Development and characterization of a canine oral mucosa equivalent in a serum-free environment Junhui Song, 1 Kenji Izumi, 1,2 Thomas Lanigan, 3 Stephen E. Feinberg 1 1 Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0018 2 Department of Oral and Maxillofacial Surgery, Niigata University, Niigata, Japan 3 Center of Gene Therapy, University of Michigan Medical Center, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0018 Received 2 April 2004; revised 3 June 2004; accepted 15 June 2004 Published online 18 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.30144 Abstract: The objectives of this study were to develop a serum-free system for culturing canine oral keratinocytes, the construction and characterization of a canine ex vivo produced oral mucosa equivalent (EVPOME), and transduc- tion green fluorescent protein (GFP) into keratinocytes as a post-grafting tracking marker. Dissociated canine buccal mucosa keratinocytes were cultured in a chemically defined serum-free medium, Epilife™. First-passage keratinocytes were transfected with the GFP gene using a lentiviral vector, sorted by flow cytometer and seeded onto a dermal equiv- alent, AlloDerm to form EVPOMEs. The EVPOME was characterized by histology and immunohistochemistry, for p63, Ki-67, and involucrin. Laser confocal microscopy was used to locate GFP-transfected keratinocytes within the EV- POME. Cultured canine oral keratinocytes grew rapidly over the first three passages and then the proliferative rate decreased. The canine EVPOME formed a well-stratified epithelial layer. The majority of p63 and Ki-67 immunopo- sitive cells were located in the basal layer whereas cytoplas- mic involucrin expression was seen in the suprabasal layers, similar to native canine buccal mucosa. Under laser confocal microscopy, significant green fluorescence was observed throughout the EVPOME. In conclusion, canine EVPOMEs were successfully fabricated in a defined serum-free system with similar characteristics to native buccal mucosa. GFP- transfected canine oral keratinocytes could be identified within the EVPOME. © 2004 Wiley Periodicals, Inc. J Biomed Mater Res 71A: 143–153, 2004 Key words: canine model; keratinocyte; serum-free cul- ture system; ex vivo produced oral mucosa equivalent (EVPOME); green fluorescent protein (GFP) INTRODUCTION Oral and maxillofacial surgeons are frequently con- fronted with a shortage of oral mucosa for reconstruc- tion of the oral cavity. Routine use of split-thickness skin grafts has disadvantages, such as presence of adnexal structures and a different pattern of keratini- zation. Cultured autologous oral keratinocyte sheets and oral mucosa equivalents constructed on collagen dermal equivalents are easily degraded and not suited for intraoral clinical applications. 1,2 We have previ- ously developed a human ex vivo produced oral mu- cosa equivalent (EVPOME) by culturing human oral keratinocytes and seeding them onto a cadaveric der- mal equivalent, AlloDerm (Lifecell Corp., Branch- burg, NJ), in a serum-free culture system without us- ing an irradiated 3T3 xenogeneic feeder cell layer. The seeded human oral keratinocytes formed a stratified epithelial layer on the dermal component of the EV- POME when it was raised to an air–liquid interface, developing into an epithelial layer that was similar to the original oral mucosa used to fabricate it. 3 Subcu- taneous grafting of human EVPOME into SCID mice showed the importance of the presence of a mature epithelia in the growth and formation of microvessels within the dermis of the EVPOME as compared with the dermal equivalent, AlloDerm, lacking an epithe- lial layer. 4 Human EVPOMEs used clinically in pa- tients to reconstruct oral defects showed the persis- tence of epithelial cells on the grafts at 7 days after transplantation by cytological smear. In addition, postoperatively, the epithelial layer of the grafted EV- POME appeared better organized, and the inflamma- tory response in underlying the dermal equivalent was less pronounced than the AlloDerm grafts lack- Correspondence to: S. E. Feinberg; e-mail: sefein@umich.edu Contract grant sponsor: Kato Memorial Bioscience Foun- dation (to KI) Contract grant sponsor: US PHS; contract grant number: NIDCR/NIH R01 DE 13417 (to SEF) © 2004 Wiley Periodicals, Inc.