Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva‐oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host‐microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof‐of‐principle “gingival crevice‐on‐chip” microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On‐chip co‐culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm‐like structures at the tissue‐microbiome interface, long‐term co‐culture, and bacterial clearance secondary to simulated GCF (s‐GCF) flow. Further, on‐chip exposure of the gingival CTEs to the toll‐like receptor‐2 (TLR‐2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s‐GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host‐microbe interactions in periodontal disease and the development of periodontal therapeutics under near‐microphysiological conditions.

Gingival and periodontal ligament fibroblasts are functionally distinct cell types within the dento-gingival unit that participate in host immune response. Their microenvironment influences the behavior and immune response to microbial challenge. We developed three-dimensional gingival and periodontal connective tissue equivalents (CTEs) using human fibrin-based matrix. The CTEs were characterized, and the heterogeneity in their innate immune response was investigated. The CTEs demonstrated no to minimal response to planktonic Streptococcus mitis and Streptococcus oralis, while their biofilms elicited a moderate increase in IL-6 and IL-8 production. In contrast, Fusobacterium nucleatum provoked a substantial increase in IL-6 and IL-8 production. Interestingly, the gingival CTEs secreted significantly higher IL-6, while periodontal counterparts produced higher IL-8. In conclusion, the gingival and periodontal CTEs exhibited differential responses to various bacterial challenges. This gives insights into the contribution of tissue topography and fibroblast heterogeneity in rendering protective and specific immune responses toward early biofilm colonizers.

Gingival connective tissue and its vasculature play a crucial role in the host's immune response against the periodontal microbiome and serve as a bridge between the oral and systemic environments. However, there is a lack of representative models that mimic the complex features of vascularized gingival connective tissue and its interaction with the periodontal microbiome, hindering our understanding of periodontal health and disease. Towards this pursuit, we present the characterization of vascularized gingival connective tissue equivalents (CTEs) as a model to study the interactions between oral biofilm colonizers and gingival tissues in healthy and diseased states. Whole-mount immunolabelling and label-free confocal reflectance microscopy of human fibrin-based matrix embedded with gingival fibroblasts and microvascular endothelial cells demonstrated the generation of bi-cellular vascularized gingival CTEs. Next, we investigated the response of the vascularized gingival CTEs to early, intermediate, and late oral biofilm colonizers. Despite colonization, the early colonizers did not elicit any significant change in the production of the cytokines and chemokines by the CTEs representative of the commensal and homeostatic state. In contrast, intermediate and late colonizers representing a transition to a diseased state exhibited connective tissue and vascular invasion, and elicited a differential immune response accompanied by increased monocyte migration. The culture supernatants produced by the vascularized gingival CTEs in response to early and intermediate colonizers polarized macrophages towards an immunomodulatory M2-like phenotype which activates and protects the host, while the late colonizers polarized towards a pro-inflammatory M1-like phenotype. Lastly, in silico analysis showed a high strength of associations between the proteins and transcripts investigated with periodontitis and vascular diseases. In conclusion, the vascularized gingival CTEs provide a biomimetic in vitro platform to study host-microbiome interactions and innate immune response in periodontal health and diseased states, which potentially paves the way toward the development and assessment of novel periodontal therapeutics.