Developing immunoresponsive tissue engineered oral mucosal equivalents to model oral inflammation and local drug metabolism.

Innate immune cells such as macrophages and dendritic cells are recruited to tissue during inflammation, where they play a key role in the detection and elimination of invading organisms and foreign molecules and help orchestrate an immune response. There has been increasing interest in local drug delivery methods to treat inflammatory-mediated and other localised diseases, especially in the oral mucosa, although little research has been undertaken to identify drug metabolism in local tissue, and the potential for recruited inflammatory immune cells to participate in local metabolism. This study quantified expression of xenobiotic metabolising enzymes (XME) in monocyte-derived immune cells and developed a tissue-engineered model of buccal mucosa containing primary macrophages to better model the immune response and to assess drug metabolism in this tissue.

Primary monocytes were isolated from peripheral blood, differentiated into monocyte-derived macrophages (MDM) or dendritic cells (MoDC) and assessed for production of XME by gene array, qPCR, and western blot where they were found to have distinct expression profiles. As immune cells work in concert with other tissue resident cells to generate an immune response, investigations were undertaken to ensure MDM were suitable for inclusion into a tissue engineered oral mucosal model, including culture in a collagen matrix and optimisation of response to stimuli, then an MDM-oral mucosal equivalent (OME) generated by incorporating MDM into a collagen hydrogel with oral fibroblasts then seeded with immortalised oral keratinocytes and cultured at an air-to-liquid interface for 10 days. These models were challenged with Escherichia coli lipopolysaccharide (LPS) ± dexamethasone to examine changes in inflammatory markers.

MDM were suitable for inclusion in a tissue engineered model, as a measurable inflammatory response was conserved in response to E. coli LPS and dexamethasone in both monolayer and within collagen hydrogel. Addition of MDM into an OME had no effect on histology, and MDM-OME were immune-positive for CD68 and epithelial makers. MDM viability was confirmed using CD11c as an MDM-specific marker. MDM-OME responded to LPS with increased gene expression of inflammatory markers, and secretion of TNF-α was increased 10-fold in LPS-treated MDM-OME compared to all other conditions. Gene expression of relevant XME was detected in the MDM-OME, although most were unaltered by treatment conditions.

The data presented in this thesis suggests a potential novel role for inflammatory MDM in local drug metabolism, and further investigations may reveal additional insights which could impact on future drug design rationale. MDM-OME were generated that responded to inflammatory stimuli by shifting to a pro-inflammatory phenotype which was inhibited by a clinically used anti-inflammatory steroid. This immunocompetent oral mucosal model will aid studies that examine efficacy of novel pharmaceuticals and biomaterials and unravel the role of immune cells in local xenobiotic metabolism.