Development and Characterisation of an Immunocompetent Three-Dimensional Oral Mucosal Infection Model

Abstract

Oral mucosal models are valuable tools for studying oral infection. Existing models have been implemented to study pathogen tissue invasion and resultant damage. These models fail to incorporate an innate immune component such as macrophages, essential for study of host response to infection. Macrophages exist within a regulatory to inflammatory polarisation spectrum, whereby the local microenvironment determines their activity, contributing to the overall behaviour of a tissue.

Three-dimensional oral mucosal models were formed using primary (POK) or immortalized (HaCaT) keratinocytes seeded upon a human gingival fibroblast (HGF) embedded type-1 collagen matrix. Later, HaCaT based models (3DOMMs) were further developed to incorporate THP-1 pro-monocyte cells into the model matrix producing a novel immunocompetent oral mucosal model (IC3DOMM). Models were characterised using a variety of histological, biochemical, and molecular techniques to detail the model microenvironment and assess their capability to detect and respond to Staphylococcus aureus and/or Candida albicans infection.

The HaCaT based 3DOMM was selected to further develop into an IC3DOMM, due to its superior epithelial barrier, and ability to upregulate both IL-6 and IL-8 in response to infection, when compared to the to the POK based P3DOMM. Model-incorporated THP-1 cells increased in size and altered morphology, suggesting differentiation into macrophages. The polarisation of THP-1-derived macrophages was not fully elucidated, as selected markers CD206 and CD163 did not sufficiently discriminate between THP-1 cells, PMA-derived, and VD3-derived macrophages.

Newly highlighted, was the inflammatory environment of the 3DOMM at early and late stage culture. High levels of IL-6 and IL-8 production strongly correlated with LDH release, hence cell death. However this did not detract from the models’ ability to upregulate pro-inflammatory cytokine production in response to infection, and it is proposed that models are used at day 14 post production.

Individual model cell types modulated IL-6, IL-8, and/or TNFα production upon stimulation with supernatant, killed cells, collagen, and differentiation reagents (PMA/VD3), demonstrating the complexity of the tissue-engineered environment. Despite this, it is necessary to consider the model as a whole, not as a sum of individual interactions, as the collective local microenvironment may modulate all cell types contained within the model. Due to the 3DOMMs intact epithelial barrier, ability to upregulate pro-inflammatory cytokine production in response to infection, and confirmed potential to incorporate THP-1-derived macrophages, it is proposed that the novel IC3DOMM is a suitable model for the study of host response to infection.