Biomaterial properties of cholecyst-derived scaffold recovered by a non-detergent/enzymatic method Thapasimuthu V. Anilkumar, Vadavanath P. Vineetha, Deepa Revi, Jaseer Muhamed, Akhila Rajan Division of Experimental Pathology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum 695012, Kerala, India Received 1 October 2013; revised 16 January 2014; accepted 18 February 2014 Published online 5 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33131 Abstract: Isolation procedures for the recovery of extracellular matrices (ECMs) from animal organs/tissues that are useful in regenerative medicine involve multiple sequential steps/stages including collection of the source organ at slaughter, their transportation to laboratory, decellularization, decontamina- tion, stabilization, and sterilization. Most of these steps require extensive use of chemicals/reagents/enzymes which may also adversely affect the quality of the scaffold. With an effort to minimize the use of chemicals/reagents/enzymes, while extracting biomaterial-grade ECM from porcine cholecyst (gall bladder), we performed preisolation ex situ incubation of the organ in a stabilizing agent that also caused in situ crosslink- ing of tissue-components and delaminated the collagen-rich ECM from the tissue-layer beneath the mucosa. The physical, chemical, and biological properties of the isolated scaffolds were similar to that of a commercially available porcine small intestinal submucosa. The cholecyst-derived scaffold not only satisfied preclinical safety-test procedures such as cytotoxicity, local response, and endotoxin load but also showed the potential to promote healing of full-thickness skin wound in a rabbit model. The procedure was also suitable for isolating scaffolds from other hollow organs such as jejunum and uri- nary bladder. It was concluded that enzyme/detergent treat- ment may be an avoidable step while isolating biomaterial- grade scaffolds from hollow organs. V C 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 1506–1516, 2014. Key Words: decellularized scaffold, acellular tissue-derived scaffold, cholecyst-derived scaffold, extracellular matrix, wound healing How to cite this article: Anilkumar TV, Vineetha VP, Revi D, Muhamed J, Rajan A. 2014. Biomaterial properties of cholecyst- derived scaffold recovered by a nondetergent/enzymatic method. J Biomed Mater Res Part B 2014:102B:1506–1516. INTRODUCTION Organs and tissue of farm animals are excellent sources of scaffolds that are useful in tissue engineering and regenerative medical applications. 1 These scaffolds are essentially extracel- lular matrices (ECMs) recovered from various organs/tissues after appropriate decellularization. Porcine-derived ECM iso- lated from the submucosa of hollow organs like small intes- tine has found extensive clinical use in regenerative medicine, mainly as xenografts. 2 Indeed, several clinical products made of biomaterial-grade ECM-scaffold are available in the mar- ket. 3 Other hollow organs such as urinary bladder 1 and chole- cyst (gall bladder) 4 are also potential sources of scaffolds. Recently, attempts for extracting whole-organ ECM from lung kidney, heart, and liver have been made. 5–8 Chemical decellu- larization, usually with detergents and enzymes, is the gold standard for isolating tissue engineering scaffolds but the pro- cedure may have deleterious effects on the quality of the scaf- fold 9 and potential end use of these scaffolds 10 irrespective of the choice of the source organs. Preparation of biomaterial-grade scaffolds is a multi- stage process. Optimized protocols for scaffold isolation have used selected chemicals and identified treatment con- ditions/procedures. 11,12 Traditionally, first, the organs/tis- sues of choice are collected from farm animal carcasses at slaughter and transferred to laboratory by procedures that cause minimal microbial contamination (by incubation in a media containing antimicrobial agents) and maximal preser- vation of organ/tissue architecture or cell viability (by incu- bation in traditional tissue/organ culture medium). Decellularization is then achieved by a combination of phys- ical methods (e.g., freezing, direct pressure, sonication, and agitation) and chemical treatment. Some of the chemicals used for decellularization are as follows: alkali/acid (e.g., acetic acid, peracetic acid, hydrochloric acid, sulfuric acid, and ammonium hydroxide); nonionic detergents (e.g., Triton X-100); ionic detergents (e.g., sodium dodecyl sulfate, Triton X-200); zwitterionic detergents (e.g., 3-[(3-cholamidopropyl) dimethylammonio]d-1-propanesulphonate, sulfobetaine-10, sulfobetaine-16); chaotropic agents (e.g., tri(n-butyl)phos- phate); chelating agents (e.g., ethylenediaminetetraacetic acid, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N 0 ,N 0 - tetraacetic acid) and hypo-/hypertonic solutions. Incubation Correspondence to: T. V. Anilkumar (e-mail: tvanilkumar@sctimst.ac.in) Contract grant sponsor: The Department of Biotechnology, Government of India; contract grant number: BT/PR15461/MED/32/167/2011 1506 V C 2014 WILEY PERIODICALS, INC.