Immuno-responsive tissue engineered oral mucosal equivalents containing macrophages

Macrophages play a key role in orchestrating the host immune response towards invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME and to date none have contained primary human macrophages. Here we report the generation of an immuno-competent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of pro-inflammatory cytokine (IL-6, CXCL8 and TNF-α) gene expression and protein secretion as compared to culture as 2D monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate a MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased pro-inflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared to OME alone. Moreover, this pro-inflammatory response was inhibited by pre-treatment with dexamethasone, showing that MDM-OME are also amenable to drug-treatment. Dual-labelled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies.