Development of a Multi-Cellular Tissue Engineered Model of Oral Lichen Planus

Oral lichen planus (OLP) is an immune-mediated mucocutaneous disease of unknown aetiology. There is no cure for OLP and the main treatment aims to relieve symptoms and control the progression. Despite advances in OLP research, investigations into the condition are hampered by the lack of effective experimental models. Tissue-engineered models have been developed, characterised and used in modelling different diseases to study their pathogenesis and test novel treatments. However, there is no OLP model, nor an oral mucosal model that incorporates polarised T cells. Therefore, this project aims to develop tissue-engineered oral mucosal equivalents (OME) containing polarised T cells to replicate OLP for use in the development of novel treatments. Initially, cell culture conditions were defined and optimised before being developed and adapted for complex 3D culture. T cells and keratinocytes were adapted to be cultured in Green’s medium without hydrocortisone with their proliferation and activation status assessed compared to culture in their original medium. T cell viability in collagen hydrogels was assessed over time using flow cytometry and a PrestoBlue metabolic assay, revealing an increase in viability 5 days post-incorporation into the 3D hydrogel. CD4+ and CD8+ T cells isolated from buffy coats showed high purity (85-95%) and viability (>90%). Upon activation with CD3/CD28 Dynabeads, CD69 levels were increased when analysed by flow cytometry. T cells were cultured in conditions to polarise naive CD4+ T cells into Th1 or Th17 and CD8+ T cells into cytotoxic T cells. Phenotypes were confirmed using flow cytometry, ELISA, qPCR and western blot. Th1 cells secreted IFN-γ and TNF-α, key cytokines for Th1 and showed a higher expression of Th1 transcriptional factor, T-bet. 92% of CD8+ T cells produced granzyme B. Full-thickness OMEs were created by seeding immortalised oral keratinocytes (FNB6) on top of normal oral fibroblast-containing hydrogels before being raised to an air-liquid interface (ALI). H&E staining of the OME revealed a multi-layered stratified squamous epithelium on top of a fibroblast-populated connective tissue. To simulate the inflammatory mucosa as in OLP, OME were cultured with IFN-γ and TNF-α. This led to a hyperproliferative epithelium and an increase in inflammatory markers, mainly chemokines relevant to T-cell chemotaxis in OLP. A chemotaxis assay was employed to investigate T cell migration towards a chemoattractant gradient and revealed that conditioned medium from TNF- and IFN--stimulated OME caused the T cells to migrate towards the chemokine gradient in a directional manner. Finally, a model of T cell-mediated inflammatory oral mucosal disease was developed in vitro by combining OME on top of a T cell-containing hydrogel, followed by stimulation with IFN-γ and TNF-α. Histological staining of the disease model revealed basement membrane destruction and basal cell layer apoptosis. 48 hours after stimulation, the recruitment of T cells towards the epithelium was evident and confirmed by immunohistochemical (IHC) staining for CD4 and CD8. Tissue destruction was shown to progress gradually over time and the OME maintained high levels of CCL5, CXCL9, and CXCL10 secretion until day 25. The disease model was validated against OLP patient biopsies with PAS staining and IHC staining for laminin, and integrin evidencing basement membrane damage and for cleaved-caspase-3 confirming the presence of apoptosis in the basal keratinocytes. In summary, this study reports the development of a complex T cell-mediated disease model that displayed basal cell apoptosis, T cell infiltration and basement membrane destruction, which are key characteristics of OLP. This novel preclinical disease model has great potential to probe for molecular mechanisms of disease and to be used as a platform to test for novel therapeutics and modes of treatment.