UNIVERSITÄTSKLINIKUM HAMBURG-EPPENDORF Institut für Experimentelle Pharmakologie und Toxikologie Direktor Prof. Dr. med. Thomas Eschenhagen Development of a Biological Ventricular Assist Device: Preliminary Data From a Small Animal Model Dissertation zur Erlangung des Grades eines Doktors der Medizin an der Medizinischen Fakultät der Universität Hamburg. vorgelegt von: Yalin Yildirim aus Marburg Hamburg 2013
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UNIVERSITÄTSKLINIKUM HAMBURG-EPPENDORF
Institut für Experimentelle Pharmakologie und Toxikologie
Direktor Prof. Dr. med. Thomas Eschenhagen
Development of a Biological Ventricular Assist Device:Preliminary Data From a Small Animal Model
Dissertation
zur Erlangung des Grades eines Doktors der Medizinan der Medizinischen Fakultät der Universität Hamburg.
vorgelegt von:
Yalin Yildirim aus Marburg
Hamburg 2013
2
Angenommen von derMedizinischen Fakultät der Universität Hamburg am: 09.09.2013
Veröffentlicht mit Genehmigung derMedizinischen Fakultät der Universität Hamburg.
Prüfungsausschuss, der Vorsitzende: Prof. Dr. med. Thomas Eschenhagen
Prüfungsausschuss, zweiter Gutachter: PD Dr. med. Ali Aydin
Prüfungsausschuss, dritter Gutachter: Prof. Dr. med. Hermann Reichenspurner
Biermann, Thomas Eschenhagen and Wolfram-Hubertus ZimmermannYalin Yildirim, Hiroshi Naito, Michael Didié, Bijoy Chandapillai Karikkineth, Daniel
Animal ModelDevelopment of a Biological Ventricular Assist Device : Preliminary Data From a Small
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Background—Engineered heart tissue (EHT) can be generated from cardiomyocytes and extracellular matrix proteins andused to repair local heart muscle defects in vivo. Here, we hypothesized that pouch-like heart muscle constructs can begenerated by using a novel EHT-casting technology and applied as heart-embracing cardiac grafts in vivo.
Methods and Results—Pouch-like EHTs (inner/outer diameter: 10/12 mm) can be generated mainly from neonatal rat heartcells, collagen type I, and serum containing culture medium. They contain a dense network of connexin 43interconnected cardiomyocytes and an endo-/epicardial surface lining composed of prolylhydroxylase positive cells.Pouch-like EHTs beat spontaneously and show contractile properties of native heart muscle including positive inotropicresponses to calcium and isoprenaline. First implantation studies indicate that pouch-like EHTs can be slipped overuninjured adult rat hearts to completely cover the left and right ventricles. Fourteen days after implantation, EHT-graftsstably covered the epicardial surface of the respective hearts. Engrafted EHTs were composed of matrix anddifferentiated cardiac muscle as well as newly formed vessels which were partly donor-derived.
Conclusions—Pouch-like EHTs can be generated with structural and functional properties of native myocardium.Implantation studies demonstrated their applicability as cardiac muscle grafts, setting the stage for an evaluation ofEHT-pouches as biological ventricular assist devices in vivo. (Circulation. 2007;116[suppl I]:I-16–I-23.)
Repairing the damaged heart is one of the major chal-lenges in modern medicine. To this end, cardiac tissue
engineers attempt to develop technologies that may allow torefurbish failing myocardium with new muscle.1,2 Therapeu-tically applied artificial myocardium would have to fulfill atleast 2 biological functions: (1) it must stabilize the failingheart to prevent further dilation and (2) it must add contractileelements to the heart to improve its systolic function. Passiveventricular restraint devices (eg, CorCap Cardiac SupportDevice; Acorn Cardiovascular Inc) have been applied to stopadverse left ventricular remodelling and dilation in failinghearts.3 Yet, the widespread use of CorCap Cardiac SupportDevices (CSD) has recently been stopped by the US Food andDrug Administration (FDA) despite positive trials in Europeand North America because of safety concerns. Indeed,pericardial constriction may occur in patients with CSDdevices necessitating reoperations that are technically chal-lenging.4 Another caveat pertaining to the cardiac restraintapproach is the lack of intrinsic contractility in the latter.Thus, development of a CSD that may not only offer restraintbut also reintroduce contractile elements, ie, cardiomyocytes,into failing hearts may eventually lead to a novel therapeuticperspective in end-stage heart failure.
Several groups have generated heart muscle constructswith functional and morphological properties of native myo-cardium in vitro (see overview in Eschenhagen and Zimmer-mann, 2005).1 Implantation studies in a rat model of myocar-dial infarction provided first proof-of-concept for atherapeutic application of engineered heart tissue (EHT) invivo.5 Most tissue engineering studies have focused on therepair of regional myocardial defects, eg, after myocardialinfarction, and not on offering passive (restraint) and active(contractility) support to the entire failing heart. We andothers have recently reported modifications of establishedcardiac tissue engineering technologies to generate complexmyocardial constructs with different geometries includingcontractile tubes and muscle networks to support failinghearts.6,7 Here, we propose a novel technology to generateEHT with a pouch-like geometry that may ultimately offerrestraint and contractile support as biological ventricularassist device (BioVAD) in vivo. Consequently, our study had2 objectives: (1) to develop a technology allowing theconstruction of myocardial pouches and (2) to investigate theapplicability of these constructs as cardiac muscle grafts inadult rats.
From the Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Germany.*Y.Y. and H.N. contributed equally to this work.Presented at the American Heart Association Scientific Sessions, Chicago, Ill, November 12–15, 2006.Correspondence to Wolfram-Hubertus Zimmermann, MD, Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical
Methods and MaterialsAll procedures were approved by the local animal protectionauthority (BWG of the Freie und Hansestadt Hamburg: #54/04) andconformed to the Guide for the Care and Use of Laboratory Animals(NIH publication 86–23, revised 1996).
Cell IsolationCardiomyocytes were isolated from neonatal Wistar rats (postnatalday 0 to 3) by a fractionated DNase/Trypsin digestion protocol asdescribed earlier.8 The resulting cell population (50% cardiomoy-cytes/50% nonmyocytes6) was immediately subjected to EHTgeneration.
Construction of a Novel Casting Mold to EngineerPouch-Like EHTWe developed a novel casting mold for the construction of pouch-like EHTs (Figure 1). Briefly, the pointed tip of a sterile 50 mLpolypropylene tube with a screw cap (#62.547.004; Sarstedt) was cutoff under sterile conditions. The tube was inverted to form the basefor a casting mold. We subsequently filled the latter with sterileagarose (1% in 0.9% NaCl), inserted a spheric glass spacer (diam-eter: 20 mm; �4.200 �L) connected to a shaft (diameter: 2 mm), andallowed the agarose to solidify around the spacer. Removal of theglass spacer created a ball-shaped recess inside the agarose block.We then inserted another spheric glass spacer (diameter: 10 mm;�520 �L) with a shaft (diameter: 2 mm) into the recess to constructa casting mold with an outer diameter of 20 mm and an innerdiameter of 10 mm. All steps were performed under sterileconditions.
Generation of Pouch-Like EHT3.7 mL EHT reconstitution mixture containing 10�106 freshlyisolated neonatal rat heart cells, solubilized collagen type I (0.8mg/mL), Matrigel (10% v/v), and concentrated culture medium (2�DMEM, 20% horse serum, 4% chick embryo extract, 200 U/mLpenicillin, 200 �g/mL streptomycin) were cast into the sphericalmold. EHTs were incubated for 1 hour at 37°C in a humidified cellculture incubator (10% CO2 and 40% O2 in room air) to facilitatehardening of the reconstitution mixture. Subsequently, culture me-dium (DMEM, 10% horse serum, 2% chick embryo extract, 100U/mL penicillin, and 100 �g/mL streptomycin) was carefully addedto not disturb the reconstitution mixture. The porous agarose moldfacilitated free diffusion of culture medium enabling unrestricted
supply with nutrients and oxygen. EHTs condensed within 3 to 7days around the central spacer and started to contract spontaneously.Beating EHTs were transferred onto flexible holders to facilitateauxotonic contractions on culture day 7. Morphological and contrac-tile properties of pouch-like EHTs were studied after 12 to 14 culturedays.
Force MeasurementsForce of contraction (twitch tension [TT]), resting tension (RT), andrelaxation time (T2: time to 50% relaxation) of pouch-like EHTswere analyzed under electrical stimulation (2 Hz) in thermostated(37°C) organ baths filled with Tyrode’s solution (mM: NaCl 120,KCl 5.4, MgCl2 1, CaCl2 0.2 to 2.8, NaH2PO4 0.4, NaHCO3 22.6,glucose 5, Na2EDTA 0.05, ascorbic acid 0.3) as previously de-scribed.8 Inotropic and lusitropic responses to calcium (0.2 to2.8 mmol/L), isoprenaline (0.1 to 1000 nmol/L), and carbachol(1 �mol/L at maximal isoprenaline) were analyzed as describedearlier.9
Morphological Evaluation of EHTFormaldehyde-fixed EHTs were sectioned or processed as wholemounts for light or confocal laser scanning microscopy as describedpreviously.9 Hematoxylin and eosin (H&E) staining was performedas described earlier.9 Pico sirius red staining was performed ondeparaffinized sections in saturated picric acid for 1 hour. Dehy-drated sections were mounted in Eukitt (Sigma) after washing inacidified water (5 mL/L acidic acid). Antibodies directed against�-sarcomeric actinin (1:1000; clone: EA-53, Sigma) and �-prolyl-4-hydroxylase (1:500; clone: 6-9H6, Chemicon) were used withappropriate secondary antibodies to identify cardiomyocytes andfibroblasts in EHTs, respectively. Phalloidin-Alexa 488 (3.3 U/mL;Sigma) and Bandeiraea simplicifolia lectin-TRITC (10 �g/mL;Sigma) were used to mark f-actin and endothelial cells, respectively.EHTs were labeled with DAPI (1 �g/mL; Molecular Probes) beforeimplantation to facilitate donor cell identification as describedpreviously.5 DRAQ5 (5 �mol/L; Alexis Biochemicals) was appliedto label nuclei in EHT sections. Confocal laser scanning microscopywas performed with a Zeiss LSM 510 META system.
EHT ImplantationEHTs were implanted in male Wistar rats (n�16; 300 to 350 g;Charles River). Anesthesia was induced in an induction chamberfilled with isoflurane (4%) and maintained after intubation andcontinuous ventilation with isoflurane (1%) supplemented room airthroughout the surgery as described earlier.5 The thoracic cavity wasopened through a left lateral thoracotomy. The hearts were exposedafter excision of the pericardium. Pouch-like EHTs were slippedover the entire left and right ventricles from apex to the base of thehearts and fixed with 2 sutures (6–0 Prolene, Ethicon) at the anteriorand lateral base of the beating hearts. Tardomyocel (12 500 IUpenicillin/kg and 15.5 mg streptomycin/kg, intramuscular injection;Bayer) and buprenorphine hydrochloride (0.1 mg/kg, intraperitonealinjection) were applied during surgery. Cyclosporine A (5 mg/kg),azathioprine (2 mg/kg), and methylprednisolone (2 mg/kg) wereadministered daily by subcutaneous injection for immune suppres-sion. Apparent signs of immune rejection or inflammatory responseswere not observed.
Statistical AnalysisData are presented as mean�SE of the mean. Statistical differenceswere determined using a repeated ANOVA (concentration responsecurves) or 1-way ANOVA with Bonferroni post hoc testing (analysisof relaxation times). P�0.05 was considered statistically significant.
Statement of ResponsibilityThe authors had full access to the data and take full responsibility forits integrity. All authors have read and agree to the manuscript aswritten.
20 mm
10 mm
EHT casting Supplementation withculture medium
EHT culturea
b c d
Figure 1. Assembly of a casting mold and culture of pouch-likeEHT. a, Schematic drawing of a casting mold and the EHT cast-ing procedure. b, Photograph of a culture medium-filled castingmold with an EHT on culture day 3. c, Pouch-like EHTs on resil-iently based stretch devices to facilitate auxotonic contractions(culture days 7 to 12). d, Pouch-like EHT in an organ bath dur-ing force measurement (culture day 12).
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ResultsConstruction of Pouch-Like EHTEHTs condensed within 3 to 7 days in the spherical castingmolds around central glass spacers forming pouch-like con-structs with an inner diameter of 10 and an outer diameter of12 mm (corresponding to a “wall thickness” of �1 mm). Wechose these dimensions to match the size of an adult rat heart(Figure 2). First visible spontaneous contractions of thepouch-like EHTs were noted as early as 2 to 3 days aftercasting. EHTs maintained their spontaneous contractions (1to 2 Hz) for at least 14 days (see supplemental video,available online at http://circ.ahajournals.org).
Contractile Properties of Pouch-Like EHTPouch-like EHTs could be stimulated electrically in a pulsedfield (50 to 100 mA, 2 Hz; Figure 3a) and demonstrated abaseline TT of 0.7�0.2 mN at 0.2 mmol/L calcium (n�4).Increasing extracellular calcium to 2.8 mmol/L or addition of
1 �mol/L isoprenaline increased TT to 1.2�0.3 mN(P�0.05) and 0.9�0.3 mN (P�0.05), respectively (n�4;Figure 3b and 3c). RT was 0.4�0.1 mN (n�4) indicatinggood compliance (TT/RT ratio �1). T2 was 59�4 ms atbaseline calcium (0.2 mmol/L). Isoprenaline shortened T2 to39�3 ms (P�0.05). Addition of the muscarinergic agonistcarbachol (1 �mol/L) reversed the inotropic (data not shown)and lusitropic isoprenaline effects (n�4; Figure 3d).
Morphological PropertiesPouch-like EHTs contained a highly interconnected networkof differentiated cardiomyocytes (Figure 4). Abundant detec-tion of connexin 43 suggested the formation of an electricalsyncytium within EHTs (Figure 4). Notably, the surface ofthe EHTs was covered by a dense epithelium consisting ofprolylhydroxylase-positive cells suggesting that fibroblast-like cells formed a pseudoepi-/endocardium (Figure 5).
Implantation StudiesPouch-like EHTs could be implanted into immune suppressedrats after mobilizing the heart through a left lateral thoracot-omy (Figure 6). All animals survived this procedure (n�16).Fourteen days after implantation, we could clearly identifythe engrafted EHTs on the hearts (Figure 7a). H&E (Figure7b) and pico sirius red (Figure 7c) staining demonstrated thepreservation of the grafts on the epicardial surface of the hosthearts. Here, it is important to note that EHTs (in vitro and invivo) consist of collagen (red stain in Figure 7c) and heartcells (yellow stain in Figure 7c). The latter formed clearlydistinguishable muscle aggregates in close proximity to thehost myocardium (Figure 7d). Confocal laser scanning anal-yses indicated that engrafted cardiomyocytes formed loosebut differentiated muscle networks (Figure 7e). EHT graftswere mostly separated from the host heart by a cell free gap(�50 to 200 �m; Figure 7f). In addition, the already in vitroobserved epithelial surface lining was still present in someareas (Figure 7g). Yet, H&E (Figure 7d) and confocal laserscanning analyses (Figure 7h) demonstrated that engrafted
Figure 2. Dimensions of pouch-like EHT. Comparison of thedimensions of a neonatal rat heart, a standard circular EHT, apouch-like EHT on a glass spacer, and an adult rat heart (fromleft to right). Scale in cm.
a1.0
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Figure 3. Contractile function of pouch-like EHT. a,Stimulated contractions at 2 Hz. b and c, Inotropicresponses of pouch-like EHTs to increasing extra-cellular calcium (b; absolute TT at baseline:0.7�0.2 mN) and isoprenaline (c; absolute TT atbaseline: 0.6�0.2 mN) concentrations (n�4). d,Lusitropic responses of pouch-like EHTs to iso-prenaline (Iso) and carbachol (CCh; n�4). *P�0.05versus baseline (0.2 mmol/L Calcium).
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muscle also formed intimate contact to the host heart.However, connexin 43 (Cx43) appeared to be less abundantand structured in the border zone comprising host myocardi-um and graft tissue (Figure 7h) when compared with theremote myocardium (Figure 7i). Importantly, we observedvascularization of implanted EHTs. Many of the newlyformed vessels were partially composed of donor cells(DAPI-label; Figure 8).
DiscussionThe present study demonstrates (1) the development ofpouch-like EHT with structural and functional properties ofnative myocardium and (2) its applicability as cardiac musclegraft in vivo. Implantation of stem cells or tissue-engineeredmyocardium are presently tested as new concepts for thetreatment of otherwise fatal myocardial defects.1,2,10,11 Earlystudies clearly demonstrated that cardiomyocytes can surviveafter implantation and integrate into host myocardium.12 In
contrast, skeletal myoblast, being the first clinically appliedcell species in the heart,13 cannot electrically couple to hostmyocytes but still appear to provide some structural sup-port.14 Recent studies suggested a therapeutic benefit afterimplantation of bone marrow–derived stem cells in patientswith myocardial infarctions.15 However, these findings werechallenged by others.16
Myocardial tissue engineering has not entered the clinicalscene, but encouraging data have recently been derived fromanimal experiments.5,17 We anticipate that first clinical trialswill be started once a scalable cell source has been identifiedthat may be clinically applicable and, secondly, when myo-cardium can be engineered at a size and with functionalproperties that may offer significant support to failing hearts.Size certainly matters and clinically applicable myocardialgrafts must not only be thick (�1 to 10 mm) but also covera large myocardial area to repair a local defect. Yet, manymyocardial diseases do not present with a localized dysfunc-
Figure 4. Organization of cardiomyo-cytes in pouch-like EHTs. Upper panels,Staining of �-sarcomeric actinin (green),f-actin (red), and nuclei (blue; DRAQ5)indicating the formation of a dense dif-ferentiated myocyte network in pouch-like EHTs in vitro. Lower panels, Stainingof f-actin (green), demonstrating sarco-meric patterning in cardiomyocytes, andconnexin 43 protein (Cx 43; red), indicat-ing the formation of gap junctionsbetween the cardiomyocytes in pouch-like EHTs. Bars�50 �m
Figure 5. Whole-mount imaging of apouch-like EHT by confocal laser scan-ning microscopy. Adjacent planes of apouch-like EHT stained for f-actin (green)and prolyl-hydroxylase (red) indicatedthat EHTs were covered by a fibroblastepithelium mimicking an epi-/endocardium-like surface structure.Bars�50 �m
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tion but with global ventricular dilation and defects inventricular systolic shortening. In these patients, implantationof an engineered myocardial pouch may not only stopventricular enlargement but also offer contractile support toglobally failing hearts. Cardiac restraint devices (eg, Acorn
CorCap Device) may prevent dilation but cannot offer activecontractile support. In contrast, pouch-like EHTs developcontractile force and can be, although technically demanding,slipped over adult rat hearts to cover the entire ventricularmyocardium as presented here. This procedure apparently did
Figure 6. Implantation of a pouch-like EHT. a,Mobilization of an adult rat heart through a left lat-eral thoracotomy. b, Implantation of a pouch-likeEHT onto the same heart. Bars�10 mm
Figure 7. Structure of pouch-like EHTs 14 days after implantation. a, Explanted heart 14 days after implantation of a pouch-like EHT(note that the atria were removed). b, H&E staining of a short axis cross section of a heart with an EHT-graft. c, Pico sirius red stainingof a short axis cross section of a heart with an EHT-graft (adjacent to section in b). d, H&E staining of an EHT-graft/host-heart borderzone; arrows highlight a cardiomyocyte aggregate within the EHT-graft. e, Cardiomyocyte networks within an engrafted EHT-pouch(actin: green). The DAPI-label (blue nuclei) indicates donor cell origin. DAPI labeled cells were not present in the recipient myocardium(inset). f, Gap (arrows) between EHT-graft and host heart. g, Prolylhydroxylase (P4H: red) positive surface linings of EHT-grafts werepartially maintained in vivo. h and i, Connexin 43 (Cx43: red) in the EHT-graft/host heart border zone (h) and within the remote myocar-dium of the same heart (i). e through i, DAPI label (blue nuclei) indicates EHT-derived cells; f-actin labeled with phalloidin-Alexa 488(green). Bars�(a) 10 mm, (b and c) 1 mm, (d and e) 100 �m, (f through i) 50 �m
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not lead to pericardial constriction and was overall welltolerated (all animals survived the procedure and 14-dayfollow-up). Pouch-like EHTs did not lose their myocardialstructure in vivo and became vascularized within the obser-vation period (14 days). Vascularization was at least partiallysupported by donor cells (DAPI-label). This finding was notsurprising given the presence of primitive capillaries in EHTsin vitro6 and the strong vascularization of EHT-grafts in a ratmodel of myocardial infarction.5 However, whether pouch-like EHTs can be applied on dilated ventricles with contrac-tile dysfunction as BioVADs remains to be evaluated.
How do we envision overcoming problems of cell sourcingand graft dimensions? Human stem cells have recently beenidentified as a putative source for cardiomyocytes.18,19 De-spite these exciting findings, the allocation of cardiomyocytesin a sufficient number to repair a large myocardial infarct (�1billion cardiomyocytes will be needed) in a reasonable periodof time (days-weeks) remains a paramount task. Geneticselection, induction of cardiomyocyte differentiation bygrowth factors, and mass culture approaches using bioreac-tors may allow up-scaling of cardiomyocyte yield, but thefeasibility of these approaches has yet to be demonstrated inhuman stem cell cultures.20,21 Moreover, it seems unlikelythat cardiomyocytes alone will be sufficient to engineeroptimal myocardial surrogate tissue in vitro. We have in factrecently demonstrated that EHTs, being equally composed ofmyocytes and nonmyocytes, are structurally and functionallysuperior to EHTs constructed from enriched cardiomyocytepopulations.6 It is very likely that nonmyocytes are necessaryfor structural and paracrine support in vitro but also facilitatecell engraftment and elicit beneficial effects on the recipientmyocardium in vivo. In contrast to cardiomyocytes, humannonmyocytes can be easily derived from cardiac biopsies or
other autologous cell sources (eg, bone marrow or fat tissue).Once human cells are available for myocardial tissue engi-neering, it will remain questionable whether they can beassembled to thick heart muscle. The present bottleneck ofsuboptimal diffusion in thick tissue structures in the absenceof vascularization will have to be overcome. Pure musclestructures in tissue engineered myocardium in vitro generallydo not become thicker than 200 �m. However, sequentialgrafting of thin cell-sheets and implantation of star-shapedEHTs, being composed of a dense network of thin musclestrands, have been applied to generate vascularized myocar-dium with a diameter of up to 1 mm in vivo.5,22 Pouch-likeEHTs were similarly vascularized and contained thick muscleaggregates in vivo. However, further improvement of musclecomposition is likely to be necessary to confer a significantamount of myocardium to support a failing heart.
Electrical coupling of implanted engineered myocardiumto the native myocardium is another important issue. We didpreviously observe anterograde and retrograde electrical cou-pling of EHT grafts to infarcted rat hearts.5 Whether Bio-VADs couple similarly well to native myocardium will haveto be assessed in more detail in further studies. We regularlyobserved a cell-free gap of 50 to 200 �m between EHT graftand native myocardium as well as a preservation of thenonmyocytes surface lining of the EHT pouches in vivo.Either finding argues against extensive EHT/host myocardi-um electrical coupling. However, we did also identify areasof intimate graft-host contacts (Figure 7d) which are likely tofacilitate undelayed impulse propagation between host anddonor myocardium if proper cell-to-cell contacts are estab-lished. The principle propensity of cardiomyocyte grafts tocouple to native myocardium has been demonstrated byseveral groups.5,23–25 We could not unequivocally identifygap junction/connexin 43 contacts between EHT grafts andhost myocardium. However, coupling through different con-nexin isoforms or even through electrotonic mechanismsinvolving myofibroblasts cannot be excluded.26 Althoughintegration of a tissue graft into the host heart appears to beideal, it may also go along with conduction abnormalities (eg,ectopic beats, conduction delay leading to reentrytachycardia). This may be controllable by parallel applicationof antiarrhythmic drugs or cardioverter-defibrillators, as per-formed in patients with myoblast implants,14 at least untilstable electrical contacts are established.5,23 Conversely, onemight speculate that electrical integration of EHT poucheswould not be desirable at all. Instead, EHTs, functioning asBioVADs, could be electrically stimulated through an inte-grated biological pacemaker or external stimulation to main-tain an electrically separate but functionally synchronizedentity. The general feasibility of external electrical stimula-tion of pouch-like EHT grafts has been demonstrated in thepresent study (contraction experiments).
Contractile performance of pouch-like EHTs was assessedby contraction experiments in the present study. Similarexperiments have been conducted previously with circularEHTs and atrial or papillary heart muscle. We have alsoperformed pressure measurements after inserting a Millar-tipcatheter through the aperture of an EHT pouch (data notshown). This was principally feasible but unreliable because
Figure 8. Vessel in pouch-like EHT. Identification of a large ves-sel in implanted EHT. Green: f-actin; red: lectin; blue: nuclei(DAPI-label indicates host origin of the respective cells). Bars:50 �m
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of technical difficulties (measurement necessitates closure ofthe EHT aperture around the catheter which was difficult toachieve). Collectively, circular and pouch-like EHTs as wellas native myocardium display similar contractile properties(eg, positive inotropic response to increasing concentrationsof extracellular calcium; positive inotropic and lusitropicresponses to isoprenaline). In fact, contractile force per EHTmuscle cell cross-sectional area (�12% to 30% of 1 mm2
total EHT cross section) was 4 to 10 mN/mm2 under maximalinotropic stimulation in the present study. This is a bit lowerthan maximal forces of optimal circular EHT cultures (20 to40 mN/mm2; unpublished data, 2006). Yet, the differencemay stem from the uniform organization of muscle bundles inthe same plane in circular EHTs versus the more randomorganization of muscle in pouch-like EHTs. Importantly,maximal force values in EHTs closely resemble optimalforces of papillary muscle preparations.27 However, calciumsensitivity in EHTs is markedly higher when compared withpapillary muscle. This may be a consequence of an immaturecalcium handling machinery in EHTs in vitro. Interestingly,Morritt et al demonstrated recently that in vivo conditionedengineered cardiac muscle, being composed mainly of neo-natal rat heart cells and Matrigel, apparently regain physio-logical calcium handling properties.28 These findings supportour own observation that in vitro engineered myocardium iscapable of terminal differentiation once the right “growthmilieu” is offered.29
ConclusionThe present study provides a new technology to generatepouch-like EHTs that may eventually be applied as BioVADsin vivo. Structure and function of pouch-like EHTs simulaterespective properties in naıve myocardium. Implantationstudies demonstrate the applicability of pouch-like EHTs invivo. However, several important issues will have to beaddressed before pouch-like EHTs can be considered as aclinically applicable BioVADs concept. These encompass:(1) providing unequivocal evidence for a therapeutic effect ofpouch-like EHTs in a clinically relevant heart failure model;(2) identification of a scalable cardiomyocyte source andallocation of cardiomyocytes as well as nonmyocytes toBioVAD engineering; (3) achieving muscle tissue dimen-sions that may offer significant support to large failingventricles; and (4) providing solutions to safety concerns (eg,arrhythmia induction, unwanted growth, immunologicincompatibilities).
Sources of FundingThis study was supported by the German Ministry for Education andResearch (01GN 0520), the Deutsche Stiftung fur Herzforschung(F29/03), the European Union (EUGeneHeart), the Novartis Foun-dation, and the LeDucq Foundation. Y.Y. was supported by theWerner Otto Stiftung.
DisclosuresThe University Medical Center Hamburg-Eppendorf has filed apatent application concerning the BioVAD-technology.
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Yildirim et al Biological Ventricular Assist Device I-23
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Oktober 2000 Universität Hamburg, HumanmedizinOktober 2002 Ärztliche Vorprüfung (Physikum)August 2003 Erstes StaatsexamenMärz 2006 Zweites Staatsexamen
Juni 2007 Drittes Staatsexamen
Wissenschaftliche Tätigkeit
Juli 2005 Dissertation im Institut für Klinische und ExperimentellePharmakologie, Prof. Dr. Wolfram Zimmermann,Prof. Dr. Thomas Eschenhagen, UKE HamburgThema: Development of a Biological Ventricular AssistDevice: Preliminary Data From a Small Animal Model
Praktisches Jahr
April 2006-August 2006 Medizinische Notaufnahme, Universitätsklinikum HamburgKardiologie, Universitätsklinikum Hamburg
August 2006-Dezember 2006 Allgemeinchirurgie, Universitätsklinikum HamburgNeurochirurgie, Upper GI, Royal North Shore HospitalSydney, Australien
Dezember 2006-März 2007 Neurochirurgie, Universitätsklinikum HamburgAssistenzarzt
Oktober 2007 Juni 2013 Herzchirurgie, Universitäres Herzzentrum Hamburg
Yalin Yildirim Hamburg, 02.05.2013
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11. Eidesstattliche Versicherung
Ich versichere ausdrücklich, dass ich die Arbeit selbständig und ohne fremde Hilfeverfasst, andere als die von mir angegebenen Quellen und Hilfsmittel nicht benutztund die aus den benutzten Werken wörtlich oder inhaltlich entnommenen Stelleneinzeln nach Ausgabe (Auflage und Jahr des Erscheinens), Band und Seite desbenutzten Werkes kenntlich gemacht habe.Ferner versichere ich, dass ich die Dissertation bisher nicht einem Fachvertreter aneiner anderen Hochschule zur Überprüfung vorgelegt oder mich anderweitig umZulassung zur Promotion beworben habe.Ich erkläre mich einverstanden, dass meine Dissertation vom Dekanat derMedizinischen Fakultät mit einer gängigen Software zur Erkennung von Plagiatenüberprüft werden kann.