Acta Biomaterialia 10 (2014) 3007–3017

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Acta Biomaterialia

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Umbilical-cord-blood-derived mesenchymal stem cells seeded ontofibronectin-immobilized polycaprolactone nanofiber improve cardiacfunction

http://dx.doi.org/10.1016/j.actbio.2014.03.0131742-7061/� 2014 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

⇑ Corresponding authors. Tel.: +82 2 880 1268; fax: +82 2 886 1268 (J.-Y. Cho).Tel.: +82 2 880 1635; fax: +82 2 888 7295 (N.S. Hwang).

E-mail addresses: nshwang@snu.ac.kr (N.S. Hwang), jeycho@snu.ac.kr (J.-Y. Cho).1 These authors contributed equally to this work.

Byung-Jae Kang a,1, Hwan Kim b,1, Seul Ki Lee c, Joohyun Kim a, Yiming Shen c, Sunyoung Jung d,Kyung-Sun Kang e, Sung Gap Im f, So Yeong Lee c, Mincheol Choi d, Nathaniel S. Hwang b,⇑, Je-Yoel Cho a,⇑a Department of Veterinary Biochemistry, BK21 Plus and Research Institute of Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Koreab School of Chemical and Biological Engineering, BioMAX Institute, Seoul National University, Seoul, Republic of Koreac Laboratory of Veterinary Pharmacology, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Koread Department of Veterinary Medical Imaging, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Koreae Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul, Republic of Koreaf Department of Chemical and Biomolecular Engineering, Korea Advanced Institute of Technology, Daejeon, Republic of Korea

a r t i c l e i n f o

Article history:Received 15 October 2013Received in revised form 8 March 2014Accepted 10 March 2014Available online 19 March 2014

Keywords:HeartUmbilical-cord-blood-derivedmesenchymal stem cellPolycaprolactone nanofiberFibronectinTransplantation

a b s t r a c t

Stem cells seeded onto biofunctional materials have greater potency for therapeutic applications. Weinvestigated whether umbilical-cord-blood-derived mesenchymal stem cell (UCB-MSC)-seeded fibronec-tin (FN)-immobilized polycaprolactone (PCL) nanofibers could improve cardiac function and inhibit leftventricle (LV) remodeling in a rat model of myocardial infarction (MI). Aligned nanofibers were uniformlycoated with poly(glycidyl methacrylate) by initiated chemical vapor deposition followed by covalentimmobilization of FN proteins. The degree of cell elongation and adhesion efficacy were improved byFN immobilization. Furthermore, genes related to angiogenesis and mesenchymal differentiations wereup-regulated in the FN-immobilized PCL nanofibers in comparison to control PCL nanofibers in vitro.4 weeks after the transplantation in the rat MI model, the echocardiogram showed that the UCB-MSC-seeded FN-immobilized PCL nanofiber group increased LV ejection fraction and fraction shortening ascompared to the non-treated control and acellular FN-immobilized PCL nanofiber groups. Histologicalanalysis indicated that the implantation of UCB-MSCs with FN-immobilized PCL nanofibers induced adecrease in MI size and fibrosis, and an increase in scar thickness. This study indicates that FN-immobi-lized biofunctional PCL nanofibers could be an effective carrier for UCB-MSC transplantation for the treat-ment of MI.

� 2014 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

1. Introduction Despite the improvements in medical and surgical treatments

Heart failure (HF) is the leading cause of death all over the world[1]. An important cause of HF is myocardial infarction (MI), which isoften caused by coronary artery obstruction leading to cardiomyo-cyte loss due to a lack of oxygen supply. The cardiomyocytes inadult mammalian hearts have limited self-regeneration capacities,and they intrinsically do not repopulate themselves when sub-jected to MI [2]. This overburdens the surviving myocardium andultimately leads to impairment of left ventricular (LV) function.Therefore, an alternative therapeutic intervention is required tocompensate for the inadequate intrinsic repair mechanism.

for MIs, therapeutic options are limited as many approaches,including reactivation of cardiomyocyte cell cycle activity andreduction of myocardial cell death, showed unsuccessful inhibitionof ventricular enlargement [3,4]. In addition, the morbidity/mortal-ity rate remains high, even after these interventions [2,5,6]. Re-cently, stem-cell-based approaches have received considerableattention for their potential to regenerate the infarcted heart andrestore cardiac function after an MI [2,6–9]. In particular, umbili-cal-cord-blood (UCB)-derived mesenchymal stem cells (MSCs),which have the capacity to differentiate into osteoblasts,chondrocytes and adipocytes, were shown to differentiate intocardiomyocytes, both in vitro and in vivo [2,10–12]. UCB-MSCsare a promising candidate for the treatment of MI due to their low-er immunogenic response compared to other stem cells, and thismay potentially allow UCB-MSCs for off-the-shelf cell source forallogeneic therapies [13].

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The functional beneficial effects of implanted stem cells are mostlikely due to the paracrine factors secreted by the cells [2,6,14].MSCs have been shown to produce stem cell homing, anti-apopto-sis, anti-inflammatory, anti-scarring and angiogenic cytokines thatsupport other cells present in the injured myocardium, and thismay be the primary mechanism for the recovery of cardiac functionand the reduction of myocardial fibrosis [2,6,10,14]. Stem cells canbe delivered into the heart by an intravenous injection into coronaryartery or by a direct injection into the myocardium. However, stemcell delivery with these techniques is limited due to low retentionand survival [2,14–17]. Typically, more than 80–90% of grafted cellsdie within 72 h after injection [18,19]. In order to improve survivaland engraftments of the implanted cells, combining cells with var-ious nanofiber-based biomaterials can be considered as a promisingapproach in cardiac tissue repair after MI [3,17,20]. Previous studiesshowed that these new strategies could improve grafted cell reten-tion and survival [3,17,21,22]. The ideal scaffold for cardiac tissueengineering should display both mechanical and biological proper-ties of native heart tissue, which can improve cell attachment andproliferation, promote cellular morphology and provide the flexibil-ity [23]. Furthermore, scaffolds for cardiac tissue engineeringshould be vascularized (or at least vascularized soon after implanta-tion) and non-immunogenic, and provide a suitable cellular interac-tion [17,20]. These properties can alter the expansion andcontraction of the heart.

Among the various methods utilized for cardiac tissue engineer-ing, electrospinning is of immense interest since it is possible tomake nonwoven meshes in nanometer scaffolds that are architec-turally similar to the native extracellular matrix (ECM) of the heart[24,25]. Furthermore, electrospun nanofibers provide a high sur-face-to-volume ratio and versatility to surface engineer for selec-tive biological activity. Electrospun nanofibers based onpolycaprolactone (PCL) have been extensively utilized for myocar-dial regeneration as three-dimensional cardiac tissue regeneration[26,27]. However, these biocompatible materials do not provideappropriate ECM-mediated biological signals for enhanced cellularattachment and migration. Thus, the presentation of appropriatechemical and physical cues onto nanofibers are required for en-hanced cellular attachment and bioactivity [28]. Fibronectin (FN)is known as an important ECM molecule for stem cell adhesion,survival and differentiation [29–31]. Furthermore, this protein isexpressed in the normal heart. Therefore, an effect of this ECMmolecule on stem cell attachment and function can be expectedif stem cells are applied once FN has accumulated onto nanofibers.

In order to strengthen the cell–ECM interactions, we immobi-lized FN onto aligned PCL nanofibers functionalized by initiatedchemical vapor deposition (iCVD) polymer films in order to modu-late UCB-MSC behavior. Specifically, we deposited poly(glycidylmethacrylate) (pGMA), which contains reactive epoxy functionalgroup that readily undergo a ring-opening reaction with peptideamine groups, onto the PCL nanofibers. The objective of the currentstudy was to investigate whether UCB-MSCs could improve cardiacfunction and inhibit left LV remodeling in a rat model of MI. In thisstudy, we demonstrate that FN immobilization on PCL nanofibersincreased the UCB-MSC adhesion. Furthermore, when FN-immobi-lized PCL nanofibers seeded with UCB-MSCs was implanted on theepicardial surface over infarcted areas, cardiac function was im-proved and LV remodeling was prevented.

2. Materials and methods

2.1. Isolation and culture of human UCB-MSCs

Human UCB-MSCs were isolated as previously described[32,33]. The human UCB-MSC isolation procedure was approved

by the Borame Hospital Institutional Review Board and Seoul Na-tional University (IRB No. 0603/001-002-07C1). Briefly, UCB sam-ples from term and preterm deliveries were harvested at thetime of birth with the mother’s informed consent (Seoul CityBorame Hospital Cord Blood Bank). The mesenchymal stem cellswere separated from the UCB using Ficoll-Paque TM PLUS (Amer-sham Bioscience, Uppsala, Sweden), and suspended in Dulbecco’smodified Eagle medium (DMEM; Gibco, Grand Island, NY, USA),containing 20% fetal bovine serum (FBS; Gibco), 100 IU ml�1 peni-cillin, 100 mg ml�1 streptomycin, 2 mM L-glutamine and 1 mM so-dium pyruvate. After 24 h, unattached cells and residualnonadherent red blood cells were removed by washing. The med-ium was changed at 48 h intervals until the cells became confluent.After cells reached 90% confluence, they were subcultured.

2.2. Preparation of aligned-cardiac nanofiber patch

PCL with a molecular weight of 43,000–50,000 g mol�1 waspurchased from Polysciences Inc. (Warington, PA, USA). PCL wasdissolved in chloroform at a concentration of 25% (w/v). The elec-trospinning process was conducted as follows: the polymer solu-tion was transferred into a 5 ml syringe (DAIHAN single-usesyringes, with rubber gasket, Korea) with an orthogonally cut-ended needle (G-22Luer, Korea). A syringe pump (Kd Scientific,Massachusetts, USA) was used to control the solution flow rate at0.5 ml h�1. To produce aligned nanofibers, a voltage of 12 kV wasapplied between the syringe needle and the rotary drum collector(NanoNC, Korea) with a rotating speed of 1000 rpm. Collected fi-bers were cut into circular 8 mm diameter shapes and further pro-cessed for surface modification.

2.3. Fabrication of functionalized cardiac nanofiber patch

The glycidyl methacrylate (GMA) monomer and the tert-butylperoxide (TBPO) initiator were purchased from Sigma–Aldrich.The pGMA films were deposited onto the nanofibers in a custom-built reactor (Daeki Hi-Tech Co. Ltd, Korea) via the iCVD processas previously described [34]. For grafting the pGMA films, theGMA monomer was vaporized in a jar, heated to 35 �C and fed intothe reactor. An initiator (TBPO) was fed to the reactor through adifferent port and mixed with the monomer in the reactor. Theflow rate of the GMA monomer was kept constant at 1.9 sccm(standard cubic centimeters per minute) while the flow rate ofTBPO was maintained at 0.8 sccm. pGMA-coated nanofibers werewashed with phosphate buffered saline (PBS) three times, and fur-ther immobilized with fluorescein-conjugated chitosan to confirmthe reactivity of pGMA. pGMA-coated PCL nanofibers were thenimmobilized with FN (10 lg ml�1) for enhanced cellular adhesions.For the nanofiber characterization, Fourier transform infrared (FT-IR, Alpha FT-IR Spectrometer, Bruker, Billerica, MA, USA) spectrawere obtained in normal absorbance mode with an average of 64scans. All spectra were baseline corrected. FT-IR bands were re-corded as spectra in the range of 400–4000 cm�1. Water contactangles were determined on a contact angle analyzer (Kruss DSA100, Germany) using the sessile drop method. Measurements wereperformed at room temperature, 30 s after deposition of the singledrops of distilled water on the sample surface. The average of threemeasurements was used as a result. For in vitro degradation ofnanofibers, the nanofiber scaffolds were immersed in 1 M sodiumhydroxide (NaOH), and washed three times with PBS. Then all sam-ples were dried and weighed for mass loss determination. The ten-sile strength of fabricated nanofibers was measured using andInstron 5583 (Instron Corporation, MA, USA) with the use of a3.35 N load cell under a cross-head speed of 3 mm min�1 at ambi-ent conditions. All nanofiber samples were prepared in the form of

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rectangular shape with dimensions of 20 � 10 mm. The thicknessof samples was 10 lm.

2.4. Cellular viability, adhesion and morphological analysis

UCB-MSCs (2.5 � 106 cells) were uniformly seeded onto FN-immobilized PCL nanofibers (8 mm in diameter). Initial cellularadhesion rates were measured by PicoGreen Assay�. Cells werecollected 48 h after seeding and DNA content was quantified usinga Quant-iT™ PicoGreen�dsDNA Assay Kit (Invitrogen™). For viabil-ity testing, a live/dead cell viability/cytotoxicity kit (MolecularProbes, L-3224) was used, following the manufacturer’s protocol.For cellular morphological analysis, cells were fixed at 4% parafor-maldehyde and stained with 406-diamidino-2-phenylindole (DAPI;Sigma–Aldrich).

2.5. Scanning electron microscopy

Cell-seeded nanofibers were fixed with a fixative composed of4.0% formaldehyde, 1.5% glutaraldehyde, 2.5% sucrose, 5 mM cal-cium chloride and 5 mM magnesium chloride in 0.1 M sodium cac-odylate for 20 min and washed with 2.5% sucrose in 0.1 M sodiumcacodylate. Fixed samples were washed with cold distilled waterand dehydrated with a graded series of cold ethanol for dehydra-tion, and samples were then incubated with 50% ethanol and 50%hexamethyldisilazane (HMDS) for 5 min and exchanged with100% HMDS for 10 min. Prior to scanning electron microscopy(SEM) imaging, nanofibers were sputtered with platinum for100 s at 20 mA. Field emission SEM images were obtained with aJEOL 6700 instrument.

2.6. Reverse transcriptase–polymerase chain reaction array

Total RNAs were extracted with Trizol, and reverse-transcribedinto cDNA using the SuperScript Synthesis System (Invitrogen™).Room temperature reactions were first denatured for 2 min at95 �C, followed by 35 cycles of 30 s denaturation at 95 �C, anneal-ing and elongation at 72 �C. PCR array analyses were performedusing human mesenchymal stem cell PCR arrays (SuperArray Bio-science) according to the manufacturer’s instructions using the Ap-plied Biosystem StepOnePlus™ Real Time PCR System (AppliedBiosystems). Internal control was normalized to housekeepinggenes, which are B2M, HPRT1, RPL13A, GAPDH, ACTB and HGDC.Gene expression fold was compared with day 1 sample.

2.7. MI model and implantation

UCB-MSC-seeded nanofibers were maintained in vitro in kerat-inocyte-serum free medium (Gibco) supplemented with 10% FBSand antibiotic–antimycotic (Gibco) at 37 �C in a 5% CO2 humidifiedatmosphere for 7 days prior to implantation. As a control, non-cell-seeded FN-immobilized PCL nanofibers were prepared by thesame method but without UCB-MSCs. Male Sprague–Dawley ratsweighing 260–300 g (Orient Bio, Seongnam, Korea) were used in theMI model. All animal procedures were performed in accordance withthe guidelines of the Institutional Animal Care and Use Committeeof Seoul National University. MI was induced by ligation of the leftcoronary artery according to the methods previously reported [35].2 weeks after induction of MI, rats were randomized into threetreatment groups, which received implantation of UCB-MSCs-seeded FN-immobilized PCL scaffold (UCB-MSC/FN-PCL group,n = 10), implantation of non-cell-seeded FN-immobilized PCL scaf-fold (FN-PCL group, n = 5) or sham operation (control group, n = 4).Cell-seeded or non-seeded FN-immobilized PCL nanofibers werefixed with fibrin sealant (TISSEL, Baxter, Deerfield, IL, USA) overthe infarcted region and adjacent normal heart. An antirejection

strategy was used according to methods previously reportedbecause a rat’s immune system could reject human cellxenograft after implantation of UCB-MSCs [36]. All the ratsreceived cyclosporine (Sandimmune�, Novartis, Basel, Switzerland)10 mg kg�1 day�1 subcutaneously for 2 weeks starting 2 days priorto grafting, followed by 100 lg ml�1 in the drinking water for thenext 2 weeks.

2.8. Functional assessment of infarcted myocardium

Cardiac function was assessed by transthoracic echocardiogra-phy before MI (normal baseline), 1 week after MI (infarct baseline)and 4 weeks after scaffold implantation. Echocardiography wasperformed using a conventional echocardiographic machine (Pro-Sound Alpha 7; ALOKA, Tokyo, Japan) with a 9 MHz linear trans-ducer by a veterinary radiologist unaware of the treatmentcondition. For analysis of LV function, LV internal diameter atend-diastole (LVIDd) and LV internal diameter at end-systole(LVIDs) were measured at the anterior wall, from the short-axisview, just below the level of the papillary muscle. LV end-diastolicvolume (LVEDV) and LV end-systolic volume (LVESV) were auto-matically calculated by the single-plane area and length method.The fractional shortening (FS) was calculated as ((LVIDd – LVIDs)/LVIDd) � 100 (%), and ejection fraction (EF) was calculated as((LVEDV – LVESV)/LVESV) � 100 (%).

2.9. Histological and immunohistochemical examinations

Hearts were harvested and fixed in 4% paraformaldehyde for24 h and then cut into four transverse slices through the infarctedarea. The slices were then embedded in paraffin and 5 lm histo-logic sections were stained with Masson’s Trichrome. The infarctsize, fibrosis and scar thickness were quantified using ImageJ soft-ware (NIH, Bethesda, MD, USA). The infarct size was defined as thepercentage of the sum of the infarcted epicardial and endocardialcircumferences divided by the sum of the LV epicardial and endo-cardial circumferences. The percentage area of fibrosis was quanti-fied by the fibrotic area divided by the sum of the fibrotic area andnon-fibrotic area. Scar thickness was measured in the center of theinfarct, at the border of the infarct and in between, and then thesefive values were averaged.

Immunohistochemical staining was performed to identify thesurvival of transplanted cells and vessel formation using the pri-mary antibodies: rabbit monoclonal anti-lamin A+C antibody(1:200, abcam�, Cambridge, MA, USA) and rabbit polyclonal anti-von Willebrand factor (vWF) antibody (1:100, abcam�). The sec-tions were incubated with primary antibodies overnight and weresubsequently exposed to biotinylated secondary antibody andstreptavidin peroxidase complex using Histostain-Plus kit (Invitro-gen™). Then, the sections were visualized by reaction with 3,30-diaminobenzidine tetrahydrochloride (liquid DAB substrate kit;Invitrogen™) and were counterstained with hematoxylin. Theanti-lamin A+C antibody reacts with human but does not reactwith mouse and rat. Lamin A+C antibody was used to identifythe survival of implanted human UCB-MSCs. The density of bloodvessels was determined in the sections stained with anti-vWF anti-body. Five high-power fields (HPFs; 200� magnification) withinthe infarcted region of each section were chosen randomly, andblood vessel density was expressed as the quantity of vessels permm2. Averages based on five HPFs from each of the two samplesper rat were calculated for comparison.

2.10. Statistical analysis

Statistical analysis was performed using SPSS version 20.0(SPSS, Chicago, IL, USA). A Kruskal–Wallis test was used to assess

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differences among the groups. A post hoc test was performed alongwith a Mann–Whitney U test. A P value less than 0.05 was consid-ered to be statistically significant.

3. Results

3.1. Fabrication of aligned-cardiac nanofiber patch

In order to immobilize FN on the PCL nanofiber, we introducedpGMA film, resulting in 50 nm thick pGMA. Fig. 1 demonstratesschematically the procedure for producing pGMA-coated and FNimmobilized pGMA-PCL nanofibers. First, pGMA films weredeposited on the aligned PCL nanofibers via iCVD. Surface coatingof pGMA functionalized nanofibers with epoxy groups. Conse-quently, pGMA-coated PCL nanofibers were immobilized with FN(10 lg ml�1) for enhanced cellular adhesions (Fig. 1A). Immobiliza-tion of FN formed new ether bonds after epoxy rings were broken.In order to assess the in vivo functionality of FN immobilized nano-fiber, we seeded UCB-MSCs onto fabricated nanofibers, then trans-planted as a patch in myocardial infarction model (Fig. 1B).

3.2. Characteristics of fabricated PCL nanofibers

SEM micrograph of electrospun fibrous scaffolds revealed uni-form aligned fibers. Furthermore, SEM images of nanofibersshowed that post modification of pGMA coating by iCVD did not af-fect the diameter or the alignment of the original fibers (Fig. 2A).The existence of epoxy groups after pGMA coating by iCVD wasconfirmed by FT-IR analysis (Fig. 2B). Compared to PCL nanofibers,a small peak appeared at 910 nm�1 for pGMA-coated PCL nanofi-bers (iCVD), which represent the epoxy group. However, the peakwas reduced after FN immobilization because new bonds wereformed (1064 nm�1) and epoxy rings were broken (Fig. 2B, as indi-cated by arrow). Binding of amine-containing and fluorescein-con-jugated chitosan (Chitosan-FITC) to the pGMA-coated PCL wasvisualized by fluorescent microscopic analysis (Fig. 2C). Conformalconjugation of chitosan-FITC to the pGMA-coated nanofibers con-firmed the reactivity of the epoxy group of pGMA on nanofibers.With a short incubation time, pGMA-PCL gave two times higherfluorescence intensity than the control nanofibers (Fig. 2D).

Fig. 1. Schematic illustration of fabrication of functionalized nanofibers for MI. (A) PCL nathe pGMA-PCL nanofibers. (B) UCB-MSCs were seeded onto the FN-immobilized PCL na

3.3. PCL nanofiber in vitro degradation

Polycaprolactone belongs to the class of aliphatic polyesters andis degraded by hydrolytic degradation [37]. PCL is a semi-crystal-line polymer. PCL is reported to degrade slowly over a period of2–4 years depending on its properties, such as molecular weightand crystallinity. To evaluate the degradation properties of PCLnanofiber, we used accelerated conditions using alkaline medium(1 N NaOH) to promote hydrolysis of the polyesters. As shown inFig. 2E, our results reveal linear degradation profiles among thePCL, pGMA-coated PCL nanofibers (iCVD), and FN-immobilizedPCL nanofiber scaffolds (FN). The PCL nanofiber showed a higherdegradation rate (rate constant K = 10.438) compared to thepGMA-coated PCL nanofiber (K = 4.8458) and FN-immobilizedPCL nanofiber (K = 4.2962). The pGMA-coated PCL nanofiber andthe FN-immobilized PCL nanofiber showed only a 26.62% and30.76% mass loss under accelerated conditions in 6 h, comparedwith a 65.3% mass loss for the control PCL nanofiber. These resultsmay indicate that the iCVD treatment to the nanofibers delayed thedegradation of the material compared to the non-coated nanofi-bers. We concluded that the degradation kinetics of iCVD-treatedmaterials were considerably slower than non-iCVD treated nanof-ibers due to its greater hydrophobicity and crystallinity by pGMAunder alkaline medium conditions. Also iCVD coating of pGMAmay have prevented the NaOH-dependent PCL degradation.

3.4. Characteristics of UCB-MSC-seeded FN-immobilized PCL nanofiber

The influences of topography and artificial surface modulationswere investigated by plotting the apparent contact angles. The con-tact angle of FN-immobilized PCL nanofiber turned out to be106.0�, which can be interpreted as being more hydrophilic com-pared to those of PCL- and iCVD-coated PCL nanofibers, which were133.5� and 131.7�, respectively. We further investigated cellular re-sponse on FN-immobilized PCL nanofibers. UCB-MSCs were seededonto FN-immobilized PCL nanofiber at 2 � 105 cells per scaffold.Even though the FN-immobilized PCL nanofiber is more hydro-philic than others, infiltration of cells into the interior of the nano-fiber was limited due to hydrophobic surface and narrow pore size(data not shown). Changes in cellular morphology usually result

nofibers were prepared and coated with pGMA by iCVD. FNs were immobilized ontonofibers and transplanted onto a MI model rat.

Fig. 2. Characterization of functionalized nanofibers. (A) SEM images of control nanofibers and pGMA-coated nanofibers. Scale bar = 50 lm. (B) FTIR spectra of control PCL,pGMA-coated nanofiber by iCVD (iCVD), and FN-immobilized nanofiber (FN). (C) Fluorescence images of control PCL nanofibers and pGMA nanofibers immobilized withchitosan-FITC. (D) Fluorescence intensity measurement of control PCL nanofibers and pGMA nanofibers immobilized with chitosan-FITC, n = 10. ⁄P < 0.05 vs. pGMA group. (E)In vitro degradation rate of control PCL, pGMA-coated nanofibers by iCVD (iCVD) and FN-immobilized nanofibers (FN).

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from varying cell–substrate interactions leading to cytoskeletalreorganizations. SEM examination showed that the morphologyof UCB-MSCs adhering to the PCL scaffolds was altered after theFN coating (Fig. 3B). The cells on the PCL nanofiber showed roundcellular morphology with abundant cellular pods. High hydropho-bicity of PCL fibers inhibits the cellular protrusion and micro-fibrillar adhesion to the nanofiber. However, FN-immobilizednanofibers provided the optimal microenvironment for UCB-MSCsattachment. Actin cytoskeletal images of UCB-MSCs cultured onFN-immobilized PCL nanofiber indicated that the UCB-MSCs ad-hered most to the FN-immobilized PCL nanofiber with fibroblasticmorphology (Fig. 3C).

3.5. Cell adhesion, proliferation and viability in scaffold

For the number of cell attachments, 2 � 105 cells were seeded ineach nanofiber construct and cultured for 24 h prior to countingcell numbers. The total number of cells attached per area(0.1 mm2) was doubled in FN-immobilized nanofibers comparedto PCL nanofibers (Fig. 4A). For the proliferation study, 5 � 105 cellswere seeded in each nanofiber and cultured for 72 h prior to per-forming a Quant-iT™ PicoGreen�dsDNA Assay. All nanofiber con-structs were digested in papainase solution to extract the DNAout of the cell. The proliferation rate, assessed by DNA content,was significantly increased in FN-immobilized nanofibers, whichwas 0.01 lg, as compared with non-coated control nanofibers,0.004 and 0.006 lg, respectively (Fig. 4B). The cytotoxicity of thescaffolds was examined by a viability assay. Cell viability, when as-sessed by live/dead assay, was not statistically different among allthe tested scaffolds over 7 days of cultivation and showed that only�10% of cultured cells were dead (Fig. 4C). These results indicatethat FN-immobilized PCL nanofibers can support the cell attach-ment and survival.

3.6. Gene expression profile of the cells seeded onto scaffolds

To determine the biological effects of immobilized FN on stemcells, we compared gene expression profiles of the human UCB-MSCs from in vitro culture depending on the FN coating usingPCR array analysis. UCB-MSCs were seeded on uncoated PCL nanof-ibers, pGMA-coated PCL nanofibers and FN-immobilized PCLnanofibers. Our result showed that the expression levels of somecellular function-related genes were increased in the UCB-MSCscultured on FN-immobilized PCL nanofibers in comparison withuncoated PCL scaffolds (Fig. 5). Some of these elevated genes areknown to be involved in regulating cellular functions of stem cells,including stem cell homing, anti-apoptosis, anti-inflammation,anti-scarring and angiogenesis. Specifically, the gene expressionlevels of the markers for stem cell homing (FGF-2), anti-apoptosis(IGF-1 and LIF), anti-inflammation (IL-6), anti-fibrosis (MMP-2)and angiogenesis (VEGF-A, KDR, JAG1, ANPEP, TGFb-1 and vWF)were higher in the FN-immobilized PCL nanofibers than those inuncoated PCL scaffolds. These results indicate that the putativeparacrine effects of UCB-MSCs cultured on the FN-immobilizedPCL nanofibers may be superior to that in uncoated PCL scaffoldsfor the treatment of MI. All the results in vitro taken together indi-cate that FN appears to be a favorable coating agent for MSC trans-plantation. Therefore, FN-immobilized PCL nanofibers were usedfor further in vivo experiments.

3.7. Assessment of cardiac functions by echocardiography

To evaluate the effect of the combined cell and scaffold therapyon LV remodeling and cardiac function, echocardiography was per-formed. LVIDd, LVIDs, EF and FS were quantified at the baseline be-fore the left anterior descending coronary artery (LAD) ligation,1 week after LAD ligation before implantation and 4 weeks after

Fig. 3. Contact angle and morphology of the cell on nanofibers. (A) Contact angle measurements of PCL nanofibers, pGMA-coated nanofibers by iCVD (iCVD) and FN-immobilized pGMA-coated nanofibers (FN). (B) SEM images of UCB-MSC morphology after 24 h in vitro on nanofibers, control PCL, pGMA-coated nanofibers by iCVD (iCVD)and FN-immobilized nanofibers (FN). Circle signifies single-cell morphology and arrow indicates linear alignment of the fiber. Scale bar = 20 lm. (C) Immunofluorescentanalysis of UCB-MSC morphology on each type of nanofiber shown with autofluorescent (green) and nuclei with DAPI (blue). Scale bar = 50 lm.

Fig. 4. Cell adhesion and viability in scaffold. (A) Initial cellular adhesion rate of UCB-MSCs onto nanofibers were measured by counting cell numbers per area (n = 3) after24 h of post seeding. (B) Proliferation rate of UCB-MSCs onto nanofibers were measured by DNA quantification assays (n = 3) after 72 h of post seeding. (C) Viability of the eachgroup was measured by live/dead viability/cytotoxicity kit (n = 3) after 48 h of post seeding. ⁄P < 0.05 vs. iCVD group. #P < 0.05 vs. FN group. All values represent mean ± SEM.

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implantation. EF and FS values were within the normal range in allrats before MI induction, but the heart function was significantlycompromised in all rats 1 week after infarction (Fig. 6A).

To assess the functional effect of cardiac patches with humanUCB-MSCs on FN-immobilized PCL nanofibers, LVIDd, LVIDs, EFand FS were calculated and compared 4 weeks after the treatment.The UCB-MSC/FN-PCL group (human UCB-MSCs on FN-immobi-lized PCL nanofibers), showed attenuation of LV dilation and dys-function compared to the control and FN-PCL groups (Fig. 6B).LVIDd values were similar in all experimental groups. However,the LVIDs value in the UCB-MSC/FN-PCL group (6.47 ± 0.95 mm)was significantly smaller than that in the control group(7.93 ± 0.33 mm), although the non-cell seeded FN-PCL group(8.02 ± 1.42 mm) was not significantly different than the controlgroup. EF in the UCB-MSC/FN-PCL group (60.2 ± 7.87%) was signif-icantly higher than those in the control (41.82 ± 8.23%) and FN-PCL(38.54 ± 7.43%) groups. Similarly, FS in the UCB-MSC/FN-PCL group

(28.9 ± 5.2%) was also significantly higher than those in tehe con-trol (18.26 ± 4.43%) and FN-PCL (16.56 ± 3.68%) groups.

3.8. Histological and histomorphometric analysis

Histological and morphometric analyses were performed toidentify LV remodeling after Masson’s trichrome staining of heartsections at 4 weeks. Masson’s trichrome staining shows that in-farcted myocardium with fibrosis appears blue, while viable myo-cardium appears red (Fig. 7A). The MI size was significantlyreduced in the UCB-MSC/FN-PCL group as compared to the controland FN-PCL groups (Fig. 7B). In addition, the percentage of ventric-ular fibrosis was also significantly reduced in the UCB-MSC/FN-PCLgroup as compared to the control and FN-PCL groups (Fig. 7B). Thescar thickness of the UCB-MSC/FN-PCL group was significantlythicker than those of the control and FN-PCL groups (Fig. 7B).

Fig. 5. RT2 profiler PCR array data analysis clustergram of the expression of 84 keygenes involved in human MSCs. cDNA expression profiling in human MSCs (RT2

Profiler). Clustergram generated from average cDNA Ct values of individual groupsof samples. For each group, average Ct values were normalized to a panel ofdifferent housekeeping genes. The range of 2�DCt values is given below the colorscale.

B.-J. Kang et al. / Acta Biomaterialia 10 (2014) 3007–3017 3013

3.9. Survival of transplanted cells

We have shown in vitro that our FN-immobilized PCL nanofi-bers support UCB-MSC survival. Thus, we tested whether the cellssurvived in vivo for 4 weeks by immunohistochemical stainingwith human cell marker lamin A+C antibody. No lamin A+C-posi-tive cells were detected in the non-cell-seeded control and FN-PCL groups. In the UCB-MSC/FN-PCL group implanted with UCB-MSCs, lamin A+C-positive cells were detected in the scaffolds orthe boundary between the scaffold and the underlying myocar-dium (Fig. 8). However, these lamin A+C-positive cells remainedisolated from each other and did not penetrate into the adjacentmyocardium or infarction area. This result may indirectly indicatethat the stem cells prevented cardiac damage, possibly by secretingparacrine factors rather than the differentiation of the implantedcells.

3.10. Blood vessel density analysis

The infarcted myocardium was immunohistochemically stainedfor vWF and the blood vessels in the infarcted region were quanti-fied. Vessel density of the infarcted area in the UCB-MSC/FN-PCLgroup was significantly higher than those in the control and FN-PCL groups (Fig. 9). This result suggests that the UCB-MSCs in theFN-immobilized PCL nanofiber promote angiogenesis by secretingor stimulating angiogenic factors such as VEGF, as demonstratedin the in vitro PCR array.

4. Discussion

In the present study, we investigated the effects of UCB-MSC-seeded FN-immobilized PCL nanofibers on improvements in car-diac function and attenuation of LV fibrotic remodeling in a ratMI model. Human UCB-MSCs were seeded onto FN-immobilizedPCL nanofibers and implanted onto the epicardial surface overthe infarcted area and adjacent infarction border zones. 4 weeksafter transplantation, the cell-seeded UCB-MSC/FN-PCL group hadsignificantly improved LV function, attenuated LV remodelingand increased neovascularization compared to the control andFN-PCL groups. These results can be explained as the outcome ofthe cardio-protective effects produced by the combined stem celland scaffold therapy. The FN-immobilized PCL scaffold alone wasnot able to prevent cardiac dysfunction and LV remodeling afterMI. This suggests that the FN-immobilized PCL nanofiber acts asa vehicle for cell delivery to improve the efficacy of stem cell ther-apy for MI.

In this study, UCB was used to obtain MSCs, and we identifiedthe cardio-protective potential of these cells. These cells, whenseeded on the FN-immobilized PCL nanofibers, expressed survivaland angiogenic factors (Fig. 5). In the present study, the FN-immo-bilized PCL nanofiber alone did not lead to angiogenesis and theimprovement of MI, but UCB-MSC implantation with our FN-immobilized PCL nanofiber induced favorable results. It can beconcluded from these findings that the observed improvementafter implantation appear by the applied UCB-MSCs not by theFN-immobilized PCL nanofiber itself. The main advantages ofUCB-MSCs are that these cells can be harvested painlessly withoutthe risk of patient morbidity. Previous studies have confirmed thatthe UCB-MSCs are immune-privileged cells with surface character-istics capable of overcoming rejection [13]. Thus, these UCB-MSCshave the potential to be utilized as off-the-shelf allogeneic cells forregenerative medicine. Our findings suggest that UCB-MSCs can bestem cell candidates for cardiac therapy, and can be used in placeof BM-MSCs because these cells have the cardio-protective poten-tial and many advantages for clinical application.

The application of biomaterials with a variety of cell types hasshown an improvement of cardiac function in animal model [38].Among various polymeric biomaterials utilized in cardiac tissueengineering applications, PCL nanofibers used in this study are espe-cially appropriate for use in the treatment of MI since they exhibitelasticity. In this study, the maximum strength of PCL nanofiberson the entire scaffold was 33.48 MPa with breaking stain at 7.9%.Furthermore, iCVD treatment did not significantly alter themechanical properties of our fibers (data not shown). When consid-ering cardiac tissue engineering, the scaffold implanted onto thecardiac surface needs to be elastic to resist injury from continuouscontraction. In addition, biomaterials for therapeutic cardiac tissueengineering should allow appropriate cellular interaction. ECM pro-tein can serve as a scaffold for cellular interaction including cellattachment, migration and proliferation [39]. In the present study,we immobilized FN as an ECM protein on PCL scaffolds to increasecell transfer efficiency and attachment. FN could be an excellentcandidate as it is normally present in the heart and plays a pivotalrole in stem cell behavior, including adhesion and proliferation. Ina previous study, stem cells attached best to FN in comparison withother ECM molecules [30]. Similarly, in the present study, FN stim-ulated in vitro UCB-MSC attachment. Electrospinning is a versatilemethod to create a biomimicking microenvironment similar to thatof natural ECM for enhanced cell adhesion and tissue growth. Theproposed scaffolds with aligned nanofiber topography are desirablefor cardiac tissue engineering due to its ability to induce cellularphenotype found in native cardiac tissue. Previously establishedmethods to surface functionalize nanofibers have been limited to

Fig. 6. Evaluation of cardiac function by echocardiography. (A) Echocardiography measurements performed before MI and 7 days after MI show that MI induces an increase inLV remodeling and LV chamber dimensions, leading to a decrease in EF and fractional FS. (B) In echocardiographic examination at 4 weeks after treatment, control and FN-PCLgroups show LV dilation and significant LV wall thinning. The UCB-MSC/FN-PCL group shows limited LV remodeling and preserved contractility. EF and FS as LV systolicfunction are significantly higher in the UCB-MSC/FN-PCL group compared to the control and FN-PCL groups. ⁄P < 0.05 vs. control group. #P < 0.05 vs. FN-PCL group. All valuesrepresent mean ± SEM.

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physical ECM coating. However, we hypothesized that permanentimmobilization of natural proteins on the nanofiber surface is re-quired for uninterrupted cell recognition that is essential for stemcell function and differentiation.

In the present study, some of the engrafted UCB-MSCs wereidentified within the seeded scaffolds and the boundary betweenthe scaffold and the underlying myocardium by immunohisto-chemical examination. However, any migration or penetration ofthese cells toward the host cardiac tissue was not detected. Never-theless, MSC-seeded FN-immobilized PCL nanofibers efficientlyprevented LV remodeling and dysfunction. These findings suggestthat the positive results seen in the UCB-MSC/FN-PCL group com-pared with the control and FN-PCL groups might be related to par-acrine activities. Likewise, previous accumulating evidence hassuggested that the main beneficial effects derived from implanted

cells are through the paracrine effects of cytokines secreted by theimplanted cells and even a few cells could activate different regen-eration pathways such as cell survival, stem cell homing, angiogen-esis and matrix remodeling if they produce crucial bioactivemediators [2,6,14,40]. To identify potential paracrine effects ofUCB-MSCs seeded in FN-immobilized PCL nanofibers, we evaluatedgene expressions of multiple paracrine factors using a PCR gene ar-ray study. The expressions of some angiogenic factors such asVEGF-A, KDR, JAG1, ANPEP, TGFb-1 and vWF were significantly in-creased in the UCB-MSCs on the FN-immobilized PCL nanofiberscompared with them on uncoated PCL scaffolds. These findingssuggest that MSCs seeded in the FN-immobilized PCL nanofiberscould modulate the expression of multiple angiogenic cytokines withthe potential to promote neovascularization. Neovascularizationcontributed to the prolonged cell survival in the FN-immobilized

Fig. 7. Evaluation of MI size, LV fibrosis and scar thickness. (A) Representative images of heart sections stained with Masson’s trichrome show fibrosis and wall thinning in theinfarcted area. Fibrotic areas are colored in blue and viable myocardium in red. (B) Infarct size, percentage of fibrosis and wall thickness are statistically compared betweendifferent groups. ⁄P < 0.05 vs. control group. #P < 0.05 vs. FN-PCL group. All values represent mean ± SEM.

Fig. 8. Representative immunostaining images from three transplant groups after 4 weeks using lamin A+C antibody to visualize the transplanted cells. Positive staining forlamin A+C is shown in only the UCB-MSC/FN-PCL group, but not in the control and FN-PCL groups. Scale bar = 100 lm.

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PCL nanofibers, and this may induce increased collateral flow andcontractility, and consequentially improvement of cardiac func-tion. In addition, stem cell homing (FGF-2), anti-apoptosis (IGF-1and LIF), anti-inflammation (IL-6) and anti-fibrosis (MMP-2)genes showed significantly higher expression levels in the

FN-immobilized PCL nanofiber group than in the uncoated scaffoldgroups. The aforementioned results indirectly indicate that UCB-MSCs seeded in the FN-immobilized PCL nanofiber can stimulatea profound neovascularization and attenuation of LV remodelingthrough their producing cytokines. The ECM provides cells with

Fig. 9. Vessel density in the MI sites. Representative images of myocardial sections from control (A), FN-PCL (B) and UCB-MSC/FN-PCL (C) groups show immunohistochemicalvWF staining of the infarcted myocardium. The vascular endothelial cells in the blood vessels are colored in brown (arrows). (D) Blood vessel densities were higher in theUCB-MSC/FN-PCL group than in the control and FN-PCL groups. Scale bar = 200 lm. ⁄P < 0.05 vs. control group. #P < 0.05 vs. FN-PCL group. All values represent mean ± SEM.

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not only structural support but also biochemical and physical cuesthat regulate cell phenotype [41]. FN used in this study as the ECMcomponent also plays a role in regulating the cell phenotype, andinteracts via integrin receptors, which regulates the cellular cyto-skeleton and cell behavior [42]. Therefore, the interaction betweenintegrin and FN may lead to improving the putative paracrine ef-fect of stem cells, although we did not yet verify and understandthe mechanisms of FN-mediated cellular signaling.

5. Conclusion

The FN-immobilized PCL nanofiber used in this study enhancedcellular adhesion, which makes it an attractive carrier for celltransplantation in the treatment of MI. Furthermore, the transplan-tation of UCB-MSCs with FN-immobilized PCL nanofiber improvedcardiac function, prevented LV remodeling and stimulated angio-genesis in the rat MI model. Therefore, the combination of FN-immobilized PCL nanofibers and UCB-MSCs may be a promisingstrategy for future cardiac clinical application.

Acknowledgements

This work was supported by the National Research Foundation(NRF) funded by the Ministry of Science, ICT & Future Planning,Republic of Korea (2012M3A9C6049716 and 20110019355).

Appendix A. Figures with essential color discrimination

Certain figures in this article, particularly Figs. 1–3 and 5–9, aredifficult to interpret in black and white. The full color images canbe found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2014.03.013.

References

[1] Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, De Simone G, et al.Heart disease and stroke statistics—2010 update: a report from the AmericanHeart Association. Circulation 2010;121:e46–e215.

[2] Segers VF, Lee RT. Stem-cell therapy for cardiac disease. Nature2008;451:937–42.

[3] Karam J-P, Muscari C, Montero-Menei CN. Combining adult stem cells andpolymeric devices for tissue engineering in infarcted myocardium.Biomaterials 2012;33:5683–95.

[4] Müller-Ehmsen J, Peterson KL, Kedes L, Whittaker P, Dow JS, Long TI, et al.Rebuilding a damaged heart long-term survival of transplanted neonatal ratcardiomyocytes after myocardial infarction and effect on cardiac function.Circulation 2002;105:1720–6.

[5] Savoye C, Equine O, Tricot O, Nugue O, Segrestin B, Sautière K, et al. Leftventricular remodeling after anterior wall acute myocardial infarction inmodern clinical practice (from the REmodelage VEntriculaire [REVE] studygroup). Am J Cardiol 2006;98:1144–9.

[6] Lu F, Zhao X, Wu J, Cui Y, Mao Y, Chen K, et al. MSCs transfected withhepatocyte growth factor or vascular endothelial growth factor improvecardiac function in the infarcted porcine heart by increasing angiogenesis andreducing fibrosis. Int J Cardiol 2012. doi: 10.1016/j.ijcard.2012.06.052.Available from: http://www.ncbi.nlm.nih.gov/pubmed/22981278.

[7] Kim B-O, Tian H, Prasongsukarn K, Wu J, Angoulvant D, Wnendt S, et al. Celltransplantation improves ventricular function after a myocardial infarction: apreclinical study of human unrestricted somatic stem cells in a porcine model.Circulation 2005;112:I-96–I-104.

[8] Yoon SJ, Fang YH, Lim CH, Kim BS, Son HS, Park Y, et al. Regeneration ofischemic heart using hyaluronic acid-based injectable hydrogel. J BiomedMater Res Appl Biomater 2009;91:163–71.

[9] Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bonemarrow cells regenerate infarcted myocardium. Nature 2001;410:701–5.

[10] Caplan AI, Dennis JE. Mesenchymal stem cells as trophic mediators. J CellBiochem 2006;98:1076–84.

[11] Wollert KC, Drexler H. Clinical applications of stem cells for the heart. Circ Res2005;96:151–63.

[12] Miyahara Y, Nagaya N, Kataoka M, Yanagawa B, Tanaka K, Hao H, et al.Monolayered mesenchymal stem cells repair scarred myocardium aftermyocardial infarction. Nat Med 2006;12:459–65.

[13] Lee OK, Kuo TK, Chen W-M, Lee K-D, Hsieh S-L, Chen T-H. Isolation ofmultipotent mesenchymal stem cells from umbilical cord blood. Blood2004;103:1669–75.

[14] Mathieu E, Lamirault G, Toquet C, Lhommet P, Rederstorff E, Sourice S, et al.Intramyocardial delivery of mesenchymal stem cell-seeded hydrogelpreserves cardiac function and attenuates ventricular remodeling aftermyocardial infarction. PLoS ONE 2012;7:e51991.

B.-J. Kang et al. / Acta Biomaterialia 10 (2014) 3007–3017 3017

[15] Marsano A, Maidhof R, Luo J, Fujikara K, Konofagou EE, Banfi A, et al. The effectof controlled expression of VEGF by transduced myoblasts in a cardiac patchon vascularization in a mouse model of myocardial infarction. Biomaterials2013;34:393–401.

[16] Siepe M, Giraud M-N, Pavlovic M, Receputo C, Beyersdorf F, Menasché P, et al.Myoblast-seeded biodegradable scaffolds to prevent post-myocardialinfarction evolution toward heart failure. J Thorac Cardiovasc Surg2006;132:124–31.

[17] Piao H, Kwon J-S, Piao S, Sohn J-H, Lee Y-S, Bae J-W, et al. Effects of cardiacpatches engineered with bone marrow-derived mononuclear cells and PGCLscaffolds in a rat myocardial infarction model. Biomaterials 2007;28:641–9.

[18] Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol2005;23:845–56.

[19] Toma C, Pittenger MF, Cahill KS, Byrne BJ, Kessler PD. Human mesenchymalstem cells differentiate to a cardiomyocyte phenotype in the adult murineheart. Circulation 2002;105:93–8.

[20] Venugopal JR, Prabhakaran MP, Mukherjee S, Ravichandran R, Dan K,Ramakrishna S. Biomaterial strategies for alleviation of myocardialinfarction. J R Soc Interface 2012;9:1–19.

[21] Araña M, Peña E, Abizanda G, Cilla M, Ochoa I, Gavira JJ, et al. Preparation andcharacterization of collagen based ADSC-carrier sheets for cardiovascularapplication. Acta Biomater 2013;9:6075–83.

[22] Liu Z, Wang H, Wang Y, Lin Q, Yao A, Cao F, et al. The influence of chitosanhydrogel on stem cell engraftment, survival and homing in the ischemicmyocardial microenvironment. Biomaterials 2012;33:3093–106.

[23] Zimmermann WH, Melnychenko I, Eschenhagen T. Engineered heart tissue forregeneration of diseased hearts. Biomaterials 2004;25:1639–47.

[24] Lin Y-D, Luo C-Y, Hu Y-N, Yeh M-L, Hsueh Y-C, Chang M-Y, et al. Instructivenanofiber scaffolds with VEGF create a microenvironment for arteriogenesisand cardiac repair. Sci Transl Med 2012;4:146ra09.

[25] Prabhakaran MP, Kai D, Ghasemi-Mobarakeh L, Ramakrishna S. Electrospunbiocomposite nanofibrous patch for cardiac tissue engineering. Biomed Mater2011;6:055001.

[26] Ishii O, Shin M, Sueda T, Vacanti JP. In vitro tissue engineering of a cardiac graftusing a degradable scaffold with an extracellular matrix-like topography. JThorac Cardiovasc Surg 2005;130:1358–63.

[27] Karikkineth BC, Zimmermann WH. Myocardial tissue engineering and heartmuscle repair. Curr Pharm Biotechnol 2013;14:4–11.

[28] Yang F, Cho S-W, Son SM, Hudson SP, Bogatyrev S, Keung L, et al. Combinatorialextracellular matrices for human embryonic stem cell differentiation in 3D.Biomacromolecules 2010;11:1909–14.

[29] Tate MC, Shear DA, Hoffman SW, Stein DG, Archer DR, LaPlaca MC. Fibronectinpromotes survival and migration of primary neural stem cells transplantedinto the traumatically injured mouse brain. Cell Transplant 2002;11:283–95.

[30] Van Dijk A, Niessen H, Ursem W, Twisk J, Visser F, van Milligen F.Accumulation of fibronectin in the heart after myocardial infarction: aputative stimulator of adhesion and proliferation of adipose-derived stemcells. Cell Tissue Res 2008;332:289–98.

[31] Wijelath ES, Rahman S, Murray J, Patel Y, Savidge G, Sobel M. Fibronectinpromotes VEGF-induced CD34 cell differentiation into endothelial cells. J VascSurg 2004;39:655–60.

[32] Seo Y, Yang S-R, Jee MK, Joo EK, Roh K-H, Seo M-S, et al. Human umbilical cordblood-derived mesenchymal stem cells protect against neuronal cell death andameliorate motor deficits in Niemann Pick type C1 mice. Cell Transplant2011;20:1033–47.

[33] Park S-B, Yu K-R, Jung J-W, Lee S-R, Roh K-H, Seo M-S, et al. BFGF enhances theIGFs-mediated pluripotent and differentiation potentials in multipotent stemcells. Growth Factors 2009;27:425–37.

[34] Kim M-J, Lee B, Yang K, Park J, Jeon S, Um SH, et al. BMP-2 peptide-functionalized nanopatterned substrates for enhanced osteogenicdifferentiation of human mesenchymal stem cells. Biomaterials2013;34:7236–46.

[35] Han TH, Lee K, Park JB, Ahn D, Park J-H, Kim D-Y, et al. Reduction in synapticGABA release contributes to target-selective elevation of PVN neuronal activityin rats with myocardial infarction. Am J Physiol Regul Integr Comp Physiol2010;299:R129–39.

[36] Matthew B. Injected versus oral cyclosporine for human neural progenitorgrafting in rats. J Stem Cell Res Ther 2012. doi: 10.4172/2157-7633.S10-003.Available from: http://www.omicsonline.org/2157-7633/2157-7633-S10-003.pdf.

[37] Lam CX, Savalani MM, Teoh SH, Hutmacher DW. Dynamics of in vitro polymerdegradation of polycaprolactone-based scaffolds: accelerated versussimulated physiological conditions. Biomed Mater 2008;3:034108.

[38] Christman KL, Lee RJ. Biomaterials for the treatment of myocardial infarction. JAm Coll Cardiol 2006;48:907–13.

[39] Wei H-J, Chen C-H, Lee W-Y, Chiu I, Hwang S-M, Lin W-W, et al. Bioengineeredcardiac patch constructed from multilayered mesenchymal stem cells formyocardial repair. Biomaterials 2008;29:3547–56.

[40] Fedak PW. Paracrine effects of cell transplantation: modifying ventricularremodeling in the failing heart. Semin Thorac Cardiovasc Surg 2008;20:87–93[Summer].

[41] Philp D, Chen SS, Fitzgerald W, Orenstein J, Margolis L, Kleinman HK. Complexextracellular matrices promote tissue-specific stem cell differentiation. StemCells 2005;23:288–96.

[42] Pimton P, Sarkar S, Sheth N, Perets A, Marcinkiewicz C, Lazarovici P, et al.Fibronectin-mediated upregulation of a5b1 integrin and cell adhesion duringdifferentiation of mouse embryonic stem cells. Cell Adh Migr 2011;5:73–82.