Perfusion-decellularized matrix: using natureaposs platform to engineer a bioartificial heartRESULTSPerfusion decellularization of cadaveric heartsProperties of the decellularized constructRecellularization of decellularized cadaveric heartsWhole-heart experimentsRe-endothelialization of decellularized constructDISCUSSIONMETHODSPerfusion decellularization of rat heartsIsolation and preparation of rat neonatal cardiocytesMedia and solutionsBiaxial passive mechanical propertiesRecellularization of decellularized rat heartsRe-endothelialization of decellularized rat heartFunctional assessment of contractile force of cross-sectionsWhole working-heart measurements on recellularized heartHeterotopic heart transplantationHistology and immunofluorescenceScanning electron microscopy and transmission electron microscopyFigure 1 Perfusion decellularization of whole rat hearts.Figure 2 Composition and characteristics of decellularized rat heart tissue.Figure 3 Vascular architecture of decellularized rat heart tissue.Figure 4 Formation of a working perfused bioartificial heart-like construct by recellularization of decellularized cardiac ECM.Figure 5 Histological analysis of recellularized rat heart constructs.ACKNOWLEDGMENTSAUTHOR CONTRIBUTIONSReferencesPerfusion-decellularized matrix: using nature’s platformto engineer a bioartificial heartHarald C Ott1, Thomas S Matthiesen2, Saik-Kia Goh2, Lauren D Black3, Stefan M Kren2, Theoden I Netoff3&Doris A Taylor2,4About 3,000 individuals in the United States are awaiting adonor heart; worldwide, 22 million individuals are living withheart failure. A bioartificial heart is a theoretical alternativeto transplantation or mechanical left ventricular support.Generating a bioartificial heart requires engineering of cardiacarchitecture, appropriate cellular constituents and pumpfunction. We decellularized hearts by coronary perfusion withdetergents, preserved the underlying extracellular matrix, andproduced an acellular, perfusable vascular architecture,competent acellular valves and intact chamber geometry. Tomimic cardiac cell composition, we reseeded these constructswith cardiac or endothelial cells. To establish function, wemaintained eight constructs for up to 28 d by coronaryperfusion in a bioreactor that simulated cardiac physiology. Byday 4, we observed macroscopic contractions. By day 8, underphysiological load and electrical stimulation, constructs couldgenerate pump function (equivalent to about 2% of adult or25% of 16-week fetal heart function) in a modified workingheart preparation.In the United States alone, nearly 5 million people live with heartfailure, and about 550,000 new cases are diagnosed annually. Hearttransplantation remains the definitive treatment for end-stage heartfailure, but the supply of donor organs is limited. Once a heart istransplanted, individuals face lifelong immunosuppression and oftentrade heart failure for hypertension, diabetes and renal failure1.Thecreation of a bioartificial heart could theoretically solve these pro-blems. Attempts to engineer heart tissue have involved numerousapproaches2. Engineered contractile rings and sheets have been trans-planted into small animals and have improved ventricular function3–5.The creation of ‘thick’ (4100–200 mm) cardiac patches has beenlimited by an inability to create the geometry necessary to support thehigh oxygen and energy demands of cardiomyocytes at a depth greaterthan B100 mm from the surface2,6. The use of channeled cardiacextracellular matrix (ECM) constructs, oxygen carriers and stackedcardiac sheets4,7,8to improve thickness has reinforced the directrelationship between perfusion and graft size or cell density6,9.To create a whole-heart scaffold with intact three-dimensionalgeometry and vasculature, we attempted to decellularize cadaverichearts by coronary perfusion with detergents, which have been shownto generate acellular scaffolds for less complex tissues, by directimmersion10–14. We then repopulated decellularized rat hearts withneonatal cardiac cells or rat aortic endothelial cells and cultured theserecellularized constructs under simulated physiological conditions fororgan maturation15. Ultimately, chronic coronary perfusion, pulsatileleft ventricular load and synchronized left ventricular stimulation led tothe formation of contractile myocardium that performed stroke work.RESULTSPerfusion decellularization of cadaveric heartsTo develop a valid perfusion decellularization protocol, we carried outantegrade coronary perfusion of 140 cadaveric rat hearts on amodified Langendorff apparatus and compared the degree of decel-lularization (that is, removal of DNA and intracellular structuralproteins) that resulted from the use of three detergent solutions(Fig. 1). The use of SDS (Fig. 1c,f) gave better results than didpolyethylene glycol (PEG; Fig. 1a,d), Triton-X100 (Fig. 1b,e)orenzyme-based protocols (data not shown) for full removal of cellularconstituents. Antegrade coronary SDS perfusion over 12 h (Fig. 1c)yielded a fully decellularized construct. Histological evaluationrevealed no remaining nuclei or contractile elements (Fig. 1g). DNAcontent decreased to less than 4% of that in cadaveric heart (Supple-mentary Fig. 1 online), whereas the glycosaminoglycan content wasunchanged. After perfusion with Triton-X100 (ref. 16) and washing,SDS levels in the decellularized myocardium could not be differen-tiated from zero in a quantitative assay (Supplementary Fig. 1).Properties of the decellularized constructCollagens I and III, laminin and fibronectin (Fig. 2a) remained withinthe thinned, decellularized heart matrix. The fiber composition(weaves, struts and coils) and orientation of the myocardial ECMwere preserved, whereas cardiac cells were removed (Fig. 2b), resultingin compressed constructs. Within the retained ventricular ECM,we saw intact vascular basal membranes without endothelial orReceived 29 May 2007; accepted 18 October 2007; published online 13 January 2008; doi:10.1038/nm16841Department of Surgery, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114, USA.2Center for CardiovascularRepair, University of Minnesota, 312 Church Street Southeast, 7-105A NHH, Minneapolis, Minnesota 55455, USA.3Department of Biomedical Engineering,University of Minnesota, 312 Church Street Southeast, 7 NHH, Minneapolis, Minnesota 55455, USA.4Department of Integrative Biology and Physiology, University ofMinnesota,
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