optimizing a spontaneously contracting heart tissue patch with rat neonatal cardiac cells on fibrin...

Optimizing a spontaneously contracting heart tissuepatch with rat neonatal cardiac cells on fibrin gelZe-Wei Tao, Mohamed Mohamed, Matthew Hogan, Laura Gutierrez and Ravi K. Birla*Department of Biomedical Engineering, Cullen College of Engineering, University of Houston, TX, USA

Abstract

Engineered cardiac tissues have been constructed with primary or stem cell-derived cardiac cells onnatural or synthetic scaffolds. They represent a tremendous potential for the treatment of injured areasthrough the addition of tensional support and delivery of sufficient cells. In this study, 1–6 million (M)neonatal cardiac cells were seeded on fibrin gels to fabricate cardiac tissue patches, and the effects ofculture time and cell density on spontaneous contraction rates, twitch forces and paced response frequen-cies were measured. Electrocardiograms and signal volume index of connexin 43 were also analysed.Patches of 1–6 M cell densities exhibited maximal contraction rates in the range 305–410 beats/min(bpm)within thefirst 4 days after plating; lowcell density (1–3M) patches sustained rhythmic contractionlonger than high cell density patches (4–6 M). Patches with 1–6 M cell densities generated contractileforces in the range 2.245–14.065 mN/mm3 on days 4–6. Upon patch formation, a paced responsefrequency of approximately 6 Hz was obtained, and decreased to approximately 3 Hz after 6 daysof culture. High cell density patches contained a thicker real cardiac tissue layer, which generated higherR-wave amplitudes; however, low-density patches had a greater signal volume index of connexin 43. Inaddition, all patches manifested endothelial cell growth and robust nuclear division. The present studydemonstrates that the proper time for in vivo implantation of this cardiac construct is just at patch forma-tion, andpatcheswith 3–4Mcell densities are the best candidates. Copyright©2014 JohnWiley&Sons, Ltd.

Received 7 May 2013; Revised 17 February 2014; Accepted 10 March 2014

Additional supporting information may be found in the online version of this article at the publisher’s web site.

Keywords tissue engineering; fibrin; cardiomyocytes; cell culture; heart muscle; cardiac constructs

1. Introduction

Engineered cardiac tissues, constructed with primary orstem cell-derived cardiac cells on a natural or syntheticscaffold, have tremendous potential to offer alternativetreatment modalities in the healing process of largeinjured areas in hearts. By embedding a sufficient numberof cells in the tissue and providing additional tensionsupport to the damaged area, they circumvent the lowrates of cell engraftment and poor cell survival that haveoccurred in present cell therapies (Sekine et al., 2011;Wei et al., 2008; Wollert and Drexler, 2010). Cardiactissue constructs have been fabricated by embeddingneonatal cardiac cells (Fujimoto et al., 2011; Kensah

et al., 2011; Zimmermann et al., 2006) with collagen type Isupplemented with Matrigel, and by embeddingpluripotent stem cell-derived cardiomyocytes withcollagen type I gel (Guo et al., 2006; Tulloch et al.,2011), poly-L-lactic acid and polylactic–glycolic acid gel(Caspi et al., 2007), omentum flap (Kawamura et al.,2013) and fibrin gel (Liau et al., 2011). Some in vivo stud-ies showed that these cardiac constructs improved cell sur-vival (Kawamura et al., 2013; Sekine et al., 2011), vascularnetwork formation (Stevens et al., 2009) and cardiac func-tion (Zimmermann et al., 2006). The twitch forces andphysical properties of available cardiac tissue constructsvaried with scaffold materials, embedded cell densitiesand culture conditions. However, with the same scaffoldmaterial, cell type and culture condition, physical charac-teristics, such as changes of spontaneous contraction rates,twitch forces and electrical pace response by culture, havenot yet been fully revealed.

*Correspondence to: Ravi K. Birla, Department of BiomedicalEngineering, Cullen College of Engineering, University ofHouston, 3605 Cullen Boulevard, Room 2005, Houston, TX77204, USA. E-mail: [email protected]

Copyright © 2014 John Wiley & Sons, Ltd.

JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE RESEARCH ARTICLEJ Tissue Eng Regen Med (2014)Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.1895

Neonatal cardiac cells possess a tremendous differenti-ation potential and regenerative capacity. The hearts of1 day-old neonatal mice can regenerate after a partialsurgical resection (Porrello et al., 2011). Fibrin is anatural, self-assembling peptide found in the body thatis used to form clots along damaged endothelium, andfibrin gels possess high seeding efficiency, uniform celldistribution and adhesion capabilities (Guyette et al.,2013; Swartz et al., 2005). They are formed by thereaction of thrombin and fibrinogen, which can beproduced from the patient’s own blood, thus reducingthe potential risk of rejection when used as a componentin clinical application. We previously described a modelfor the self-organization of primary cardiac cells on alaminin substrate to form a cardioid (Baar et al., 2005),which exhibited physiological performance metricscomparable to normal mammalian cardiac tissue. Thenan engineered heart muscle was developed by seedingor embedding 2–3 day-old neonatal rat cardiac cells onthe surface of, and within, a fibrin gel (Huang et al.,2007). In the present study, we fabricated a cardiac tissuepatch by seeding neonatal rat heart cells on fibrin gel,optimized natural spontaneous contraction by evaluatingcell densities and length of culture, and determined thechanges of twitch force and electrical pace response. Thephysical characteristics of cardiac tissue patches will beapplied and the optimized patch will be used for in vivografting in future studies.

2. Materials and methods

All animal protocols were approved by the Institutional Ani-mal Care and Use Committee (IACUC) at the University ofHouston, in accordance with the Guide for the Care and Useof Laboratory Animals (NIH Publication No. 86–23, 1996).All materials were purchased from Sigma-Aldrich (St. Louis,MO, USA) unless otherwise specified.

2.1. Isolation of primary cardiac cells

Cardiac cells were isolated from the hearts of 2–3 day-oldneonatal Sprague–Dawley rat pups, using an establishedmethod (Huang et al., 2007). Each heart was cut intothree or four pieces in an ice-cold phosphate bufferconsisting of 116 mM NaCl, 20 mM HEPES, 1 mM

Na2HPO4, 5.5 mM glucose, 5.4 mM KCl and 0.8 mM

MgSO4. After the blood cells had been rinsed out, theheart pieces were transferred to a dissociation solutionconsisting of 0.32 mg/ml collagenase type 2-filtered(Worthington Biochemical Corporation, Lakewood, NJ,USA) and 0.6 mg/ml pancreatin in phosphate buffer.The hearts were cut into 1 mm3 pieces and thentransferred to an orbital shaker and maintained at 37°Cfor 30 min at 60 rpm. At the end of the digestion process,the supernatant was collected in 3 ml horse serum toneutralize the enzyme and centrifuged at 1000 rpm for

5 min at 4°C. The cell pellet was resuspended in 5 mlhorse serum and kept in an incubator at 37°C, suppliedwith 5% CO2. Fresh dissociation solution was added tothe partially-digested tissue and the digestion processwas repeated a further two or three times. Cells from allthe digests were pooled, centrifuged and suspendedin culturemedium (CM) consisting of M199 (Life Technolo-gies, Grand Island, NY, USA) with 20% F12k (Life Technol-ogies), 10% fetal bovine serum, 5% horse serum, 1%antibiotic–antimycotic, 40 ng/ml hydrocortisone and 100ng/ml insulin. Cell viability was analysed by Trypan blue(4%) staining, according to the manufacturer’s protocol.

2.2. Fabrication of artificial cardiac patch

The method of fabricating the cardiac patch is shown inFigure 1A–H. A 35 mm tissue culture plate was coatedwith 2 ml SYLGARD (PDMS, type 184 silicone elastomer;Dow Chemical, Midland, MI, USA). The plate was air-dried for 2 weeks and sterilized with 80% ethanol beforeuse. Four minutien pins (Fine Science Tools, Foster City,CA, USA), 0.1 mm diameter, were placed in the cultureplate to form a 2×2 cm square. The fibrin gel was madeby plating 1 ml CM containing 10 U/ml thrombin, adding500 μl saline containing 20 mg/ml fibrinogen and mixingwell to promote gel formation within 10 min. Primarycardiac cells were diluted in CM at a pre-set density, and2 ml of the cell suspension was transferred to the cultureplate. Aminocaproic acid (2 mg/ml) was added to theculture plate to inhibit fibrinolysis by endogenousproteases. The cells were cultured in an incubator at 37°Cand 5% CO2 with CM changes every other day.

2.3. Spontaneously beating cardiomyocytes,patch formation and contraction rate

Sixteen hours after cell plating, spontaneously beatingcardiomyocyteswere countedmanually from five×200 fields(centre and top, right, left and bottom-most from the centreof the patch) under an inverted phase-contrast microscope(Olympus, Centre Valley, PA, USA). The total beating cellsfromone patch of one densitywere averaged. The contractionof cultured cardiac constructs and fibrin gel detachment fromculture plates were observed from day 1 to day 6; the patchgrowth progress was captured in still photographs andvideos, using a camera (Lumenera, Ottawa, ON) mountedon an inverted phase-contrast microscope. The videos werereplayed slowly and the contraction rates manually counted.

2.4. Contractile twitch force andelectrocardiogram (ECG)

From the first day of patch formation, spontaneous and elec-trical paced twitch force (10 V, 0.1 s) were measured withina thermostatted (37°C) water bath using a high-sensitivityisometric force transducer (MLT0202, ADInstruments,

Z.-W. Tao et al.

Copyright © 2014 John Wiley & Sons, Ltd. J Tissue Eng Regen Med (2014)DOI: 10.1002/term

Colorado Springs, CO, USA) connected to a quad bridgeamplifier (FE224, ADInstruments). ECG signal wasmeasured using Octal Bio Amp (ML138, ADInstruments)(Figure 1I, J). Data were acquired through a 16-channelPowerLab system (PL3516/P, ADInstruments). As shownin Figure 1I, J, the contractile twitch force was measuredby attaching the force transducer arm to one free-cornerof the square patch, while the other three ends of the squarepatch were held fixed by pins. In order to obtain the Frank–Starling relationship for themeasured twitch force, pre-ten-sion was adjusted using a micro-manipulator (Radnoti LLC,Monrovia, CA, USA) and measurements of spontaneouscontraction were recorded. We defined a spontaneous con-traction rate of> 120 bpm as high-rate contraction and aspontaneous contraction rate of< 20 bpm as low-rate con-traction. The ECG of the patch was measured, as shown inFigure 1I, by inserting a needle cathode (MLA1213,ADInstruments) into the centre of the patch and a needleanode in one of the four patch corners. The mediumimmersing the patch was used as ground. LabChart

(ADInstruments) was used for data analysis. The peak anal-ysis module was used to calculate the maximal twitch forceand baseline force (pre-tension). The ECG analysis modulewas used to calculate the R-wave amplitude. Electrical pac-ing was performed by attaching the stimulating electrodes(MLA0320, ADInstruments) on the edge of the patch tissueat frequencies of 0.25, 0.5 and 1–8 Hz (in 1 unitincrements) at 2, 4 and 6 days after patch formation.

2.5. Morphology

Seven days after plating, the formed patches were cutdiagonally. The diagonal edge of the triangular blockwas aligned with the edge of a slide. The cross-sectionand surface of the triangular block were photographedand the images were traced using ImageJ 1.47d (WayneRashand, National Institutes of Health, USA) to obtainthe area of the cross-section, the thickness and theheight of the triangular block, and then this triangular

Figure 1. Schematic methods for artificial cardiac tissue patch fabrication, and ECG and contractile twitch force measurements

Spontaneously contracting cardiac patch fabricated on fibrin gel


block was weighed. From the central part of the patch,two 0.5×0.5 cm blocks were taken, placed in a peel-awaydisposable embedding mold (VWR International, Radnor,PA, USA), frozen in liquid N2 and then immediately im-mersed in Tissue Tek OCT compound (VWR International,Radnor, PA, USA) and placed in a �80°C freezer. Once theOCT compound solidified, each sample was sliced using acryostat (Thermo Fisher Scientific, Waltham, MA, USA).Tissue cross-sections and planar sections were cut at a thick-ness of 10 or 6 μm. The sections were placed on VWR®Microslides for preparation of morphological and immuno-fluorescence examinations. Images from cross-sections of6 μm thickness were taken directly under a phase-contrastlight microscope (Olympus, Centre Valley, PA, USA). Formeasurement of the ‘real cardiac layer’ (a layer of cells andnaturally produced extracellular matrix forming on top ofthe fibrin gel scaffold) thickness, 10 μm thick cross-sectionswere stained with Masson’s trichrome, according to themanufacturer’s protocol, and images were taken under alight microscope. The distinct tissue layers were traced andthicknesses calculated using ImageJ.

2.6. Immunofluorescence

For observation of endothelial cell growth, nucleardivision and collagen type I distribution in the patch, freshtissue patches were directly fixed in ice-cold acetone for10 min; 1.0 × 1.0 cm tissue patch blocks from the centrewere trimmed, non-specific epitope antigens were blockedand the cell membranes permeated with 10% goat serum/0.5% Triton X-100 at room temperature for 45 min. Tissuepatch blocks were then incubated in mouse anti-α-actininantibody (1:200; Sigma, A7811), rabbit anti-collagen typeI (1:100; Abcam, ab34710), rabbit anti-von Willebrandfactor (vWF; 1:750; Abcam, ab6994), rabbit anti-ki 67(1:100; Abcam, ab66155) or rabbit anti-connexin 43(Cx43; 1:100) at room temperature for 2 h. Subsequently,tissue blocks were treated with goat anti-mouse and goatanti-rabbit secondary antibodies (1:400; AlexaFluor 488,546 and 633, Life Technologies) at room temperaturefor 1 h. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI; 2.5 μg/ml) for 5 min at roomtemperature. Fluorescent images were obtained using aNikon C2+ confocal laser scanning microscope (NikonInstruments, Melville, NY, USA). For measurement ofthe volume indices of gap junctions, collagen fibres andmyofibrils, signal volumes of Cx43, collagen type I andα-actinin expressions were examined within 6 μm cross-sections. Two Z-stack scans from each sample wereacquired, with a signal depth of 8 μm over 33 frames.After determining specific thresholds for Cx43, collagentype I and α-actinin, signal volumes for each sample weremeasured. The signal volume indices of Cx43 (or collagentype I) for different cell densities were expressed as:

Cx43 signal volume index ¼ Cx43 signal volume=

α� actinin signal volumeþ Cx43 signal volumeð Þand averaged for each sample.

2.7. Statistics

Results are presented as mean± standard deviation(SD). χ2 analysis was used to test frequency variables.Comparisons among groups were made with a one-wayanalysis of variance (ANOVA) followed by the Bonferronipost hoc comparison test. In all tests, differences wereconsidered statistically significant at p< 0.05.

3. Results

3.1. Patch formation

By the present established isolation method, cell viabilitywas 81.0±2.2% (n=16). The time required for patchformation was a function of the initial plating density.On day 4, formation was complete for 28.0% (n=7/25),56.3% (9/16) and 40.0% (6/15) of patches with 1, 3 and5 M densities, respectively (Pearson’s χ2 test, p=0.195).On day 6, formation was complete for 68.0% (17/25),87.5% (14/16) and 60.0% (9/15) of patches with 1, 3 and5 M densities, respectively (Fisher’s exact test, p=0.232).

3.2. Beating cells and contraction rate

After cell plating, live cardiac cells (Figure 2A, greenarrowheads) scatter and attach to fibrin gels with aspindle-shaped morphology; however, dead cardiac cells(Figure 2A, red arrowheads) congregate with a sphere-shaped morphology. Each beating cardiomyocyte(Figure 2A–C, white arrows) contracts at its own rate.The density of beating cells counted 16 h after platingwas found to correlate significantly with the total celldensity of the patches. There were 9.4±3.6 (n=7),16.9±2.8 (n=7) and 23.1±2.5 (n=8) beating cells infive ×200 fields in dishes with 1, 3 and 5 M densities,respectively (Figure 2D). Movie S1 (see supportinginformation) was taken from a 3 M density dish at 36 hafter plating; it shows uneven pacing from differentbeating cells which compete with each other.

On day 2 after plating, spontaneous tissue contractionswere observable under a microscope for 1–3 M densities.The highest contraction rates appeared on days 4, 3 and3, and they were 305±54 (n=7), 330±49 (n=8) and374±69 (n=9) bpm for 1, 2 and 3 M densities, respec-tively. For the 4–6 M densities, spontaneous contractionswere observable 1 day after plating. Their highestcontraction rates were attained on day 2; the highest con-traction rates for 4, 5 and 6 M densities were 365±52(n=11), 405±34 (n=11) and 410±33 (n=12) bpm,respectively. The highest contraction rates for 1, 3 and5 M patches are illustrated in Figure 2E. Compared with1 M density, the highest contraction rates in 3 and 5 Mdensities were significantly greater (p< 0.05 or p< 0.01).More-over, on day 6, the contraction rate in 1 M (243±71 bpm,n=7) was significantly greater than that in 3 M

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(32±23, n=9; p< 0.01) and 5M (43±78, n=11;p< 0.01) densities. Movie S2 (see supporting information)shows a representative contraction taken from a patch with6 M density on day 3 (contraction rate 448 bpm).

3.3. Contractile twitch force and ECG

High-rate (> 120 bpm) and low-rate (< 20 bpm) twitchforces were recorded from formed patches on days 4–6.For 1, 2 and 3 M patches, high-rate rhythmic contractionswere detected throughout the entire recording period(starting at the onset of pre-tension). Figure 3A showsrhythmic contraction from a 3 M patch and Figure 3Bdemonstrates its synchronous ECG. The largest twitchforce was recorded when the pre-tension was set in therange 1000–3000 μN. However, for 4, 5 and 6 M patches,high-rate arrhythmic contractions were detected for a fewseconds after pre-tension loads were applied, after whichlow-rate contractions were observed. Figure 4C, D showthe arrhythmic contraction and its synchronous ECG froma 5 M patch.

The high-rate contractile force recorded with a pre-tension range of 1000–3000 μN for 1–6 M densities variedfrom 550±120 μN (n=11) to 2806±916 μN (n=10),with the greatest high-rate contractile force at 4443 μNfrom a 6 M patch. The low-rate contractile force recordedwith a pre-tension range of 1000–3000 μN for 1–6 Mpatches varied from 970±132 μN (n=11) to3346±932 μN (n=10), with the greatest low-ratecontractile force at 4917 μN from a 6 M patch. Whencompared with 2 M density patches (1463±576 μN,n=11), the high-rate contractile force of 4 M(2602±638 μN, n=9) and 6 M (3346±932 μN,

n=10) patches were significantly greater (p< 0.01)(Figure 3G). Likewise, the low-rate contractile forces of2 M patches (1710±575 μN, n=10) were also signifi-cantly lower than those of 4 M (3086±804 μN, n=11)and 6 M (3346±932 μN, n=10) patches (p< 0.01;Figure 3H).

Electrocardiograms of the patches exhibited naturalQRS complex patterns, as evidenced by the represen-tative signals in 3 and 5 M patches in Figure 3B, D.Figure 3G shows that the R-wave amplitudes duringhigh-rate contraction of 4 M (24.4± 8.7 μV, n=10)and 6 M (28.1±6.4 μV, n=5) patches were greaterthan those from 2 M (8.9±8.1 μV, n=8) patches(p< 0.01).

3.4. Effect of culture time on contractiletwitch force

The effects of culture time on patch twitch force wereexamined for 4 M density patches at 0, 2, 4 and 6 daysafter formation. High- and low-rate twitch forcesdecreased during culture (Figure 4A, B). High-rate twitchforces on days 0, 2 and 4 were 2602±638 (n=9),1912±898 (n=7) and 1530±463 (n=5) μN,respectively. On day 6 high-rate twitch forces were notdetectable. Low-rate twitch forces on days 0, 2, 4 and 6were 3068±804 (n=11), 1879±822 (n=11),1509±538 (n=8) and 1318±279 (n=6) μN, respec-tively. A significant difference in high-rate twitch force(p< 0.05) was obtained between days 0 and 4(Figure 4C); a significant difference in low-rate twitchforce (p< 0.01) was obtained between days 0 and 2, 4and 6 (Figure 4D).

Figure 2. Beating cardiomyocytes at 16 h after plating and spontaneous contraction rates at 1–6 days with 1, 3 and 5 M density patchtissues: (A) 1 M, (B) 3 M and (C) 5 M densities: green arrowheads, live cardiac cells; red arrowheads, dead cardiac cells; arrows, beat-ing cells. (D) Number of beating cells of five ×200 fields with 1, 3 and 5 M densities at 16 h after plating. (E) Spontaneous contractionrates at 1–6 days with 1, 3 and 5 M densities. Values are mean±SD; *p<0.05, **p<0.01



Figure 3. (A) Representative plot showing the rhythmic contraction from a 3 M patch; (B) the synchronous ECG of (A). (C) Represen-tative plot showing the arrhythmic contraction from a 5 M patch; (D) the synchronous ECG of (C). (E) Average high-rate contractileforces for cell densities of 2, 4 and 6 M. (F) Average low-rate contractile forces for cell densities of 2, 4 and 6 M. (G) Average R-waveamplitude of high-rate contractions for cell densities of 2, 4 and 6 M. Values are mean±SD; *p<0.05, **p<0.01

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Contractile response frequencies of 1–6 M densitypatches were synchronous with electrical pacing up toeach patch’s maximal spontaneous contraction rate.Figure 4A shows that the representative maximalsynchronous response frequency was 8 Hz, obtained froma 4 M patch at its formation. The average maximal syn-chronous response frequency for 4 M patches on days 0,2, 4 and 6 were 5.9±0.8 (n=9), 5.6±0.9 (n=9),5.0±0.8 (n=8) and 3.2±1.0 (n=7) Hz, respectively.Maximal synchronous response frequency decreased dur-ing culture (Figure 4B) and was significantly smaller onday 6 relative to the other days (p< 0.01, Figure 4E).The contractile forces generated by 4 M density patchespaced at 0.25 Hz (corresponding to low-rate contraction)and 2 Hz (corresponding to high-rate contraction)

decreased during culture (Figure 4F, G). No significantdifferences in contractile force generated by 4 M densitypatches paced at 0.25 or 2 Hz were observed when com-pared with low- and high-rate spontaneous contractileforces of 4 M density patches at the same time points.

3.5. Morphology

The physical properties of fibrin gel scaffolds of theformed patches were examined on day 7. The cross-sectional area of the triangular patch tissue with 1–6 Mdensity was 32.5±2.8 mm2 (n=10), its thickness was0.94±0.13 mm (n=10), its height was 15.1±2.1 mm(n=10) and its weight was 494±43 mg (n=10); there

Figure 4. Effects of culture time on contractile force and response frequency due to electrical pacing. (A) Representative plotsshowing the change of twitch force and pre-tension with pacing frequencies upon patch formation; arrowhead, pacingmarker. (B) Representative plots showing the change of twitch force and pre-tension with pacing frequencies on day 6 afterpatch formation; arrowhead, pacing marker. (C, D) Graphs showing the changes of spontaneous high- and low-ratecontractile forces with culture time. (E) Graphs showing the change of electrical response frequency during culture. (F, G)Graphs showing the changes of contractile twitch force due to pacing with 0.25 and 2.0 Hz during culture. Values are mean±SD;*p<0.05, **p<0.01



were no significant differences in the cross-sectionalareas, patch thicknesses, heights and weights of thetriangular blocks with 1–6 M densities (p> 0.05). Thehigh- and low-rate contractile twitch forces are generatedby one corner of patch tissue (half of the patch, atriangular block); therefore, for a 6 M density patch thehigh-rate mean contractile force/mm3 volume or high-rate mean contractile force/g wet weight was calculatedas 2086 μN ÷ (0.5×32.5 mm2×15.1 mm)=8.514 mN/mm3, or as 2086 μN ÷ 494 mg=4220 mN/g. By the sameformula, the low-rate mean contractile force for the 6 Mdensity was 14.065 mN/mm3 or 6770 mN/g; the high-ratemean contractile force for 1 M was 2.245 mN/mm3 or

1110 mN/g, and its low rate mean contractile force was3.959 mN/mm3 or 1960 mN/g.

A layer of cardiac cells and self-produced extracellular ma-trix proteins, whichwe call the ‘real cardiac layer’, formed ontop of thefibrin gel scaffold network (arrowheads, Figure 5A)and the planar networks of fibrin gel scaffold are shown inFigure 5B. The thickness (arrowheads, Figure 5C) of the realcardiac tissue layer was further revealed by Masson’strichrome staining, although they were physically alteredby the fixing process. Positive staining for vWF (red;Figure 5D) suggests that endothelial cells were growingand some endothelial cell grew together (Figure 5D, arrow);positive staining for ki67 (white, Figure 5E) suggests the

Figure 5. Patch morphology: (A) cross-section and (B) planar section directly from frozen samples illustrate the composition of asample patch obtained with a light microscope; arrowheads in (A) indicate the real cardiac layer. (C) Cross-section image fromMasson’s trichrome staining; arrowheads indicate the real cardiac layer. (D) Image from constructs by immunofluorescence stainingshowing growth of heart muscle (α-Actinin), endothelial cells (vWF); arrow indicates a congregation of endothelial cells. (E) Imageshowing nuclear division (Ki67). (F) Image showing the distribution of collagen type I within the constructs. (G) Detailedimage showing a congregation of endothelial cells (vWF). (H) Detailed image showing a nucleus in karyokinesis (Ki67; arrow). (I)Image showing gap junction protein (Cx43) and endothelial cells (vWF) in the tissue

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presence of robust nuclear division, and positive staining forthe collagen type I (purple, Figure 5F) indirectly illustratesthe distribution of cardiac fibroblasts within the cardiac tis-sue. A larger endothelial cell congregation within the patchis shown in Figure 5G and a dividing cardiac nucleus in kar-yokinesis is shown in Figure 5H (arrow). Positive stainingfor connexin 43 (yellow, Figure 5I) indicates intercellularcoupling via gap junctions between cardiomyocytes.

The real cardiac layers and parts of the support fibrinscaffold stained with Masson’s trichrome from 2, 4 and 6M patches are shown in Figure 6A–C. The representativeexpressions of myofibrils by α-actinin staining and gapjunctions by Cx43 staining for 2, 4 and 6 M patches areshown in Figures 6D–O. The average thicknesses of realcardiac layers for 2, 4 and 6 M patches were 18.2±2.4(n=13), 21.4±1.4 (n=14) and 20.6±2.4 (n=12)μm, respectively; there were significant differences when

comparing 4 and 6 M (p< 0.05 or p< 0.01) with 2 M(Figure 6P). The signal volume index of Cx43 was greaterfor 2M (0.182±0.051, n=13) than for 4M (0.132±0.039,n=13) and 6 M (0.126±0.038, n=16; p< 0.05 orp< 0.01) patches (Figure 6Q). The signal volume index ofcollagen type I was also calculated to be 0.221±0.065(n=9), 0.209±0.070 (n=8) and 0.196±0.050 (n=15)for 2, 4 and 6M, respectively; however, there were no signif-icant statistical differences (p> 0.05).

4. Discussion

In the present study, cardiac tissue constructs werefabricated by seeding 1–6 M neonatal heart cells on fibringel. Patches of 1–6 M cell densities exhibited maximal con-traction rates in the range 305–410 bpm and generated

Figure 6. Real cardiac layer thickness and gap junctions in the patch. (A–C) Cross-sections showing real cardiac layer thickness andsupport scaffold fibrin network in the patch by Masson’s trichrome staining for cell densities of 2, 4 and 6 M. (D–F) Cross-sectionsshowing growth of heart muscle (α-Actinin) and gap junctions (Cx43) for 2, 4 and 6 M patches. (G, J, M) Total signal volumes;(H, K, N) signal volumes of Cx43; (I, K, O) signal volumes of α-actinin for 2, 4 and 6 M. (P) Graph showing differences in real cardiaclayer thickness. (Q) Signal volume indices of Cx43 for 2, 4 and 6 M patches. Values are mean±SD; *p<0.05, **p<0.01



contractile forces in the range 2.245–14.065 mN/mm3.Lower cell density patches (1–3 M) with a thinner layerof real cardiac tissue that generated lower R-waveamplitudes, and with a greater signal volume index ofconnexin 43, sustained longer rhythmic contractionthan higher cell density patches (4–6 M). In addition, allpatches manifested endothelial cell growth and a robustnuclear division. The contractile forces and responsesto electrical pacing of the constructed cardiac patchesdecreased by culture.

Fibrin, alone or in combination with other materials,has been used as a biological scaffold for stem andprimary cells to regenerate adipose tissue, bone, cardiactissue, cartilage, liver, nervous tissue, ocular tissue, skin,tendons and ligaments (Ahmed et al., 2008). The fibrinsupport scaffold in the present study was developed withhuman fibrinogen and thrombin. A thin layer of realcardiac tissue on the top of fibrin gel network varied withthe plated cell density, with the 4 and 6 M patches havinga significantly thicker layer than the 2 M patches(Figure 6P). Besides the plating cell density, the growthrate of each cell type within the patch might be responsi-ble for the differences. Freshly isolated neonatal cardiaccells consist of cardiac fibroblasts, cardiomyocytes,smooth muscle cells, endothelial cells and cardiac stemcells. We used 2–3 day-old rat heart cells to construct thispatch. Endothelial cell marker vWF-positive staining indi-cates the presence of endothelial cells and endothelial cellcongregations (Figure 5D, E, G, I). A positive staining forvWF demonstrated that there were potential angiogenesisbuds, which would be suitable to grow and connect tohost micro-blood vessels and bring nutrients into thepatch during in vivo applications. There were signs ofrobust cell proliferation in these patches, which wererevealed by nuclear division and marked by ki67 positivestaining (Figures. 5E, H). The Cx43-positive stainingindicates that cardiomyocytes in the present cardiac patchexhibit electromechanical coupling (Figure 5I, 6D–F). Thedetected ECG signal and the natural, adult-heart-like,QRS complex revealed that these patches can sustainelectrical propagation with speeds that would be close tonative tissues. The R-wave amplitudes increased withthickness of real cardiac tissue.

Higher cell density patches (4–6 M) began localizedspontaneous contractions earlier (day 1) than lower celldensity ones (1–3 M). The range of highest contractionrates for 1–6 M density patches was 251–443 bpm. Thereason why the contraction rate was associated withinitial plating densities was that the higher the density,themore spontaneously beating cardiomyocytes (Figure 2).Freshly isolated cardiomyocytes demonstrate spontane-ous contraction and this phenomenon may result fromthe overdrive disruption of sinoatrial pacemaker cells byenzyme dissociation (Wollenberger, 1964; Mark andStrasser, 1966; Yang and Murray, 2011). Movie S1 (seesupporting information) was taken from a 3 M densitypatch and shows, 36 h after cell plating, the spontaneouslybeating cardiomyocytes emerged from the initial platingcells and becoming the majority; they paced at different

frequencies that definitely initiated signal competition. Af-ter a few more days of incubation, the various frequenciescoalesced into a single contraction frequency for the major-ity of the patch; this result adheres to a synchronizationphenomenon that is dependent on the number of initialspontaneously beating cardiomyoctes and the conductionof their action potentials (Kryukov et al., 2008; Maedaet al., 2005). Once sufficient connections are formed, thefastest beating cells can initiate whole-tissue contraction.High-density cardiac cell plating supplies more high-speedbeating cells, cardiac fibroblasts and endothelial cells, sothey needed a shorter time than low-density plating to at-tain their maximal contraction rates (Figure 2). The 3 Mdensity dishes may have possessed a more ideal proportionof cardiomyocytes to cardiac fibroblasts and endothelialcells than the 1 M or 5 M density dishes. Thus, thisfavourable cellular proportion may explain the superiorrates of successful patch formation of 3 M density patcheson days 4 and 6 to 1 M and 5 M density patches.

A synchronized contraction relies on the appropriateproportion of cardiomyocytes to cardiac fibroblasts, aswell as endothelial cells and smooth muscle cells. In thepresent study, patches with 1–3 M densities presumablymaintained the appropriate cell-type proportion thatenhanced the synchronization and increased the contrac-tion rate. Cardiac fibroblasts and endothelial cells prolifer-ate faster than cardiomyocytes. The overpopulation of thecardiac support cells (i.e. mismatch of contraction cellswith extracellular matrix), the ageing and apoptosis ofthe spontaneously beating cells, and the space shrinkagewith the process of patch formation are responsible forthe decrease in contraction rate. The above reasons mayalso partially explain why steady contractions weredetected from 1–3 M patches, while more arrhythmiccontractions were detected from 4–6 M patches.

The strength of a muscle’s contraction is influenced bythe number of fibres within the muscle that have interac-tions of myosin cross-bridges with actin, the rate ofcontraction and the relaxed length of the muscle fibres.The present 1–6 M density cell patches generated meancontractile forces in the range 2.245–14.065 mN/mm3 or1110–1960 mN/g. By calculating contractile force overthe cross-sectional area of the constructs, our previousbioengineered heart muscle (Huang et al., 2007)generated 12–15 mN/mm2 and the bioartificial cardiactissue constructed by Kensah et al. (2011) generated4.49–4.65 mN/mm2; both are still well below the contrac-tion force of ~50 mN/mm2 exerted by the papillary mus-cle of native rat myocardial tissue (Han and Ogut, 2010).However, it is reasonable to compare contractile force bycross-sectional area when they have the same shape.

The real cardiac tissue layers of 4 and 6 M patches werethicker than those of 2 M; however, the signal volume indi-ces of collagen type I were not significantly different. Thissuggests that the myofibril content in 4–6 M patches maybe higher than that in 2 M patches. As such, the differencein myofibril content may explain the greater twitch forcesgenerated by patches with higher densities. Based on theresults for percentage of complete formation of patches

Z.-W. Tao et al.


on days 4–6, contractile force and thickness of the real car-diac layer, a 3 or 4 M patch is the optimal choice for futurein vivo study. Decreases in maximal response frequency un-der electrical pacing and average twitch forces presumablyderived from the aging and apoptosis of cardiomyocytesand an inappropriate proportion of cardiomyocytes tocardiac fibroblasts. Our results revealed a statistical dif-ference in Cx43 signal volume indices in 4 and 6 Mpatches when compared with 2 M (Figure 6Q). Cx43 isthe major protein of cardiac ventricular gap junctionsand is crucial to cell–cell communication and cardiacfunction (Boengler et al., 2006). Recent studies havereported that changed expression of Cx43 might contrib-ute to a higher level of arrhythmogenicity (Salamehet al., 2009; Severs et al., 2008).

The present cardiac patch could beat spontaneously inthe range 251–443 bpm and generated mean contractileforces in the range 2.245–14.065 mN/mm3. We suggestthat the proper time for in vivo implantation of this con-struct is just at patch formation, based on the contraction

rate, contractile force and response to electrical pacedecreasing by culture. The 3–4 M density cardiac patchesshow superior rates of successful patch formation andadult heart beat-like contraction rates, sustain a longerrhythmic contraction and have moderate contractileforce; thus, they are the most favourable candidates.However, the thickness, longevity and synchronicity ofthe present patch model are limited, due to low levels ofnutrient supply within the scaffold network.

Conflict of interest

The authors have declared that there is no conflict ofinterest.

Acknowledgements

This study was supported by a grant from the National Institutesof Health, entitled ‘Fabrication of 3D Cardiac Patches forMyocardial Regeneration’ (Grant No. R01-EB011516).

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optimizing a spontaneously contracting heart tissue patch with rat neonatal cardiac cells on fibrin...

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