Engineering of a vascularized scaffold for artificial tissue and organ generationIntroductionMethodsGraft harvestingMatrix preparation and acellularizationDNA isolation and quantificationCulture of bone marrow-derived progenitor cellsReendothelization of the vascular structuresCSFE labeling of reseeded cellsPositron emission tomographyImmunohistologyStatisticsResults and discussionVascularized biological scaffoldReendothelialization of vascular structuresEndothelial differentiation of bioartificial capillary networkVitality of vascularized scaffoldResponse to hormonal stimuliLimitationsConclusionsAcknowledgmentsReferencesBiomaterials 26 (2005) 6610–6617Engineering of a vascularized scaffold for artificial tissueand organ generationHeike Mertschinga,b,, Thorsten Wallesb, Michael Hofmannc,Johanna Schanza, Wolfram H. KnappcaFraunhofer Institute for Interfacial Engineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart, GermanybTissue Engineering Network, Hannover Medical School, Podbielskistr. 380, 30659 Hannover, GermanycDepartment of Nuclear Medicine, Molecular Imaging and Therapy Group, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, GermanyReceived 16 December 2004; accepted 4 April 2005Available online 23 June 2005AbstractTissue engineering is an emerging field in regenerative medicine to overcome the problem of end-stage organ failure. However,complex tissues and organs need a vascular supply to guaranty graft survival and render bioartificial organ function. Here wedeveloped methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structureswithin these matrices with allogeneic porcine endothelial progenitor cells. Cellular adherence and vitality was characterized byquantitative 2-[18F]-fluoro-20-desoxy-glucose (FDG) positron emission tomography (PET) and subsequent immunohistologicalwork up. The generated matrices showed insulin-dependent FDG uptake predominantly in the region of the former vascularstructures. Stain for vitality and the specific endothelial markers CD31, VE-Cadherin and Flk-1 matched this functional finding.Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation, these results indicate thatour generated matrix allows the generation of complex bioartificial tissues and organs for experimental and future clinicalapplication.r 2005 Elsevier Ltd. All rights reserved.Keywords: Bioprosthesis; Vascularized scaffold1. IntroductionTissue engineering was originally developed as analternative therapy for the treatment of tissue loss orend-stage organ failure resolving the shortage in tissuesand organs for transplantation therapy [1,2]. It repre-sents a biology-driven approach by which biologicaltissues are engineered through combining materialtechnology and biotechnology [3]. Significant progresshas been made in recent years; however, one of themajor obstacles in tissue engineering of thick, complextissues is to keep the construct viable in vitro (duringcultivation and formation of the tissue) as well as in vivo(on implantation) [4]. In vivo, the construct must bevascularized immediately to allow for its survival andlater integration since the host’s vascularization is notsufficient to feed the implant [4,5].Our research focuses on overcoming the problem ofmissing graft vascularization. Recently we have devel-oped methods for the generation of a primary vascular-ized biological scaffold for tissue engineering thataffords vascular anastomosis of any bioartificial con-struct to the recipient blood supply. Porcine smallintestine segments are harvested, sterilized and decel-lularized and the remainder of the vessels are inoculatedwith bone marrow-derived endothelial progenitor cells(bmEPC) resulting in a biological vascularized scaffoldARTICLE IN PRESSwww.elsevier.com/locate/biomaterials0142-9612/$ - see front matter r 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.biomaterials.2005.04.048Corresponding author. Fraunhofer Institute for Interfacial En-gineering and Biotechnology, Nobelstrasse 12, 70569 Stuttgart,Germany. Tel.: +49 711 970 4117; fax: +49 711 970 4047.E-mail address: [email protected](H. Mertsching).(BioVaMs) [14]. Here we describe the morphologicalcomposition of the vascularized scaffold and thefunctional characterization of the vascular networkemploying quantitative positron emission tomography(PET) using picomolar amounts of 2-[18F]-fluoro-20-desoxy-glucose (FDG). We demonstrate the differentia-tion of the seeded bmEPC into endothelial cells (EC)forming a functional vascular network. Cellular dis-tribution in the vascularized scaffold approximatesfindings in native small bowl segments and the cellsare viable and respond to physiological hormonalstimuli. These results indicate the practicability togenerate vascularized scaffolds for tissue engineeringapplications.2. MethodsAll reagents were purchased from Merck (Darmstadt,Germany), Sigma-Aldrich (Mu¨nchen, Germany) and allexperiments were done at room temperature unless indicatedotherwise. Cell culture media and supplements were fromPromocell (Heidelberg, Germany). The applied antibodieswere obtained at Dako (Hamburg, Germany).2.1. Graft harvestingGerman landrace pigs (n ¼ 10; age: 3 months; body weight18–25 kg) were obtained from a local dealer (TierzuchtanstaltMariensee, Germany) and they underwent scaffold harvestingunder sterile conditions. All animals received human care incompliance with the Guide for Care and use of LaboratoryAnimals published by the National Institutes of Health (NIHpublication No. 85–23, revised 1996) after approval from ourinstitutional animal protection board (Experiment #02–504).General anesthesia was induced by continous trapanal andfentanyl infusion. A median laparotomy was used to isolate a10–15 cm long segment of jejunum, including its artery andvein pedicle. Following systemic administration of Heparin(300 IE/kg) the feeding artery was cannulated with a 6-Frenchcatheter and flushed with 100 ml NaCl 0.9%. The draining veinwas cannulated with an 8-French catheter and venous back-flow was controlled macroscopically. The intestinal lumen wasflushed with 500 ml NaCl 0.9% at 4 1C containing antibioticsolution (neomycin 3250 IE and bacitracin 250 IE), immedi-ately after explantation. The fourth lumbar vertebral bone waspunctured and 50 ml bone marrow was gathered to isolateporcine bone marrow-derived
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