TISSUE ENGINEERING Volume 9, Number 1, 2003 © Mary Ann Liebert, Inc. Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice* KENJI IZUMI, D.D.S., Ph.D., 1 STEPHEN E. FEINBERG, D.D.S., M.S., Ph.D., 1 HIROTO TERASHI, M.D., Ph.D., 2 and CYNTHIA L. MARCELO, Ph.D. 2 ABSTRACT The aim of this study was to determine the optimal stage of development at which transplant hu- man ex vivo-produced oral mucosa equivalents (EVPOMEs) in vivo. EVPOMEs were generated in a serum-free culture system, without the use of an irradiated xenogeneic feeder layer, by seeding human oral keratinocytes onto a human cadaveric dermal equivalent, AlloDerm. EVPOMEs were cultured for 4 days submerged and then for 7 or 14 days at an air–liquid interface to initiate strat- ification before transplantation into SCID mice. AlloDerm, without epithelium, was used as a con- trol. Mice were killed on days 3, 10, and 21 posttransplantation. Epithelium of the transplanted EVPOMEs was evaluated with the differentiation marker keratin 10/13. Dermal microvessel in- growth was determined by immunohistochemistry with a mouse vascular marker, lectin binding from Triticum vulgaris. The presence and stratification of the epithelium were correlated with revas- cularization of the underlying dermis. The microvessel density of AlloDerm without epithelium was less than that of EVPOMEs with an epithelial layer. Microvessel density of the dermis varied di- rectly with the degree of epithelial stratification of the EVPOMEs. The EVPOMEs cultured at an air–liquid interface for 7 days had the optimal balance of neoangiogenesis and epithelial differenti- ation necessary for in vivo grafting. 163 INTRODUCTION S INCE O’CONNOR AND OTHERS 1 first reported the use of cultured autologous epithelial sheets in patient care, there have been numerous clinical trials to study the treat- ment of large wounds with cultured epidermal cell sheets. 2–4 These investigators reported that cultured au- tografts of keratinocytes had persistent problems, which included blistering, wound contracture, susceptibility to infection, and varying graft “take” rate. The problems were most likely due to the lack of a dermal component and of a mature dermoepidermal junction. 5,6 To over- come these adverse events, artificial dermal substitutes have been used to assist in keratinocyte growth. 7–12 One dermal analog used for treatment of full-thickness wounds is an acellular human cadaver dermis, AlloDerm (LifeCell, Branchburg, NJ). AlloDerm is a durable acellular dermis, which retains extracellular matrix proteins and an intact basement membrane structure, while being surgically manageable. Several investigations have reported on the use of either AlloDerm or deepidermized human dermis in the devel- 1 Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, Ann Arbor, Michigan. 2 Section of Plastic and Reconstructive Surgery, University of Michigan Medical Center, Ann Arbor, Michigan. *This investigation was performed at the Sections of Oral and Maxillofacial and Plastic and Reconstructive Surgery, Depart- ment of Surgery Laboratories, University of Michigan Health Science Center, Ann Arbor, Michigan. Parts of this investigation were presented at the American Association of Oral and Maxillofacial Surgeons Annual Meeting in Boston, September 29, 1999.