Our long-term goal is to produce a """"""""smart"""""""" oral mucosal graft, ex vivo, that will be used for reconstruction of major oral defects that are seen secondary to oncologic resection, traumatic events or developmental disturbances. At present, oral mucosa is in limited supply for use in major reconstructive procedures. Other alternatives such as skin introduce disadvantages, such as the presence of adnexal structures and a different pattern of keratinization that can lead to a compromise in oral function. This proposal plans to harvest human oral keratinocytes from keratinized mucosa, expand them in vitro, and seed them onto a cellular, non- immunogenic dermal equivalent, AllodDerm/TM (already approved for human use), to produce a mucosal equivalent with similar anatomic and handling properties as native oral mucosa. The ex vivo produced oral mucosal composite (EVPOMC) will be developed in an environment free of serum and transformed irradiated feeder cells so that it can be used for grafting back into autogenous human recipients. The oral keratinocytes of the EVPOMC will be transfected with a plasmid, using a novel approach to alter cell membrane viscosity, to cause them to over secrete vascular endothelial growth factor (VEGF), thus making this a """"""""smart"""""""" oral mucosal grafting device. We will evaluate vascular ingrowth into the EVPOMC, by histology and immunohistochemistry, to assess the functionality of the inserted plasmid in the device. The safety and efficacy of the device, which is necessary for FDA approval, will be tested in SCID mice and minipigs.
Our specific aims are: 1. Characterize the ex vivo produced human oral mucosal composite. 2. Define optimal parameters for transfection of oral keratinocytes with a cDNA plasmid for VEGF. 3. Develop animal models to a) demonstrate the safety of the transfected oral mucosal device (SCID mouse), and b) show that the transfected oral mucosal device can be grafted successfully to an intra-oral site (minipig). The transfection and grafting of oral mucosal device, in this investigation, will allow us to gather the necessary preclinical data, to use for submission to regulatory agencies, for the translation of this technology into early Phase I human clinical trials.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Special Emphasis Panel (ZDE1-YA (32))
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Kousvelari, Eleni
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University of Michigan Ann Arbor
Schools of Dentistry
Ann Arbor
United States
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Winterroth, Frank; Kato, Hiroko; Kuo, Shiuhyang et al. (2014) High-frequency ultrasonic imaging of growth and development in manufactured engineered oral mucosal tissue surfaces. Ultrasound Med Biol 40:2244-51
Winterroth, Frank; Hollman, Kyle W; Kuo, Shiuhyang et al. (2013) Characterizing morphology and nonlinear elastic properties of normal and thermally stressed engineered oral mucosal tissues using scanning acoustic microscopy. Tissue Eng Part C Methods 19:345-51
Izumi, Kenji; Neiva, Rodrigo F; Feinberg, Stephen E (2013) Intraoral grafting of tissue-engineered human oral mucosa. Int J Oral Maxillofac Implants 28:e295-303
Izumi, Kenji; Marcelo, Cynthia L; Feinberg, Stephen E (2013) Enrichment of oral mucosa and skin keratinocyte progenitor/stem cells. Methods Mol Biol 989:293-303
Marcelo, Cynthia Luz; Peramo, Antonio; Ambati, Amala et al. (2012) Characterization of a unique technique for culturing primary adult human epithelial progenitor/""stem cells"". BMC Dermatol 12:8
Peramo, Antonio; Marcelo, Cynthia L; Feinberg, Stephen E (2012) Tissue engineering of lips and muco-cutaneous junctions: in vitro development of tissue engineered constructs of oral mucosa and skin for lip reconstruction. Tissue Eng Part C Methods 18:273-82
Winterroth, Frank; Hollman, Kyle W; Kuo, Shiuhyang et al. (2011) Comparison of scanning acoustic microscopy and histology images in characterizing surface irregularities among engineered human oral mucosal tissues. Ultrasound Med Biol 37:1734-42
Winterroth, Frank; Lee, Junho; Kuo, Shiuhyang et al. (2011) Acoustic microscopy analyses to determine good vs. failed tissue engineered oral mucosa under normal or thermally stressed culture conditions. Ann Biomed Eng 39:44-52
Winterroth, Frank; Hollister, Scott J; Feinberg, Stephen E et al. (2011) Non-linear stress-strain measurements of ex vivo produced oral mucosal equivalent (EVPOME) compared to normal oral mucosal and skin tissue. Conf Proc IEEE Eng Med Biol Soc 2011:286-9
Tobita, T; Izumi, K; Feinberg, S E (2010) Development of an in vitro model for radiation-induced effects on oral keratinocytes. Int J Oral Maxillofac Surg 39:364-70

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