Nanoengineering gold particle composite fibers for cardiac tissue engineering

Journal ofMaterials Chemistry B

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View Article OnlineView Journal

aThe Laboratory for Tissue Engineering an

Molecular Microbiology and Biotechnology

Tel Aviv University, Tel Aviv 69978, Israel. EbCenter for Nanoscience and Nanotechnolo

IsraelcSchool of Chemistry, Raymond and Beverly

University, Tel Aviv 69978, Israel

† Electronic supplementary informa10.1039/c3tb20584c

‡ Equal contribution.

Cite this: DOI: 10.1039/c3tb20584c

Received 23rd April 2013Accepted 10th June 2013

DOI: 10.1039/c3tb20584c

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

Nanoengineering gold particle composite fibers forcardiac tissue engineering†

Michal Shevach,‡ab Ben M. Maoz,‡bc Ron Feiner,a Assaf Shapiraa and Tal Dvir*ab

Gold nanostructures can be incorporated into macroporous scaffolds to increase the matrix conductivity

and enhance the electrical signal transfer between cardiac cells. Here we report a simple approach for

fabricating 3-dimensional (3D) gold nanoparticle (NP)-based fibrous scaffolds, for engineering functional

cardiac tissues generating a strong contraction force. A polycaprolactone–gelatin mixture was

electrospun to obtain fibrous scaffolds with an average fiber diameter of 250 nm. In a facile method,

gold NPs were evaporated on the surface of the fibers, creating nanocomposites with a nominal gold

thickness of 2, 4, and 14 nm. Compared to pristine scaffolds, cardiac cells seeded on the nano-gold

scaffolds assembled into more elongated and aligned tissues. The gold NPs on the fibers were able to

maintain the ratio of cardiomyocytes to fibroblasts in the culture, to encourage the growth of

cardiomyocytes with significantly higher aspect ratio, and promote massive cardiac sarcomeric actinin

expression. Finally, engineering cardiac tissues within gold NP-based scaffolds exhibited significantly

higher contraction amplitudes and rates, as compared to scaffolds without gold. We envision that

cardiac tissues engineered within these gold NP scaffolds can be used to improve the function of the

infarcted heart.

Introduction

Transplantation of engineered cardiac patches is a promisingstrategy for improving the function of diseased hearts.1–4 In thisapproach cardiac cells are seeded within 3-dimensional (3D)biomaterial scaffolds to induce quick assembly into a func-tioning cardiac tissue. Then, these cardiac patches are trans-planted to replace the injured tissue and regain function.5 Inrecent years engineering strategies were focused on inducingcardiac specic morphogenesis, maintaining cell viability, andpromoting proper function of the engineered tissue.6–9 Thus,bioreactors providing enhanced mass transfer increased cellsurvival and electrical and mechanical signals were able topromote the formation of a typical cardiac ultrastructuralmorphology.10–13 In an attempt to induce cell–matrix interac-tions within the engineered 3D microenvironments, the surfaceof scaffolds was chemically modied with ECM proteins or withtheir functional motifs.14 Such modication has led to the

d Regenerative Medicine, Department of

, George S. Wise Faculty of Life Science,

-mail: [email protected]

gy, Tel Aviv University, Tel Aviv 69978,

Sackler Faculty of Exact Sciences, Tel Aviv

tion (ESI) available. See DOI:

Chemistry 2013

formation of elongated and aligned cells, resembling themorphology of cells of the natural myocardium.15 Micro andnanofabrication processes were developed to create 2-dimen-sional (2D) substrates with various topographies for inducingcell adhesion, and for controlling cardiac cell assembly andmorphogenesis.16–19

Recently it has been suggested that incorporation of inor-ganic structures, such as nanoparticles (NPs), nanowires ornanorods, into tissue engineering scaffolds may play a key rolein affecting the mechanical and adhesive properties of thematerial, inducing tissue morphogenesis and directing cell self-assembly in 3D.5,20–22 Gold nanostructures are consideredpromising candidates in biology, including tissue engineering20

and biosensing,23–26 as they are biocompatible,27 inert to thecells,20 and show localized surface plasmon resonance (LSPR) inthe visible range. More importantly, the electrical properties ofgold nanostructures were utilized to improve electricalcommunication between adjacent cardiac cells.20,28 In a recentstudy gold nanowires were embedded within the pore walls ofmacroporous alginate scaffolds to increase the spatial andoverall conductivity of the matrix.20 When cardiac cells werecultured in these nanowired scaffolds they exhibited increasedexpression of contractile and electrical coupling proteins. Thesenanowired matrices improved the propagation of the electricalsignal within the biomaterial, resulting in a synchronouslybeating cardiac patch.20 In a different study, cardiomyocyteswere cultured in hybrid hydrogel scaffolds based on sphericalgold NPs, homogeneously distributed throughout a polymeric

J. Mater. Chem. B

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Journal of Materials Chemistry B Paper

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gel. The cells exhibited increased expression of connexin 43, aprotein located between cardiac cells, responsible for electricalsignal transfer.28

Overall, the incorporation of different nanoscale structures ofgold into biomaterials improved the engineered cardiac tissuestructure and function. However, the scaffolds used in thesestudies did not mimic the brous structure of the natural micro-environment, which induces a proper organization of the cells.

To recapitulate the natural ECM topography and 3D struc-ture, electrospinning was used to fabricate brous scaffoldsfrom various polymers.29 In this inexpensive and facile method,an electric eld is used to deposit high aspect ratio bers,ranging in diameter from tens of microns to tens of nanome-ters, on a target substrate.30 These bers resemble themorphology of the native ECM and ber-based scaffolds can bemanipulated to provide signals for the assembly of elongatedand aligned cardiac tissues.31

In this study, we introduced a facile and inexpensiveapproach to incorporate gold NPs on the surface of nanoscaleelectrospun bers. The nanocomposites were produced byevaporating a thin layer of gold on polycaprolactone (PCL)–gelatin bers. The thickness of the evaporated gold lm on thebers allowed control of the island size.32,33 Here, we rstinvestigated the effect of the gold NP nominal thickness on theoptical and structural properties of the biomaterial. Next, theeffect of the different nanocomposites on cardiac tissueassembly and function was examined. Overall, the obtaineddata suggested that the incorporation of gold NPs onto thesurface of bers comprising the scaffolds was benecial forimproving the assembly of a functional cardiac tissue.

Materials and methodsElectrospinning

Electrospun matrices were fabricated as previously described.34

Briey, 10% gelatin (Sigma, St. Louis, MO) and 10% PCL (Sigma,St. Louis, MO) were separately dissolved in 2,2,2-triuoroethanol(Acros, Belgium) overnight at room temperature. The next day,the solutions were mixed in the ratio of 1 : 1. Using a syringepump (Harvard apparatus, Holliston, MA), the polymer solutionwas delivered through a stainless steel 20G capillary at a feedingrate of 0.5 mL h�1. A high voltage power supply (Glassman highvoltage inc.) was used to apply a 10 kV potential between thecapillary tip and the collector, positioned 10 cm beneath the tip.

Gold NP preparation

Aluminum foils covered with electrospun bers were mountedin a VST e-beam evaporator. Homogeneous deposition wasobtained by moderate rotation of the substrate plate. Au lms(2.0 to 14 nm thick) were prepared by evaporation of Au(99.999%) from a tungsten boat at 1–3 � 10�6 torr at a deposi-tion rate of 0.5 A s�1.

Reection measurements

Due to the thickness of the scaffolds and therefore their limitedtransparency we chose to detect LSPR by reectance and not

J. Mater. Chem. B

absorbance. Reectionmeasurements were carried out using anOcean Optics reection probe model R00-7 VIS/NIR and quartztungsten halogen (QTH) lamp. The data were collected using anOcean Optics Red Tide 650 diode-array spectrophotometer(spectral range: 380–900 nm) at an angle of incidence of 90�.The reection probe was centrally placed ca. 5 mm from the topedge of the slide, at a vertical distance of 8 mm from the planarsubstrate. The measured spot was ca. 4 mm in diameter.

Scanning electron microscopy

A Quanta 200 FEG Environmental Scanning Electron Micro-scope (ESEM) with a eld-emission gun (FEG) electron sourcewas used. Imaging was carried out under low vacuum with ahigh tension of 20 kV and a working distance of 7.3 mm.

High resolution transmission electron microscopy

Cross-sections were prepared by embedding the polymer inepoxy glue followed by polishing to obtain the desired size. Allsamples were deposited on carbon coated copper grids (SPI) fortransmission electron microscopy (TEM) analysis. Images wererecorded using a Philips FEI Tecnai F20 FEG-TEM.

Cardiac cell isolation, seeding and cultivation

The procedure for cell isolation employed in this study wasapproved by the Animal Care and Use Committee of Tel AvivUniversity in Israel (L-11-053). Neonatal ventricle myocytes (1- to3-day-old Sprague-Dawley rats) were harvested and cells wereisolated as described previously.12 Briey, isolated ventricleswere cut into �1 mm3 pieces and incubated repeatedly (6–7times) in buffer containing collagenase type II (95 U mL�1;Worthington, Lakewood, NJ) and pancreatin (0.6 mg mL�1;Sigma, St. Louis, MO). Aer each digestion round, the mixturewas centrifuged (600g, 5 min, 25 �C) and the cell pellet was re-suspended in cold M-199 medium (Biological Industries, BeitHaemek, Israel) supplemented with 0.5% (v/v) fetal calf serum(FCS, Biological Industries). The pooled cells were centrifugedand resuspended twice in culture medium (M-199 mediumsupplemented with 5% FCS, 0.6 mM copper sulphate penta-hydrate, 0.5 mM zinc sulphate heptahydrate, 500 U mL�1

penicillin, and 100 mg mL�1 streptomycin (Biological Indus-tries)). Aer two rounds of preplating for 40 min, the cells werecounted and seeded on the scaffolds using a single droplet. Thecell-seeded constructs were cultivated at 37 �C in a 5% carbondioxide humidied incubator.

Immunostaining

The cellular constructs were xed and permeabilized in coldmethanol and blocked for 8 minutes at room temperature inSuper Block (ScyTek laboratories, US). Aer three PBST washes,the samples were incubated with primary mouse monoclonalanti a-actinin (1 : 750, Sigma). Aer incubation, the sampleswere washed and incubated for 1 h with goat anti-mouse AlexaFluor 647 (1 : 500, Jackson, West Grove, PA). For nuclei detec-tion, the cells were incubated for 5 min with 5 mg mL�1 Hoechst33258 (Sigma).

This journal is ª The Royal Society of Chemistry 2013


Fig. 1 Schematic overview of the study. (A) Fabrication of PCL–gelatin fiber scaffolds by electrospinning. (B) Evaporation of gold NPs onto the fibers to createnanocomposite scaffolds. (C) Cardiac cells are seeded in the nanocomposite scaffolds for engineering a functional cardiac tissue.

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Analyses of tissue function

Contraction of the cardiac cell constructs was recorded using aninverted microscope (Nikon Eclipse TI) and NIS element so-ware. The contraction amplitude was analyzed using ImageJsoware (NIH). The contraction rate was manually counted.

Statistical analysis

Statistical analysis data are presented as means � SEM.Univariate differences between the pristine scaffold and goldNP scaffolds were assessed with Student's t-test. All analyseswere performed using GraphPad Prism version 5.00 forWindows (GraphPad Soware). p < 0.05 was consideredsignicant.

Fig. 2 Gold NP fiber scaffolds. (A) Macroscopic pictures of the scaffolds withvarying nominal thickness of gold. Upper left and clockwise: no gold, 2 nm, 4 nm,and 14 nm. (B) eSEM image of the 4 nm scaffold. Gold NPs were evaporated onthe lower left part (bright part) while the upper right part is without gold. Asshown, the morphology of the fibers was not affected due to the introduction ofgold. Bar ¼ 20 mm. (C–F) Fiber morphology by a light microscope. (C) No gold. (D)2 nm. (E) 4 nm. (F) 14 nm. Bar ¼ 5 mm.

Results and discussion

Electrical coupling between cardiomyocytes is essential forengineering functional cardiac tissues.35 Therefore, our goal inthis study was to engineer a cellular microenvironment thatencourages coupling of cardiac cells and the formation of acontracting tissue.

In the myocardium, cardiac cells, including cardiomyocytesand broblasts, are arranged in-between an intricate network ofprotein bers, such as brillar collagens, ranging from 10 toseveral hundreds of nanometers.5 The mesh is covered withnanoscale proteins that provide specic binding sites for celladhesion and cues for proper cell assembly. Here, we rstfocused on recapitulating the morphology of the native matrix.Fibrous scaffolds with an average ber diameter of 250 nm werefabricated by electrospinning of PCL and gelatin. The PCLimproved the mechanical strength of the scaffold,29 while thebio-inductive gelatin contributed to its biological activity,promoting cell adhesion.36,37

Recently it was shown that gold nanowires were used tointeract with cardiomyocytes and to increase electrical signaltransfer between the cells.20 Therefore, we sought to exploit thisphysiological phenomenon and incorporate gold NPs onto thesurface of the bers. We hypothesized that by using thisapproach the NPs can serve as couplers between cells andpromote functional assembly of a cardiac tissue. As a facileapproach for gold incorporation into the bers, the functional


groups in the gelatin were utilized as binding sites for theevaporated gold NPs (Fig. 1). By changing evaporation condi-tions we were able to fabricate bers with various nominal goldthicknesses, creating scaffolds with typical pink to marooncolors (Fig. 2A). As judged by SEM and optical microscopyimages, the process did not change the brous morphology ofthe scaffolds (Fig. 2B–F). As shown in Fig. 3, the NPs weredeposited homogeneously on the bers creating polymer–goldnanocomposites. Furthermore, the samples were characterizedby TEM and AFM to evaluate the coverage by the particles and

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the composite topography (Fig. 4). As shown, the NPs weredistributed on the bers without affecting their brousmorphology (Fig. 4D). The scaffolds were washed several timesand treated with adhesive tape to ensure strong gold NPbinding.

Due to the formation of small gold NPs, LSPR could beobserved (Fig. 5A). Once the correlation between the reectanceand NP size was found, a simple spectrometer may be used toidentify the size of the NPs. Since the NPs are located on thesurface of the bers (Fig. 3) the LSPR could be used to observechanges in the surrounding medium, and in theory also fordetecting cell adhesion or enhanced secretion of biomoleculessuch as proteins and hormones.25,26 For example, the system

Fig. 3 eSEM micrographs of the scaffolds. Upper and lower panels are low and higNominal thickness of 4 nm. (C) Nominal thickness of 14 nm. Bars: upper panels left

Fig. 4 NPs on the fibers. (A) Illustration of a fiber cross-section. (B) Cross-sectional TEcovered with gold NPs. (D) Topography of gold NP fibers by AFM. (E) Topography o

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could be modied to detect troponins in the engineered tissuemicroenvironment, indicating cardiomyocyte stress.38 More-over, LSPR is known to enhance some optical activities such asRaman (SERS), circular dichroism and uorescence, and thuscan be used to measure these effects at very low moleculeconcentrations.25,26,39,40 As shown, cell seeding on the goldscaffolds (4 nm) resulted in a shi in the maximum reectanceof the LSPR (Fig. 5B), indicating that the nanocomposite scaf-folds can be used to evaluate the existence of cells on thematrix,seeding efficiency and cell viability.

To investigate the effect of gold NPs in the scaffolds on celland tissue assembly, we engineered cardiac cell constructs.Cardiac cells isolated from neonatal rat hearts were seeded by a

h magnifications, respectively. (A) Scaffolds with a nominal thickness of 2 nm. (B)to right ¼ 4 mm, 6 mm, 5 mm. Lower panels ¼ 300 nm.

M of the fibers coated with gold NPs. Bar¼ 100 nm. (C) Illustration of a single fiberf gold NPs (4 nm) on a single fiber.



Fig. 5 Reflectance measurements. (A) An LSPR peak can be seen in the 2 nm and4 nm samples. The 14 nm sample has a broad peak as expected. (B) A plasmonshift due to the cells located on the gold scaffolds.

Fig. 6 Cardiac cell organization on the scaffolds. Cardiac sarcomeric actininimmunostaining on day 7. (A) Cardiac cells cultured on pristine scaffolds withoutgold mostly exhibited a rounded morphology. (B) Cardiac tissue engineered in2 nm gold NP scaffolds. (C) Cardiac tissue engineered in 4 nm gold NP scaffolds.(D) Cardiac tissue engineered in 14 nm gold NP scaffolds. In the 14 nm goldscaffolds the cells exhibited an elongated and aligned morphology. Bar¼ 100 mm(left), 20 mm (right). Actinin – pink, nuclei – blue.

Fig. 7 Cell characteristics in the scaffolds. (A) Cell aspect ratio. (B) The ratio ofcardiomyocytes to cardiac fibroblasts in different scaffolds. * indicates p ¼ 0.003,** indicates p ¼ 0.0004.


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single droplet on the different scaffolds. To evaluate cardiactissue assembly, cell constructs were immunostained forcardiac sarcomeric actinin, a protein responsible for cardiac cellcontraction. On day 7, cardiac cells cultured on pristine PCL–gelatin scaffolds without gold NPs mostly exhibited a roundedmorphology (Fig. 6A), while cells cultured on gold NP scaffoldswere elongated with massive striation, suggesting strongcontraction potential (Fig. 6B–D). Furthermore, cells cultivatedon the 14 nm gold NP scaffolds were aligned to each other,exhibiting morphology resembling that of native heart bundles(Fig. 6D).41 Analysis of cell elongation revealed a signicantlyhigher aspect ratio (p < 0.0001) in cells grown on the gold NPbers (Fig. 7A). This phenomenon indicates that the gold NPslocated on the surface of the bers encouraged the assembly ofan anisotropic tissue with typical cardiac cell morphology.42

Matrix stiffness and nanostructure morphology are twoparameters that have been shown to affect cellular behavior.43

Recently it was reported that the matrix stiffness affected DNAcontent, cell proliferation, cell apoptosis, and spreading.44 Inthe context of cardiac tissues, Marsano and colleagues haveshown that changes in mechanical properties affect thecontractile properties of the engineered cardiac tissue.45 Morerecently it was shown that incorporation of NPs into electrospunbers altered their elasticity.22 Since cardiomyocytes, the con-tracting cells of the heart, are terminally differentiated cells, it isessential in cardiac tissue engineering to maintain their ratiocompared to non-contracting cells in the culture (i.e. bro-blasts). Otherwise, the broblasts can take over the culture,shiing it towards a non-contracting culture. In our approach,the ability to control the nominal thickness of the NPs and theirhomogeneous distribution on the bers may allow a facileapproach for tuning the mechanical properties of the matrixaccording to our needs. For example, the matrix stiffness can beoptimized to obtain a scaffold that on one hand may attenuatebroblast proliferation in co-culture and, on the other hand,may enhance cardiomyocyte contractility performances. Thus,we analyzed the ratio of cardiomyocytes to cardiac broblastson different scaffolds, and evaluated the potential of the NPs toattenuate the proliferation of broblasts. Fig. 7B reveals asignicantly higher ratio of myocytes to broblasts in culturesgrown on gold NP bers, suggesting higher potential to main-tain the contractility of the engineered tissue.

Finally, to evaluate the contribution of gold NPs for engi-neering functional cardiac tissues, we investigated the perfor-mances of the engineered tissues. Since a strong contraction of


an engineered tissue is essential for creating an effective heartpatch,35 contraction amplitudes of the cell constructs wereevaluated by image analysis. While on day 3, no contraction was

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Fig. 8 Engineered cardiac tissue function. (A) Contraction amplitude on day 3.(B) Contraction rate on day 3. (C) Contraction amplitude on day 7. (D) Contractionrate on day 7. n $ 5 in each group.


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observed within tissues engineered without gold NPs, highcontraction amplitudes were observed in all gold NP cellconstructs (Fig. 8 and ESI 1–4†). Tissues that were engineeredwithin 4 and 14 nm gold NP constructs revealed signicantlyhigher amplitudes, compared to tissues engineered withinpristine scaffolds (Fig. 8A). Furthermore, on day 3, these engi-neered tissues exhibited signicantly higher contraction rates,as compared to the ones engineered without gold NPs (Fig. 8B).On day 7, contraction was observed within tissues engineered inthe pristine scaffolds as well. However, their contractionamplitude and rate were signicantly lower than the gold NPtissues (Fig. 8C and D and ESI 5–8†). The weak contraction forcemay be attributed to the rounded morphology of the cells andthe lack of tissue anisotropy. Recently it was shown that goldnanowires can transfer the electrical signal between adjacentcardiac cells.20 This unique transfer phenomenon is importantsince post-isolation and seeding, cardiac cells lose their elon-gated morphology and acquire an immature roundedmorphology.21 At that point, connexin 43 proteins, gap junctionmolecules responsible for transferring the electrical signalbetween cells, are internalized and the cells are required toestablish new couplings. We believe that the NPs in thesescaffolds were used to quickly promote cell–cell interactions,and thus improve the transfer of the electrical signal, enhancingthe overall contractility of the engineered tissue.

Conclusions and future prospects

In this article we report the incorporation of gold NPs onto thesurface of brous scaffolds for engineering functional cardiactissues. The facile and inexpensive technique we used for goldNP incorporation relied on immediate conjugation of the NPs tothe functional groups of the protein comprising the bers. GoldNP ber scaffolds induced quick formation of elongated andaligned tissues with a morphology resembling that of cardiac

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cell bundles in vivo. Furthermore, we have shown that thisstructure has led to improved function, resulting in engineeredtissues capable of generating a strong contraction force.

Future studies should focus on optimizing the space neededbetween the incorporated NPs, and investigating the mecha-nism by which cardiomyocytes interact with gold NPs. In vivoexperiments should be performed to investigate the potential ofthe gold NP cardiac patches to improve the function of theinfarcted heart.

Another interesting study would be to evaluate the potentialof the LSPR to serve as a sensor for the state of the cultivatedcells. For example, antibodies specic for a certain molecule,known to be secreted by cells at stress could be conjugated tothe gold NPs. Post-secretion from stressed cells, these mole-cules may bind to the antibodies, triggering a shi in the typicalplasmon resonance.46

Acknowledgements

T.D. acknowledges support from the European Union FP7program, Alon Fellowship, and the Nicholas and ElizabethSlezak Super Center for Cardiac Research and BiomedicalEngineering at Tel Aviv University. M.S. thanks the MarianGertner Institute for Medical Nanosystems Fellowship. B.M.M.was supported by The Tel Aviv University Center for Nano-science and Nanotechnology. The work is part of the doctoralthesis of M.S. at Tel-Aviv University. We would like to thankProf. Gil Markovich and Dr. Tzahi Cohen-Karni for their usefulcomments.

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Nanoengineering gold particle composite fibers for cardiac tissue engineering

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