides with the lengthof the in vivo experiments (1 day of preculture and 3 daysof follow-up after implantation) Interestingly expressionof early cardiac markers such as TBX5 (T-box transcrip-tion factor 5) MEF2C (Myocyte Enhancer Factor 2C) andsome definitive cardiac markers such as ACTN1 (Actininalpha1) cTnT (Troponin T2) and GJAI (Connexin-43) can


SubATDPCs

RAD16-I

Elastomeric membranelowast

Figure 1 RECATABI concept scheme Elastomeric membrane pores completely filled with the self-assembling peptide RAD16-I andsubATDPCs growing on and inside the composite lowastThe pores of the scheme are spherical but different architectures can be used

be observed Additionally expression of CDH1 (E-Cadherin)and SNAI1 (Snail Family Zinc Finger 1 a natural repressor ofCDH1) were analysed

No important differences were detected between day 1and day 4 on gene expression except for cTnT which wasdownregulated in the composite but was upregulated againin PCLMA at 4 days of culture In addition the expressionof CDH1 in both composite systems used was downregu-lated All these results indicate that the composite is notcardioconductive since no difference between materials canbe observed but it is able to maintain the cardiac fate ofsubATDPCs

36 In Vivo Implantation With the intention of monitoringcell survival and distribution of the implanted cells afterthe bioactive patch implantation a noninvasive biolumi-nescence imaging (BLI) system was used Cells within thebioimplant were previously labelled with CMVRlucRFPttkphotoprotein reporters for BLI and fluorescence detectionPhoton counts quantification showed human subATDPCssurvival and thoracic location three days after implantationof PEA and PCLMA bioimplants (supplementary data)Moreover whole heart excision and histology examinationdemonstrated correct position of the bioactive implant on theinfarcted area (Figures 5(A)ndash5(D))

Three days after implantation subATDPCs within thePEA bioimplant were homogeneously distributed givingsome of them an elongated shape (Figure 5(E)) On the otherhand PCLMA bioimplant cells were mainly distributed inthe borders of the myocardium (My) or inner membrane(im) and only few were found in the outer membrane (om)(Figure 5(I)) Both bioactive implants were attached to theheart (Figures 5(C) 5(D) 5(F) and 5(I)) and few cells alreadystarted migration to the damaged myocardium (Figures 5(G)and 5(J))

4 Discussion

The high impact of MI worldwide leads a lot of researchersto focus their attention on cardiac tissue engineering (CTE)with the aim to obtain an effective approach for necrotic tissuerepair CTErsquos success highly depends on the choice of theappropriate cell source [7 8] and the biomaterial [61] usedas a vehicle to graft the cells in the infarcted tissue Here wepropose a combination of an elastomeric membrane and aself-assembling peptide combining their intrinsic propertiesand obtaining a more adaptable patch Different types ofmaterial-based approaches have been developed with theaim to improve cardiac function after myocardial infarctionto avoid heart failure [62] One of these approaches is theuse of biomaterials to constrain the post-MI failing heartpreventing it from further remodeling and dilatation [63ndash66] Our design aims to avoid ventricle dilatation using anondegradable or semidegradable material and at the sametime provides the cells a biological biophysical andmechan-ical support for their growth function self-renewal anddifferentiation thanks to RAD16-I peptide We think that thecombination of elastomeric membranes with self-assemblingpeptide RAD16-I solves both issues (see Figure 1) Frommacroscopically point of view the elastomeric membranescan fit the implantation site and do not affect negatively theremaining performance of the heart due to their intrinsicelasticity These materials are elastic enough to not restrainthe remaining contractility of the heart but at the same timecan avoid the negative dilatation due to matrix remodelingAdditionally they maintain their original dimensions afterimplantation (do not swell) and have mechanical modulisimilar to soft tissues On the other hand RAD16-I peptidecould provide an adequate interface between the elastomericmembranes and the cells enhancing their binding signallingand proper interaction


(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 2 Elastomeric membrane and composite characterization Microscopic images of the ((a)ndash(d)) PEA scaffolds with cylindricalorthogonal pores and ((e)ndash(h)) PCLMA scaffolds with spherical pores ((a) and (e)) SEM images cross section and ((b) and (f)) SEM imagessurface ((c) and (g)) Congo Red staining and ((d) and (h)) cryoSEM images of the scaffolds loaded with 015 RAD16-I and gelled with PBSsurface White arrows point out the elastomeric membrane while the black ones point out the self-assembling peptide structure after gel

Two chemically and architecturally different elastomericmembranes were tested PEA scaffolds with cylindricalorthogonal pores and PCLMA ones with spherical poresBoth membranes are elastic enough to withstand the stressesarising from heartbeat and ensure stable placement of seededcells under those conditions as it can be observed in Figure 5With the aim to provide an adequate microenvironmentthe membranesrsquo pores were filled with RAD16-I nanofiberpeptide since previously it was demonstrated that the useof RAD16-I peptide inside the elastomeric membrane porousincreases the seeding efficiency [45] The peptide was intro-duced into both porous structures simply by pressure (seeFigure 1) One additional advantage of these macroporousscaffolds is their capacity to allow the development ofmicrovasculature in vivo [67] Unfortunately as the animalmodel used is analyzed at a really short term (3 days) theformation of vessels is not plausible and for this reason it wasnot analyzed In vitro experiments at day 7 show a limitedcell distribution with the cells remaining in the surface ofthe scaffold but 3 days after implantation cell distributionand penetration were dramatically enhanced In vitro thecell distribution remains mainly limited to the surface ofthe composite provably due to the difficulty of oxygen andnutrients diffusion inside the structure After implantationof the patch on the infarcted heart the conditions changedramatically mainly due to the new environment which isdifferent from the previous culture system Since it is a modelof acute MI immediately after the intervention the necroticheart tissue creates an unfriendly environment which causesthe implanted cells to migrate towards the contrary direction(inside the composite)

From an electrical standpoint it is well known that theelectrical resistivity is lower and less frequency dependentin necrotic than in healthy myocardium [68 69] Accordingto the values reported in Table 2 it can be observed thatthe resistivity values for the composites are below infarcted

nontransmural and transmural myocardium resistivity butthe resistivity value of normal myocardium is larger com-pared to infarcted myocardium (250Ωsdotcm at 10 kHz) [51]We think that the resultant equivalent resistance comingfrom the parallel combination of the infarcted tissue andthe scaffold will actually benefit the electrical coupling withnative myocardium tissue Based on the simple circuit theoryprinciple which confirms that the equivalent resistance oftwo resistances in parallel is equal or lower than the lowestresistance the scaffold will provide a low-resistance pathwaythat should contribute to facilitating the electrical signalpropagation from the native heart tissue into the scaffoldThus we speculate that both composites which have similarelectrical resistivity will equally facilitate the propagation ofelectrical pulses throughout the contact area between thecomposites and the infarcted zone

Since both materials presented are candidates to beused for cell therapy they were loaded with subATDPCs(see Figure 1) The identity of progenitor cells during earlycardiogenesis is regulated by tightly coordinated spatiallyand temporally active signaling pathways and molecularmechanisms This leads cells to a progressive restriction ofundifferentiated progenitors to the different cardiovascularlineages The molecular identity of these inductive signals isnot well understood but various transcription factors thatmay regulate cardiac commitment and differentiation havebeen isolated [70]

The in vitro genetic profiles of such cells cultured in bothcomposites were analysed before their in vivo implantationInterestingly TBX5 [71 72] and MEF2C [73] early cardiacmarkers and definitive cardiac markers such as ACTN1cTNT and GJAI were expressed [74] TBX5 is a memberof T-box gene family [75] critical for the development ofthe heart Its expression is critical to the formation of theelectrical system that coordinates contractions of the heartchambers It has been shown to interact with MEF2C to


PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin

Figure 3 Bioimplant characterization Microscopic images of subATDPCs growing in developed composite after 7 seeding days (a) Surfaceof PEA scaffold (b) surface of PCLMA scaffold ((c) (d)) close up of top PEA and PCLMA scaffold respectively (e) PEA scaffold cross section(f) PCLMA scaffold cross section and ((g) and (h)) Dapi amp Phalloidin staining of subATDPCs in PEA and PCLMA scaffold In images (a)to (f) white arrows indicate the cells growing in the bioactive implant black arrows note the nanofibers of self-assembling peptide RAD16-Iand red arrows signalize the structure of porous elastomeric membrane White arrows in image (g) and (h) indicate scaffolds surface whileorange arrows indicate cells migrating inside the composite Scale bars (a) 30 120583m (b) (E)ndash(H) 100 120583m (c) 3 120583m and (d) 5 120583m

synergistically activate target genes expression in cardiomy-ocytes [72] MEF2C is a MDS-box transcription factor whichplays a key role in myogenesis [76] specifically controllingthe differentiation of cardiomyoblasts into cardiomyocytes

[77] (see Figure 6) On the other hand a decrease of CDH1expression with respect to cells growing in 2D culture wasobserved although SNAI1 repression factor was maintainedCDH1 and GJAI genes encode for cell connection proteins


2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp

Figure 4 Expression of cellular markers in developed bioimplant by RT-PCR Cardiogenic and general markers expressed by subATDPCscultured in developed patch of PEA and PCLMA after 1 (d1) and 4 (d4) days of culture in vitro compared to two-dimensional (2D) culturesNC negative control (PCR without template)

CDH1 [78] is involved in mechanisms regulating cell-celladhesions mobility and proliferation while GAJ1 providesa route for the diffusion of low molecular weight materialsfrom cell to cell having a crucial role in the synchronizedcontraction of the heart As suggested by the results shownin Figure 5 these cells are capable of migrating either insidethe membrane or to the infarcted area after implantationIt is important to mention that most of the studied genespresented the same pattern for all conditions but cTNTwas only expressed in the PCLMA bioactive implant after4 days in vitro Therefore it seems that subATDPCs aremaintaining their gene profile which indicates that they tendto preserve their cardiogenic potential lineage at least invitro These results lead us to conclude that the proposedmaterials combined with RAD16-I are not cardioconductivematerials but the cardiomyogenic potential of subATDPCs ismaintained [60]

The in vitromodel gives an overview of the patch poten-tial but in any case these results could be extrapolated to thein vivo scenario Here a proof of concept of short time pointswas developed to analyse the patch feasibility in terms ofintegration and cell viability Interestingly we notice that bothpatches intimately attach to the myocardium Additionallyboth remained in the position at the infarction area wherethey were placed suggesting some resilience to the heartmechanical forces and in addition 3 days after implantationthe cells remained alive and mostly at the thoracic area(Figure 2 supplementary data) Although both bioactiveimplants were attached to the myocardium the PCLMAbioimplants showed greater cell immobilization and betterintegration in the infarcted area than the PEA ones Cellsgrowing inside the PEA bioimplants were homogenously

distributed acquiring some elongated shape (Figures 5(E)and 5(F)) while in the PCLMA ones cells were mainlydistributed at the interface between the bioimplant and themyocardium (Figure 5(I)) We did not expect to observeprofusely cell migration at such short time assay but wespeculate that the cellsmight start to contribute to a paracrineeffect by secreting specific factors phenomena previouslyreported using MSC and cardiomyocytes in rat and micemodels respectively [79ndash81] Importantly in previous studiesan increase of vascularization and a reduction of infarct sizeafter ATDPCs injection were reported [45] Therefore theirbetter retention onto the infracted tissue using this platformmight lead to positive benefits by the stimulation of vesselformation Further in vivo studies at longer time points willbe performed with the aim to analyse the beneficial role ofthese bioactive patches in vivo

5 Conclusions

PEA- and PCLMA-based bioimplants developed providea good platform for cell therapy aiming to assist tissuerestoration of the infarcted ventricular area Importantly thescaffold plus peptide gel composite devices brings a suitablemicroenvironment where cells can be retained alive at theimplanted site which is an improvement of the proposed todate direct cell injection therapies Moreover these patchespermit cells to maintain their genetic profile enhancing theirtherapeutic potential at the time of being implanted Finallyall these characteristics could improve the cell capacity toprovide a positive paracrine effect on resident cells in thehost tissue whichmight improve the recovery of the infarctedzone


PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)

Figure 5 General view of the PEA and PCLMA bioactive implants on mice heart after 3 days (a) Whole heart excision and macroscopicexamination ((A) and (B)) Macroscopic view of mouse heart and bioactive implant (outlined) at 3 days after implantation and ((C) and(D)) Massonrsquos trichrome staining of heart with bioactive implant cross sectionThe dotted zone in black corresponds to the bioactive implantand the dotted red area to the infarcted zone (b) Microscopic view of the bioactive implant ((E) and (H)) Detail of elongated subATDPCs(red) inside the PEA (E) and PCLMA (H) bioactive implants ((F) and (I)) views of the PEA (F) and PCLMA (I) bioactive implants attachedto the myocardium (implant and myocardium are limited by dotted lines (Phalloidin staining in red)) and ((G) and (J)) migration of thesubATDPCs (white arrows) to the myocardium (cTnI staining in white) in PEA (G) and PCLMA (J) groups Constitutive expression of RFP(red) in subATDPCs Nuclei were counterstained withHoescht 33342 (blue) Scale bars 1mm ((A)ndash(D)) 50 120583m ((E)ndash(H) and (J)) and 100 120583m(I)


Pluripotentstem cell

Cardiacprogenitor

cell

Immaturecardiomyocyte

Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5

cTnTcTnIGJA1ACTN1MHC

Myofibrillar organizationElectrical transmissionCalcium handling

Figure 6 Sequential steps in cardiac differentiation in vitro from pluripotent stem cells to functional cardiomyocytes [70] Cardiacdevelopment starts with the commitment of undifferentiated pluripotent stem cells of the inner cell mass of blastocysts to mesodermalrestricted derivatives during embryonic development Typical markers and characteristics for the different cell types are indicated Structuraland functional maturation is not well understood Maturation might be provoked by hormones electrical and mechanical stimulation andorganization in 3D engineered heart tissues (EHTs)

Abbreviations

3D Three-dimensionalACTN1 Actinin alpha 1ASCs Adult stem cellsBLI Bioluminescence imagingCDH1 E-cadherinCTE Cardiac tissue engineeringcTnI Cardiac Troponin IESCs Embryonic stem cellsGJA1 Gap junction protein alpha 1 (Connexin 43)LAD Left anterior descending arteryMEF2C Myocyte enhancer factor 2CMI Myocardial infarctionMSCs Mesenchymal stem cellsPCI Percutaneous coronary interventionPCLMA poly(caprolactone 2-(methacryloyloxy)ethyl

ester)PEA poly(ethyl acrylate)RT-PCR Reverse transcription polymerase chain

reactionSNAI1 Snail family zinc finger 1subATDPCs Subcutaneous adipose tissue derived

progenitor cellsTBX5 T-box transcription factor 5cTnT Troponin T2

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

C Castells-Sala A Valles-Lluch and C Soler-Botija con-tributed equally to this work

Acknowledgments

The authors wish to thank the Department of CardiacSurgery (Hospital Germans Trias i Pujol Badalona) for theircollaboration in obtaining human samples Dr Bago for hiskind contribution in the cell transduction process and BLIanalysis and Joan Gilabert from Biomaterials Laboratory(GEMAT IQS-School of Engineering) who kindly helpedthem with wettability measurements The research leadingto these results has received funding from the EuropeanUnion Seventh Framework Programme (FP72007ndash2013)under Grant agreement no 229239 This work was alsosupported by Grants fromMinisterio de Educacion y Ciencia(SAF2011-30067-C02-01 andM Arnal-Pastor FPU 2009-1870grant) Red de Terapia Celular-TerCel (RD1200190029) RedCardio-vascular (RD1200420047) and Fundacio LaMaratode TV3 (122232)

References

[1] C Stamm B Nasseri Y-H Choi and R Hetzer ldquoCell therapyfor heart disease great expectations as yet unmetrdquo Heart Lungand Circulation vol 18 no 4 pp 245ndash256 2009

[2] J-P KaramCMuscari andCNMontero-Menei ldquoCombiningadult stem cells and polymeric devices for tissue engineering in


infarcted myocardiumrdquo Biomaterials vol 33 no 23 pp 5683ndash5695 2012

[3] S Fernandes S Kuklok J McGonigle H Reinecke and C EMurry ldquoSynthetic matrices to serve as niches for muscle celltransplantationrdquo Cells Tissues Organs vol 195 no 1-2 pp 48ndash59 2011

[4] A N Patel and J A Genovese ldquoStem cell therapy for thetreatment of heart failurerdquo Current Opinion in Cardiology vol22 no 5 pp 464ndash470 2007

[5] J A Genovese M Cortes-Morichetti E Chachques G FratiA N Patel and J C Chachques ldquoCell based approaches formyocardial regeneration and artificial myocardiumrdquo CurrentStem Cell Research andTherapy vol 2 no 2 pp 121ndash127 2007

[6] K O Lui L Bu R A Li and C W Chan ldquoPluripotentstem cell-based heart regeneration from the developmental andimmunological perspectivesrdquo Birth Defects Research Part CmdashEmbryo Today vol 96 no 1 pp 98ndash108 2012

[7] L S Abdelli H Merino C M Rocher and D K SinglaldquoCell therapy in the heartrdquo Canadian Journal of Physiology andPharmacology vol 90 no 3 pp 307ndash315 2012

[8] J Hoover-Plow and Y Gong ldquoChallenges for heart disease stemcell therapyrdquo Vascular Health and Risk Management vol 8 no1 pp 99ndash113 2012

[9] KGOldroydC Berry and J Bartunek ldquoMyocardial repair andregeneration bone marrow or cardiac stem cellsrdquo MolecularTherapy vol 20 no 6 pp 1102ndash1105 2012

[10] M Gnecchi P Danieli and E Cervio ldquoMesenchymal stem celltherapy for heart diseaserdquo Vascular Pharmacology vol 57 no 1pp 48ndash55 2012

[11] J Liu Z Zhang Y Liu et al ldquoGeneration characterizationand potential therapeutic applications of cardiomyocytes fromvarious stem cellsrdquo Stem Cells and Development vol 21 no 12pp 2095ndash2110 2012

[12] J R Venugopal M P Prabhakaran S Mukherjee R Ravichan-dran K Dan and S Ramakrishna ldquoBiomaterial strategies foralleviation ofmyocardial infarctionrdquo Journal of the Royal SocietyInterface vol 9 no 66 pp 1ndash19 2012

[13] D M Clifford S A Fisher S J Brunskill et al ldquoLong-termeffects of autologous bone marrow stem cell treatment in acutemyocardial infarction factors that may influence outcomesrdquoPLoS ONE vol 7 no 5 Article ID e37373 2012

[14] R H Lee B Kim I Choi et al ldquoCharacterization andexpression analysis of mesenchymal stem cells from humanbone marrow and adipose tissuerdquo Cellular Physiology andBiochemistry vol 14 no 4-6 pp 311ndash324 2004

[15] A A Qayyum M Haack-Soslashrensen A B Mathiasen EJoslashrgensen A Ekblond and J Kastrup ldquoAdipose-derived mes-enchymal stromal cells for chronic myocardial ischemia (MyS-tromalCell Trial) study designrdquo Regenerative Medicine vol 7no 3 pp 421ndash428 2012

[16] X Bai J Ma Z Pan et al ldquoElectrophysiological properties ofhuman adipose tissue-derived stem cellsrdquo American Journal ofPhysiology Cell Physiology vol 293 no 5 pp C1539ndashC15502007

[17] N Tandon B Goh A Marsano et al ldquoAlignment and elonga-tion of human adipose-derived stem cells in response to direct-current electrical stimulationrdquo in Proceedings of the AnnualInternational Conference of the IEEE Engineering in Medicineand Biology Society (EMBC rsquo09) pp 6517ndash6521 2009

[18] M Rigol N Solanes J Farre et al ldquoEffects of adipose tissue-derived stem cell therapy after myocardial infarction impact of

the route of administrationrdquo Journal of Cardiac Failure vol 16no 4 pp 357ndash366 2010

[19] V Planat-Benard C Menard M Andre et al ldquoSpontaneouscardiomyocyte differentiation from adipose tissue stroma cellsrdquoCirculation Research vol 94 no 2 pp 223ndash229 2004

[20] BWeber S M Zeisberger and S P Hoerstrup ldquoPrenatally har-vested cells for cardiovascular tissue engineering fabrication ofautologous implants prior to birthrdquo Placenta vol 32 no 4 ppS316ndashS319 2011

[21] M Cortes-Morichetti G Frati O Schussler et al ldquoAssociationbetween a cell-seeded collagen matrix and cellular cardiomy-oplasty for myocardial support and regenerationrdquo Tissue Engi-neering vol 13 no 11 pp 2681ndash2687 2007

[22] J C Chachques ldquoDevelopment of bioartificial myocardiumusing stem cells and nanobiotechnology templatesrdquo CardiologyResearch and Practice vol 2011 Article ID 806795 7 pages 2011

[23] J C Chachques M M Pradas A Bayes-Genis and C SeminoldquoCreating the bioartificial myocardium for cardiac repair chal-lenges and clinical targetsrdquo Expert Review of CardiovascularTherapy vol 11 no 12 pp 1701ndash1711 2013

[24] B C Karikkineth andW-H Zimmermann ldquoMyocardial tissueengineering and heart muscle repairrdquo Current PharmaceuticalBiotechnology vol 14 no 1 pp 4ndash11 2013

[25] B Demirbag P Y Huri G T Kose A Buyuksungur and VHasirci ldquoAdvanced cell therapies with and without scaffoldsrdquoBiotechnology Journal vol 6 no 12 pp 1437ndash1453 2011

[26] C Soler-Botija J R Bago and A Bayes-Genis ldquoA birdrsquos-eye view of cell therapy and tissue engineering for cardiacregenerationrdquo Annals of the New York Academy of Sciences vol1254 no 1 pp 57ndash65 2012

[27] M Perez Olmedilla N Garcia-Giralt M M Pradas et alldquoResponse of human chondrocytes to a non-uniform distri-bution of hydrophilic domains on poly (ethyl acrylate-co-hydroxyethyl methacrylate) copolymersrdquo Biomaterials vol 27no 7 pp 1003ndash1012 2006

[28] A J Campillo-Fernandez S Pastor M Abad-Collado et alldquoFuture design of a new keratoprosthesis Physical and biolog-ical analysis of polymeric substrates for epithelial cell growthrdquoBiomacromolecules vol 8 no 8 pp 2429ndash2436 2007

[29] A J Campillo-Fernandez R E Unger K Peters et al ldquoAnal-ysis of the biological response of endothelial and fibroblastcells cultured on synthetic scaffolds with various hydrophilichydrophobic ratios influence of fibronectin adsorption andconformationrdquo Tissue Engineering Part A vol 15 no 6 pp1331ndash1341 2009

[30] M Soria C Marti M Salmero et al ldquoSurvival and differen-tiation of embryonic neural explants on different biomaterialsrdquoJournal of BiomedicalMaterials Research Part A vol 79 pp 495ndash502 2006

[31] C Martınez-Ramos A Valles-Lluch J M G Verdugo et alldquoChanneled scaffolds implanted in adult rat brainrdquo Journal ofBiomedical Materials Research Part A vol 100 no 12 pp 3276ndash3286 2012

[32] P Rico J C R Hernandez D Moratal G Altankov M MPradas and M Salmeron-Sanchez ldquoSubstrate-induced assem-bly of fibronectin into networks influence of surface chemistryand effect on osteoblast adhesionrdquo Tissue Engineering Part Avol 15 no 11 pp 3271ndash3281 2009

[33] J M Soria M Sancho-Tello M A G Esparza et al ldquoBiomate-rials coated by dental pulp cells as substrate for neural stem celldifferentiationrdquo Journal of Biomedical Materials Research PartA vol 97 no 1 pp 85ndash92 2011


[34] J L Escobar Ivirico E Costa Martınez M Salmeron SanchezI Munoz Criado J L Gomez Ribelles andMMonleon PradasldquoStructure and properties of methacrylate-endcapped capro-lactone networks with modulated water uptake for biomedicalapplicationsrdquo Journal of Biomedical Materials Research Part BApplied Biomaterials vol 83 no 1 pp 266ndash275 2007

[35] J L E Ivirico M Salmeron-Sanchez J L G Ribelles et alldquoProliferation and differentiation of goat bone marrow stromalcells in 3D scaffolds with tunable hydrophilicityrdquo Journal ofBiomedical Materials Research Part B Applied Biomaterials vol91 no 1 pp 277ndash286 2009

[36] J E Ivirico G G Ferrer E Novella-Maestre A Ruiz-SauriM M Pradas and C Carda ldquoIn vivo response of methacrylateendcapped caprolactone scaffoldsrdquo Regenerative Medicine vol2 no 5 p 616 2007

[37] I R Degano L Quintana M Vilalta et al ldquoThe effect of self-assembling peptide nanofiber scaffolds on mouse embryonicfibroblast implantation and proliferationrdquo Biomaterials vol 30no 6 pp 1156ndash1165 2009

[38] J Kisiday M Jin B Kurz et al ldquoSelf-assembling peptide hydro-gel fosters chondrocyte extracellular matrix production and celldivision implications for cartilage tissue repairrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 99 no 15 pp 9996ndash10001 2002

[39] C E Semino J R Merok G G Crane G Panagiotakosand S Zhang ldquoFunctional differentiation of hepatocyte-likespheroid structures fromputative liver progenitor cells in three-dimensional peptide scaffoldsrdquo Differentiation vol 71 no 4-5pp 262ndash270 2003

[40] D A Narmoneva R Vukmirovic M E Davis R D Kammand R T Lee ldquoEndothelial cells promote cardiac myocytesurvival and spatial reorganization implications for cardiacregenerationrdquo Circulation vol 110 no 8 pp 962ndash968 2004

[41] E Genove C Shen S Zhang and C E Semino ldquoThe effectof functionalized self-assembling peptide scaffolds on humanaortic endothelial cell functionrdquo Biomaterials vol 26 no 16 pp3341ndash3351 2005

[42] E Garreta E Genove S Borros and C E Semino ldquoOsteogenicdifferentiation of mouse embryonic stem cells and mouseembryonic fibroblasts in a three-dimensional self-assemblingpeptide scaffoldrdquo Tissue Engineering vol 12 no 8 pp 2215ndash2227 2006

[43] L Quintana T FernandezMuinos E GenoveM DMOlmosS Borros and C E Semino ldquoEarly tissue patterning recreatedby mouse embryonic fibroblasts in a three-dimensional envi-ronmentrdquo Tissue EngineeringmdashPart A vol 15 no 1 pp 45ndash542009

[44] J Wu N Marı-Buye T F Muinos S Borros P Favia and C ESemino ldquoNanometric self-assembling peptide layers maintainadult hepatocyte phenotype in sandwich culturesrdquo Journal ofNanobiotechnology vol 8 article 29 2010

[45] A Valles-Lluch M Arnal-Pastor C Martınez-Ramos etal ldquoCombining self-assembling peptide gels with three-dimensional elastomer scaffoldsrdquo Acta Biomaterialia vol 9 no12 pp 9451ndash9460 2013

[46] C E Semino ldquoCan we build artificial stem cell compartmentsrdquoJournal of Biomedicine and Biotechnology vol 2003 no 3 pp164ndash169 2003

[47] J C Rodrıguez Hernandez A Serrano Aroca J L GomezRibelles and M Monleon Pradas ldquoThree-dimensional nano-composite scaffolds with ordered cylindrical orthogonal poresrdquo

Journal of Biomedical Materials Research Part B Applied Bio-materials vol 84 no 2 pp 541ndash549 2008

[48] J M Estelles I Krakovsky J C R Hernandez A MPiotrowska and M M Pradas ldquoMechanical properties ofporous crosslinked poly(ethyl-acrylate) for tissue engineeringrdquoJournal ofMaterials Science vol 42 no 20 pp 8629ndash8635 2007

[49] MArnal-Pastor AValles-LluchMKeicher andMM PradasldquoCoating typologies and constrained swelling of hyaluronicacid gels within scaffold poresrdquo Journal of Colloid and InterfaceScience vol 361 no 1 pp 361ndash369 2011

[50] R Brıgido Diego M Perez Olmedilla A Serrano Aroca etal ldquoAcrylic scaffolds with interconnected spherical pores andcontrolled hydrophilicity for tissue engineeringrdquo Journal ofMaterials Science Materials in Medicine vol 16 no 8 pp 693ndash698 2005

[51] J CincaMWarren A Rodrıguez-Sinovas et al ldquoPassive trans-mission of ischemic ST segment changes in low electricalresistance myocardial infarct scar in the pigrdquo CardiovascularResearch vol 40 no 1 pp 103ndash112 1998

[52] Y Salazar R Bragos O Casas J Cinca and J RosellldquoTransmural versus nontransmural in situ electrical impedancespectrum for healthy ischemic and healed myocardiumrdquo IEEETransactions on Biomedical Engineering vol 51 no 8 pp 1421ndash1427 2004

[53] A Bayes-Genis C Soler-Botija J Farre et al ldquoHuman pro-genitor cells derived from cardiac adipose tissue amelioratemyocardial infarction in rodentsrdquo Journal of Molecular andCellular Cardiology vol 49 no 5 pp 771ndash780 2010

[54] O M Martınez-Estrada Y Munoz-Santos J Julve M Reinaand S Vilaro ldquoHuman adipose tissue as a source of Flk-1+ cellsnew method of differentiation and expansionrdquo CardiovascularResearch vol 65 no 2 pp 328ndash333 2005

[55] M Vilalta C Jorgensen I R Degano et al ldquoDual luciferaselabelling for non-invasive bioluminescence imaging of mes-enchymal stromal cell chondrogenic differentiation in deminer-alized bone matrix scaffoldsrdquo Biomaterials vol 30 no 28 pp4986ndash4995 2009

[56] Q-Z Chen S E Harding N N Ali A R Lyon and AR Boccaccini ldquoBiomaterials in cardiac tissue engineering tenyears of research surveyrdquo Materials Science and Engineering RReports vol 59 no 1ndash6 pp 1ndash37 2008

[57] W Hiesinger M J Brukman R C McCormick et al ldquoMyocar-dial tissue elastic properties determined by atomic forcemicroscopy after stromal cell-derived factor 1120572 angiogenictherapy for acute myocardial infarction in a murine modelrdquoJournal of Thoracic and Cardiovascular Surgery vol 143 no 4pp 962ndash966 2012

[58] G Xu L Zhang G Ren et al ldquoImmunosuppressive propertiesof cloned bone marrowmesenchymal stem cellsrdquo Cell Researchvol 17 no 3 pp 240ndash248 2007

[59] M Dominici K Le Blanc I Mueller et al ldquoMinimal crite-ria for defining multipotent mesenchymal stromal cells TheInternational Society for Cellular Therapy position statementrdquoCytotherapy vol 8 no 4 pp 315ndash317 2006

[60] A Llucia-Valldeperas B Sanchez C Soler-Botija et al ldquoElec-trical stimulation of cardiac adipose tissue-derived progenitorcells modulates cell phenotype and genetic machineryrdquo Journalof Tissue Engineering and Regenerative Medicine 2013

[61] C Castells-Sala and C E Semino ldquoBiomaterials for stem cellculture and seeding for the generation and delivery of cardiacmyocytesrdquo Current Opinion in Organ Transplantation vol 17no 6 pp 681ndash687 2012


[62] M H van Marion N A M Bax A C C van SpreeuwelD W J van der Schaft and C V C Bouten ldquoMaterial-based engineering strategies for cardiac regenerationrdquo CurrentPharmaceutical Design vol 20 no 12 pp 2057ndash2068 2014

[63] A Shafy T Fink V Zachar N Lila A Carpentier and J CChachques ldquoDevelopment of cardiac support bioprostheses forventricular restoration andmyocardial regenerationrdquo EuropeanJournal of Cardio-Thoracic Surgery vol 43 no 6 pp 1211ndash12192013

[64] M A Laflamme and C E Murry ldquoHeart regenerationrdquoNaturevol 473 no 7347 pp 326ndash335 2011

[65] J C Chachques J C Trainini N Lago M Cortes-MorichettiO Schussler and A Carpentier ldquoMyocardial Assistance byGrafting a New Bioartificial Upgraded Myocardium (MAG-NUM trial) clinical feasibility studyrdquo Annals of ThoracicSurgery vol 85 no 3 pp 901ndash908 2008

[66] L Qian W Shim Y Gu et al ldquoHemodynamic contributionof stem cell scaffolding in acute injured myocardiumrdquo TissueEngineering A vol 18 no 15-16 pp 1652ndash1663 2012

[67] M Arnal-Pastor C Martınez Ramos M Perez Garnes MMonleon Pradas and A Valles Lluch ldquoElectrospun adherent-antiadherent bilayered membranes based on cross-linkedhyaluronic acid for advanced tissue engineering applicationsrdquoMaterials Science and Engineering C vol 33 no 7 pp 4086ndash4093 2013

[68] L A Geddes and L E Baker ldquoThe specific resistance ofbiological materialmdasha compendium of data for the biomedicalengineer and physiologistrdquoMedical and Biological Engineeringvol 5 no 3 pp 271ndash293 1967

[69] MA FallertM SMirotznik SWDowning et al ldquoMyocardialelectrical impedance mapping of ischemic sheep hearts andhealing aneurysmsrdquoCirculation vol 87 no 1 pp 199ndash207 1993

[70] L Cyganek ldquoCardiac progenitor cells and their therapeuticapplication for cardiac repairrdquo Journal of Clinical amp Experimen-tal Cardiology S11 article 8 2013

[71] T K Ghosh E A Packham A J Bonser T E Robinson S JCross and J D Brook ldquoCharacterization of the TBX5 bindingsite and analysis of mutations that cause Holt-Oram syndromerdquoHuman Molecular Genetics vol 10 no 18 pp 1983ndash1994 2001

[72] C Wang D Cao Q Wang and D-Z Wang ldquoSynergisticactivation of cardiac genes bymyocardin and Tbx5rdquo PLoS ONEvol 6 no 8 Article ID e24242 2011

[73] MM Lockhart E EWirrig A L Phelps et al ldquoNorris R a andWesselsA 2013Mef2c regulates transcription of the extracellularmatrix protein cartilage link protein 1 in the developing murineheartrdquo PLoS ONE vol 8 no 2 Article ID e57073 2013

[74] M Peran J A Marchal E Lopez et al ldquoHuman cardiactissue induces transdifferentiation of adult stem cells towardscardiomyocytesrdquo Cytotherapy vol 12 no 3 pp 332ndash337 2010

[75] I P G Moskowitz A Pizard V V Patel et al ldquoThe T-Box transcription factor Tbx5 is required for the patterningand maturation of the murine cardiac conduction systemrdquoDevelopment vol 131 no 16 pp 4107ndash4116 2004

[76] J Wilson-Rawls J D Molkentin B L Black and E N OlsonldquoActivated Notch inhibits myogenic activity of the MADS-Boxtranscription factor myocyte enhancer factor 2Crdquo Molecularand Cellular Biology vol 19 no 4 pp 2853ndash2862 1999

[77] C Karamboulas G D Dakubo J Liu et al ldquoDisruption ofMEF2 activity in cardiomyoblasts inhibits cardiomyogenesisrdquoJournal of Cell Science vol 119 no 20 pp 4315ndash4321 2006

[78] H Ninomiya R David E W Damm F Fagotto C MNiessen and R Winklbauer ldquoCadherin-dependent differentialcell adhesion in xenopus causes cell sorting in vitro but not inthe embryordquo Journal of Cell Science vol 125 no 8 pp 1877ndash18832012

[79] M Gnecchi H He N Noiseux et al ldquoEvidence supportingparacrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvementrdquoTheFASEB Journal vol 20 no 6 pp 661ndash669 2006

[80] I Chimenti R R Smith T-S Li et al ldquoRelative roles of directregeneration versus paracrine effects of human cardiosphere-derived cells transplanted into infarcted micerdquo CirculationResearch vol 106 no 5 pp 971ndash980 2010

[81] A H S Kumar and N M Caplice ldquoClinical potential ofadult vascular progenitor cellsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 30 no 6 pp 1080ndash1087 2010

Submit your manuscripts athttpwwwhindawicom

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embryonic stem cells (ESCs) and adult stem cells (ASCs)[2 6ndash12] ASCs are present in various organs and tissuesincluding bone marrow [13 14] adipose tissue [14ndash19] andumbilical cord [20 21] Their principal advantages are basedon the fact that they can be isolated from the own patient(autologous) and present a low risk of generating an immuneresponse and tumour development The benefits reportedfor cardiomyoplasty lead cells-based therapies to turn intonew hope in regenerative medicine and are undergoingexperimental and clinical trials Unfortunately until nowcell transplantation has not achieved clear hemodynamicbenefits for myocardial diseases [22 23] The main obstacleis that MI is an ischemic event followed by inflammatoryreaction cytokines and growth factors secretion Thereforethe transplantation of unprotected cells into this environmentresults in significant cell death and low cell bioretentionand engraftment [24] In this context it seems suitable toprovide a safe environment (niche) for cell proliferation anddifferentiation [23] On the other hand extracellular matrixthat gives structural strength to the LV is pathologicallymodified (collagen type I decreased from 80 to 40) and afibrous scar is formed [22] Therefore cardiomyocytes deathand scar formation modulate cardiac remodeling whichrefers to the changes in size shape structure and physiologyof the heart The regional structural changes lead to globalLV geometric change (dilated cardiomyopathy) altering fiberdirection and diminishing function This contributes to anincrease in LV wall stress and mitral valve regurgitation [23]

Nowadays a definitive statement about TE and bioma-terials fields cannot be done but there are some reliableremarks referred to as materials-based platforms they pro-vide mechanical support to the damaged tissue while itregenerates can be modified to supply the bioactive agentsnecessary for regeneration and are able to restrict the cells tothe defect site guiding cell growth through surface chemistryand topography [25] RECATABI consortium proposes anapproach to address both the death of cardiomyocytes and theremodelling of the heart muscle with the aim to avoid heartfailureThe strategy consists in the development of a bioengi-neering platform that contains three main components (1)elastomeric microporous membrane which provides biome-chanical support (2) self-assembling peptide nanofiber gelwhich assures an adequatemicroenvironment and (3) subcu-taneous adipose tissue derived progenitor cells (subATDPCs)[26] Each of the components used to develop the patch is notnovelty by themselves but the right combination of them is

Since the activity of heart muscle involves both con-traction and dilation the use of elastomeric membranesmay provide restoration forces In addition it is conceivablethat the transmitted stresses to the engrafted cells allowdeep structural and functional biointegration With the hopeto introduce a new insight materials used for the firsttime in this field were examined Complete biodegradationof grafted scaffolds may compromise long-term limitationof postischemic ventricular remodeling For this reasonnondegradable poly(ethyl acrylate) (PEA) with cylindricalorthogonal pores and semidegradable poly(caprolactone 2-(methacryloyloxy) ethyl ester) (PCLMA) with intercon-nected spherical pores)membranes were tested [23] PEA has

mechanical properties in the range of those of soft biologicaltissues and it has shown excellent biological performancein vitro with different cell types [27ndash33] On the otherhand PCLMA also elastomeric at body temperature hasa biologically stable skeleton with a biodegradable lateralchain of caprolactone It has been chosen to carry out thesepreliminary in vivo experiments for its outstanding biologicalbehaviour tested with several architectures and cell typesboth in vivo and in vitro [34ndash36] The elasticity of thesepolymers at physiological temperature is expected to permittheir adaptation to the curved ventricle over their entiresurface and follow its mechanical deformation This elasticproperty is crucial to not disturb the synchronous beatingand at the same time to avoid further dilatation It is alsoexpected to allow the transfer of cells from the patch to themyocardium due to its good tissue-material biointegration

The RAD16-I self-assembling peptide nanofibers (provedto be nontoxic in vivo [37]) were chosen to provide a suitableenvironment to the implanted cells It has been proved tobe a good candidate to culture different cell types in a 3Denvironment [38ndash43] but its soft nature is a drawback forits direct implantation as a cell vehicle Nonetheless it hasalso been described to improve cell attachment in polymericscaffolds with respect to bare ones [44ndash46] Here we proposethat the combination of RAD16-I and elastomeric scaffoldsshould overcome the shortcomings of the gel combining theadvantages of both materials

In the present work we study the mechanical and bio-logical properties of the developed bioactive patch as wellas a preliminary assessment of their early response 3 daysafter implantation in small animal model (mice) of acuteMI As stated the elastomeric scaffolds employed possessdifferent architectures and compositions and thus slightlydifferent mechanical properties degradability and biologicalperformance It is not intended to compare both structuresand chemistries nor to study their effects in terms of cardiactissue regeneration but to develop ldquoproof of conceptrdquo anddemonstrate their suitability for the proposed application interms of manipulability and applicability onto the infarctedarea in the affected ventricle

2 Materials and Methods

21 Preparation of the Elastomeric Membranes (MacroporousScaffolds) Poly(ethyl acrylate) (PEA) scaffolds with inter-connected cylindrical orthogonal pores were obtained bythe template leaching method described before [47ndash49] Onthe other hand poly(caprolactone 2-(methacryloyloxy)ethylester) (PCLMA) scaffolds with spherical interconnectedpores were obtained by a procedure analogous to that fol-lowed by Escobar Ivirico et al [34] For detailed methodsplease refer to the Supplementary Materials and Methodssection in the Supplementary Material available online athttpdxdoiorg1011552015804017 Finally both materialswere washed with water and dried PEA and PCLMA scaf-folds were cut into samples having 5mm in diameter and1mm thickness Bulk films of both materials obtained bypolymerization in flat glass moulds were prepared to be






2critical point






24 Cell Culture


2) in 120572-MEM






2)















3 Results
























SubATDPCs

RAD16-I







4 Discussion



(a) (b) (c) (d)

(e) (f) (g) (h)








PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials








2critical point






24 Cell Culture


2) in 120572-MEM






2)















3 Results
























SubATDPCs

RAD16-I







4 Discussion



(a) (b) (c) (d)

(e) (f) (g) (h)








PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





2)















3 Results
























SubATDPCs

RAD16-I







4 Discussion



(a) (b) (c) (d)

(e) (f) (g) (h)








PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























SubATDPCs

RAD16-I







4 Discussion



(a) (b) (c) (d)

(e) (f) (g) (h)








PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b) (c) (d)

(e) (f) (g) (h)








PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




PEA PCLMA

Surface

Close up of top

Section

DAPI and

(a) (b)

(c) (d)

(e) (f)

(g) (h)

phalloidin





2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




2D PEA d1

PEA d4

PCLMA d4

PCLMA d1

NC

TBX5

MEF2C

ACTN1

cTnT

GJA1

CDH1

SNAI1

169bp

139bp

188 bp

207 bp

92bp

216 bp

164bp





5 Conclusions



PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




PEA PCLMA

(A) (B)

(C) (D)

(a)

PEA

PCLMA

(E) (F) (G)

(H) (I) (J)

(b)




Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials





Cardiacprogenitor

cell


Maturecardiomyocyte

Nkx25GATA4MEF2CTBX5




Abbreviations









Acknowledgments


References































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials































































































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Development of Bioactive Patch for...

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