Development and Characterization of a Human Tissue-Engineered Oral Mucosa

Abstract

Open wounds in the mouth should be covered by a graft to prevent microbial infection, excessive fluid loss, foreign material contamination, or relapse (secondary to wound contracture). Currently oral mucosal or skin grafts are used for this purpose; however, both of these grafts require a second surgical procedure and have disadvantages in intraoral use. Split thickness skin grafts may contain adnexal structures, and express a different pattern of surface keratinization. The elective nature of the majority of oral and maxillofacial surgical procedures should allow the flexibility and timing to develop a tissue-engineered oral mucosa. As a tissue-engineered skin has been developed to treat patients suffering burn wounds, chronic ulcers, etc, there is a similar need for the development of a tissue-engineered oral mucosa suitable for intraoral grafting procedures. This graft would be used after major trauma, surgical resections, and for maxillofacial prosthetic surgery. Our research team has been successful in developing an ex vivo produced oral mucosal equivalent (EVPOME) fabricated in an environment free of serum, transformed irradiated feeder cells and pituitary extract. Our EVPOME consists of autologous oral mucosa keratinocytes and an acellular, non-immunogenic dermal equivalent, AlloDerm. The EVPOME was cultured at an air-liquid interface for up to two weeks, resulting in a well-differentiated, parakeratinized epithelial layer similar to native oral mucosa. The expression pattern of differentiation and proliferation markers showed an active and hyperproliferative state. The ultrastructure of EVPOME demonstrated hemi-desmosome-like structures were formed at the interface (basement membrane) of basal keratinocytes and a dermal layer by day 7 at an air-liquid interface. Tissue-engineered cell-based products usually fall under the auspices of the Center for Biological Evaluation and Research (CBER). They are considered a combination product by the Food and Drug Administration (FDA), and need to comply with the regulatory standards for determining safety and efficacy. Animal models are important to determine for conventional safety and efficacy. The SCID mice transplantation model (small animal) allowed us to investigate how EVPOME behaves in an in vivo environment and to determine the optimal stage of the EVPOME for clinical use. A large animal model is also required by the FDA prior to human clinical use; thus we developed a canine EVPOME fabrication protocol that was identical to our human EVPOME protocol. Our canine model enables us to evaluate the fate of EVPOME grafts in situ. Furthermore, the guidelines of CBER require us to demonstrate “dose and potency” of cell-based products, as is seen with drugs. Dose and potency appear to be representative of viable cell numbers and relevant biological function/activity of EVPOME, which provides a quantitative measurement of product quality. These quantitative measurements are challenging and specific for each cell-based product and need to be addressed.