Development and Characterization of a Tissue-engineered Human Oral Mucosa Equivalent Produced in a Serum-free Culture System

Abstract

A problem maxillofacial surgeons face is a lack of sufficient autogenous oral mucosa for reconstruction of the oral cavity. Split-thickness or oral mucosa grafts require more than one surgical procedure and can result in donor site morbidity. Skin has disadvantages of adnexal structures and a different keratinization pattern than oral mucosa. In this study, we successfully assembled, ex vivo, a human oral mucosa equivalent, consisting of epidermal and dermal components, in a defined, essential-fatty-acid-deficient, serum-free culture medium without a feeder layer, that could be used for intra-oral grafting in humans. Autogenous oral keratinocytes were seeded onto a cadaveric dermis, AlloDerm. The oral mucosa equivalent was cultured at an air-liquid interface for 2 wks. The resulting equivalent had a well-stratified parakeratinized epithelial layer similar to native oral keratinized mucosa. Expression of differentiation markers, filaggrin and cytokeratin 10/13, suggested a premature keratinized state. The presence of proliferation markers, proliferating cell nuclear antigen (PCNA) and Ki-67, suggested a state of hyperproliferation. Fatty acid composition of the equivalent was similar to that of in vitro cultured oral keratinocytes but differed from the that of in vivo native tissue, showing a lower content of 18:2 and 20:4, and a higher content of 16:1 and 18:1 fatty acids, respectively. The keratinocytes of the equivalent appeared to be in a more active and proliferative state than native keratinized mucosa. The dynamic nature of the cell population on the oral mucosa equivalent may be beneficial for intra-oral grafting procedures and for transfection of the keratinocytes.

Evaluation of Transplanted Tissue-Engineered Oral Mucosa Equivalents in Severe Combined Immunodeficient Mice

“…18 Briefly, human oral keratinocytes were generated and amplified from trypsinized discarded human oral mucosa obtained by routine dentoalveolar surgical procedures. AlloDerm (LifeCell) was rehydrated in phosphate-buffered saline (PBS) 1 h before use.…”

“…[13][14][15][16][17] We reported on the characteristics of an ex vivo-produced oral mucosa equivalent (EVPOME) utilizing AlloDerm in a serum-free, defined medium, without an irradiated xenogeneic feeder layer. 18 In preparation for human clinical trials it was necessary to optimize the culture protocol for the in vivo grafting of EVPOME. In this investigation, we evaluated EVPOME by transplanting it into immunologically compromised (SCID, severe combined immunodeficient) mice, using the technique of Barrandon et al, 19 which places the graft into a subcutaneous dorsal skin pouch of athymic mice.…”

“…18 For detection of mouse vascular endothelial cells, immunohistochemical staining for Triticum vulgaris (wheat germ agglutinin, WGA) lectin binding was used to determine revascularization by counting the number of microvessels within the dermal component. 20 Sections were first treated with 2% hydrogen peroxide in methanol for 30 min to inhibit endogenous peroxidase, followed by 10% bovine serum albumin (BSA; Sigma, St. Louis, MO) for 1 h. The sections were then incubated with biotinylated WGA antibody (Sigma), diluted 1:20 in 1% BSA, for 1 h at 37°C and washed in phosphate-buffered saline (PBS), followed by the ABC method (Vector Laboratories, Burlingame, CA) for 10 min.…”

Strategies for Oral Mucosal Repair by Engineering 3D Tissues with Pluripotent Stem Cells

“…36 Oral tissues can be generated by harvesting cells from the oral cavity, expanding them, and growing them in a 3D tissue before engraftment. 37,38 Recently, 3D, skin-like tissues have been approved for clinical use in the treatment of periodontal disease. 39 Levenberg et al first established proof-ofconcept that ectodermal and mesenchymal cells differentiated from hESCs could assemble into tissues displaying in vivo-like features and could integrate into the host vasculature.…”

Engineered Keratinized Oral Mucosa Decreased C. albicans Transition Through the Production of Keratins 10, 14, 16, and 19 by Oral Epithelial Cells

“…Tissue was grown until the epithelial cells reached confluence. To mimic the keratinized palatal mucosa tissue, EHOM were then raised to an air-liquid interface for 5 more days to allow the epithelium to stratify [27,28]. Following this process, as we have shown previously, the EHOM was a well-organized and stratified tissue in which epithelial cells expressed proliferating keratins such as Ki-67, K14, and K19 and also differentiating keratin K10.…”