ARTICLE IN PRESS Biomaterials 26 2005 6610 6617 www elsevier com locate biomaterials Engineering of a vascularized scaffold for arti cial tissue and organ generation Heike Mertschinga b Thorsten Wallesb Michael Hofmannc Johanna Schanza Wolfram H Knappc a Fraunhofer Institute for Interfacial Engineering and Biotechnology Nobelstrasse 12 70569 Stuttgart Germany b Tissue Engineering Network Hannover Medical School Podbielskistr 380 30659 Hannover Germany c Department of Nuclear Medicine Molecular Imaging and Therapy Group Hannover Medical School Carl Neuberg Str 1 30625 Hannover Germany Received 16 December 2004 accepted 4 April 2005 Available online 23 June 2005 Abstract Tissue engineering is an emerging eld 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 bioarti cial organ function Here we developed methods to decellularize porcine small bowl segments and repopulate the remaining venous and arterial tubular structures within these matrices with allogeneic porcine endothelial progenitor cells Cellular adherence and vitality was characterized by quantitative 2 18F uoro 20 desoxy glucose FDG positron emission tomography PET and subsequent immunohistological work up The generated matrices showed insulin dependent FDG uptake predominantly in the region of the former vascular structures Stain for vitality and the speci c endothelial markers CD31 VE Cadherin and Flk 1 matched this functional nding Providing evidence for vitality up to 3 weeks post reconstitution and typical endothelial differentiation these results indicate that our generated matrix allows the generation of complex bioarti cial tissues and organs for experimental and future clinical application r 2005 Elsevier Ltd All rights reserved Keywords Bioprosthesis Vascularized scaffold 1 Introduction Tissue engineering was originally developed as an alternative therapy for the treatment of tissue loss or end stage organ failure resolving the shortage in tissues and organs for transplantation therapy 1 2 It represents a biology driven approach by which biological tissues are engineered through combining material technology and biotechnology 3 Signi cant progress has been made in recent years however one of the major obstacles in tissue engineering of thick complex Corresponding author Fraunhofer Institute for Interfacial Engineering and Biotechnology Nobelstrasse 12 70569 Stuttgart Germany Tel 49 711 970 4117 fax 49 711 970 4047 E mail address heike mertsching igb fraunhofer de H Mertsching 0142 9612 see front matter r 2005 Elsevier Ltd All rights reserved doi 10 1016 j biomaterials 2005 04 048 tissues is to keep the construct viable in vitro during cultivation and formation of the tissue as well as in vivo on implantation 4 In vivo the construct must be vascularized immediately to allow for its survival and later integration since the host s vascularization is not suf cient to feed the implant 4 5 Our research focuses on overcoming the problem of missing graft vascularization Recently we have developed methods for the generation of a primary vascularized biological scaffold for tissue engineering that affords vascular anastomosis of any bioarti cial construct to the recipient blood supply Porcine small intestine segments are harvested sterilized and decellularized and the remainder of the vessels are inoculated with bone marrow derived endothelial progenitor cells bmEPC resulting in a biological vascularized scaffold ARTICLE IN PRESS H Mertsching et al Biomaterials 26 2005 6610 6617 BioVaMs 14 Here we describe the morphological composition of the vascularized scaffold and the functional characterization of the vascular network employing quantitative positron emission tomography PET using picomolar amounts of 2 18F uoro 20 desoxy glucose FDG We demonstrate the differentiation of the seeded bmEPC into endothelial cells EC forming a functional vascular network Cellular distribution in the vascularized scaffold approximates ndings in native small bowl segments and the cells are viable and respond to physiological hormonal stimuli These results indicate the practicability to generate vascularized scaffolds for tissue engineering applications 2 Methods All reagents were purchased from Merck Darmstadt Germany Sigma Aldrich Mu nchen Germany and all experiments were done at room temperature unless indicated otherwise Cell culture media and supplements were from Promocell Heidelberg Germany The applied antibodies were obtained at Dako Hamburg Germany 2 1 Graft harvesting German landrace pigs n 10 age 3 months body weight 18 25 kg were obtained from a local dealer Tierzuchtanstalt Mariensee Germany and they underwent scaffold harvesting under sterile conditions All animals received human care in compliance with the Guide for Care and use of Laboratory Animals published by the National Institutes of Health NIH publication No 85 23 revised 1996 after approval from our institutional animal protection board Experiment 02 504 General anesthesia was induced by continous trapanal and fentanyl infusion A median laparotomy was used to isolate a 10 15 cm long segment of jejunum including its artery and vein pedicle Following systemic administration of Heparin 300 IE kg the feeding artery was cannulated with a 6 French catheter and ushed with 100 ml NaCl 0 9 The draining vein was cannulated with an 8 French catheter and venous back ow was controlled macroscopically The intestinal lumen was ushed with 500 ml NaCl 0 9 at 4 1C containing antibiotic solution neomycin 3250 IE and bacitracin 250 IE immediately after explantation The fourth lumbar vertebral bone was punctured and 50 ml bone marrow was gathered to isolate porcine bone marrow derived endothelial precursor cells At the end of the operation the animals were killed by an anesthesia overdose The specimen were stored at 4 1C until further processing 2 2 Matrix preparation and acellularization The scaffold was decellularized after mechanical removal of the small bowl mucosa using a modi ed method of Meezan 6 and kept in cell type speci c medium at 37 1C until reseeding Modi cations were as follows For decellularization the small bowel segment was incubated in 1 sodium azide solution 2 h 6611 at 4 1C under shaking conditions To remove the cellular proteins an incubation in 1 M sodium chloride solution containing 2000 U DNase Type I 2 h at 37 1C followed To dissolve
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