Immunoresponsive Tissue-Engineered Oral Mucosal Equivalents Containing Macrophages

Abstract

Macrophages play a key role in orchestrating the host immune response toward 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. In this study, we report the generation of an immunocompetent 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 proinflammatory cytokine (IL-6, CXCL8, and TNF-α) gene expression and protein secretion compared with culture as two-dimensional monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate an 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 proinflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared with OME alone. Moreover, this proinflammatory response was inhibited by pretreatment with dexamethasone, showing that MDM-OME are also amenable to drug treatment. Dual-labeled 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. Impact statement Three-dimensional in vitro models of the oral mucosa have been used extensively to investigate the host response to pathogens, but, to date, few have contained primary leukocytes. In this report, we describe the successful incorporation of primary human macrophages into oral mucosal equivalents (OME). These macrophage-containing models were histologically similar to the oral mucosa and immunoresponsive to bacterial lipopolysaccharides by upregulation of key proinflammatory markers. These advanced OME will significantly aid research into host–pathogen interaction, biomaterial toxicity, and drug delivery studies where the presence of an immune cell component is critical to better represent host oral tissue.

An organotypic oral mucosal infection model to study host-pathogen interactions

“…69 Alternative models utilising primary cells, or h-TERT immortalised cells have achieved stratification that is more comparable to their in vivo counterpart. 66,74 Due to the reduced keratinisation, these models better represent the buccal mucosa as opposed to the hard palate as intended. 75 Considering that the common site for DS infection is that underlying the denture plate, in some cases, such as to model initial DS onset, it may be necessary to consider alternative keratinocyte cell types to better represent the keratinised barrier of a healthy oral mucosa, prior to the introduction of colonised denture-mimics.…”

“… 69 Alternative models utilising primary cells, or h-TERT immortalised cells have achieved stratification that is more comparable to their in vivo counterpart. 66 , 74 …”

“…Those that have been developed included Macrophages and Langerhans cells, however to the best of our knowledge, none have been further implemented for the study of pathogen-host interactions. 62 , 64 – 67 Models with the ability to recognise and effectively respond to infection would be informative tools to determine pathogen-host interactions. It is therefore of particular interest to identify any barriers in the current field that may hinder the effective development and implementation of an immunocompetent oral mucosal infection model.…”

Investigating local and systemic Innate Immune Responses in Skin and Oral Mucosa in vitro

“…Geen enkel van deze modellen bevat immuun cellen. Sivard et al en Ollington et al beschrijven veelbelovende orale mucosale modellen voor het testen van stoffen en biocompatibiliteit met respectievelijk ingebouwde uit navelstrengbloed-en monocytafgeleide DC ( Ollington, Colley en Murdoch 2021;Sivard et al 2003). Zowel logistiek als donorvariatie zijn aanzienlijke beperkingen van deze modellen.…”