Introduction: Tissue-engineered oral mucosa (TEOM) is increasingly being used to assess drug delivery and toxicity, as well as for modeling oral diseases. Current TEOM models are constructed using primary oral fibroblasts and keratinocytes that display donor-to-donor variability and whose widespread use is restricted by availability and ethic limitations. To address these issues, an attractive approach is the development of TEOM using immortalised cells.
Aim: This study aimed to construct and characterise TEOM based on TERT2-immortalised oral keratinocytes (FNB6) cells and use these TEOM to assess the toxicity and delivery of corticosteroids using a novel electrospun-based oral patch.
Methods: TEOM were constructed by culturing immortalised FNB6 oral keratinocytes on top of a normal oral fibroblast (NOF)-populated collagen type 1 hydrogel in tissue culture transwell inserts at an air-to-liquid interface (ALI) for up to 14 days. The TEOM were characterised using histological, immunohistological, ultrastructural (TEM), tissue viability (AlamarBlue), trans-electrical resistance (TEER), and permeability (FITC-dextran) analysis. Cytotoxicity assessment of seven corticosteroids was performed using MTT assay on monolayer cultures (FNB6 and NOF cells) and TEOM. Novel mucoadhesive bilayer patches containing clobetasol 17-propionate (CP) were subjected to morphological, physicochemical, drug release, swelling and cytotoxicity analysis. In vitro permeation studies of the corticosteroids against TEOM was measured using HPLC. The immunosuppressive effect of delivered CP against activated Jurkat T-cells was assessed by measuring changes in interleukin-2 (IL-2) release.
Results: Histologically, TEOM mimicked native oral mucosa displaying a stratified epithelium, fibroblast-containing connective tissue and basement membrane. IHC revealed the expression markers for differentiation (cytokeratin 4,13,14), proliferation (Ki-67), cell adhesion (E-cadherin, claudin-4). Furthermore, TEM confirmed the presence of desmosomes and hemidesmosomes in the epithelium. Maximal TEOM viability was found up to day 25 and maximal TEER value was exhibited at day 20 (155.8 Ω.cm2). Permeability analysis showed that only small molecules (3 kDa) could pass through the epithelium. Differential drug sensitivity of corticosteroids against monolayer cultures was ranked as follow; CP > BU > BD > BV > TA > HV > HB by IC50 value, and this was similar for TEOM although IC50 values were higher for 3D models. Novel mucoadhesive bilayer patches containing CP exhibited good physicochemical characteristics and drug release profiles. Toxicity testing to the OECD standard revealed that patch delivered CP was considered a non-irritant. Oral mucosal delivered CP using liquid or patch formulation into the TEOM tissue or receptive medium was both dose and time-dependent. In addition, both liquid and patch delivered CP significantly reduced the secretion of IL-2 by activated Jurkat T cells in a TEOM model replicating an oral inflammatory disease.
Conclusion: FNB6 TEOM models are able to mimic the native oral mucosa and have the potential to be used for drug delivery and toxicity evaluation. Oral patch-delivered CP was able to cross the TEOM and inhibit the IL-2 secretion of activated T cells, suggesting that this mode of drug delivery could be used to treat oral inflammatory diseases.
