Understanding the ecology and communication mechanisms between the human host and its microbiota in health and disease states is still a challenge in both clinical and in vitro applications. Among the diverse ecological niches in the human body, the periodontal pocket of the human gingiva is unique in its characteristics. Microenvironmental stimuli (salivary flow, drinking, chewing) and cyto-anatomical features (tissue organization and gradients of oxygen, pH, metabolites) in the periodontal pocket promote the formation of distinct microenvironments in which polymicrobial communities organize into compositionally different biofilms forming the microbiome. Within the pocket, these communities establish mutualistic and coevolutionary interactions with the host and are critical to preserve oral health. Despite research efforts, gingival host-microbiome interactions are still facing several challenges when investigated in vitro. Traditional tissue engineering strategies (e.g., 2D or 3D planar models) are not able to replicate in a single model the structural, physical, and metabolic conditions present in the gingival pocket, which are essential to support long-term in vitro investigations of host-microbiome interactions. Being able to mimic and decipher these interactions is of uttermost importance, as when a biofilm shifts towards a dysbiotic and pathogenic state, unbalanced interactions with the host occur causing oral diseases (gingivitis, periodontitis). If healthy interactions are not reestablished, the pocket can become a reservoir of opportunistic pathogens potentially contributing to non-oral diseases (Inflammatory Bowel Disease, Alzheimer’s Disease, arthritis), therefore, affecting overall human health.

To fill this gap, our group in collaboration with the ADA Forsyth Institute has tooled up to develop the first in vitro oral tissue model (OTM) able to mimic host (gingiva, periodontal pocket) and healthy patient-derived subgingival plaque interactions for seven days. In this work, we provide a new set of tissue engineering tools by combining structural biopolymer design and advanced nanomanufacturing techniques to mimic a living replica of the gingival tissue. Our OTM features a complex three-dimensional architecture of the human gingiva, establishing long-term interactions between the gingival sulcular epithelium and the subgingival plaque microbiome isolated from periodontally healthy human patients. The key elements of the OTM are the reproduction of the cyto-anatomical features of the periodontal pocket, the maintenance of the whole subgingival microbiome rather than selected species, the mimicking of salivary flow and composition by artificial saliva formulation in a custom-made bioreactor, and the incorporation of mouthwash rinses as an oral hygiene practice. 

The present study provides an analysis on both host and microbiome. We successfully preserved long-term (7 days) host viability and functional response (barrier integrity, immune regulation) in presence of a complex microbiome by supporting host-microbiome equilibrium via mouthwash rinse, pH buffering and oral clearance via salivary formulation and shear dynamic. In response to the bacterial challenge, we profiled initial (pro-inflammatory) and prolonged (anti-inflammatory) host’s response similar to that in vivo, further suggesting the capability of the OTM to be integrated with immune cells. Establishment of homeostatic conditions were also supported by the ability of the OTM to preserve a complex (diverse and eubiotic) human oral microbiome in vitro, with species segregating according to their oxygen tolerance. Lastly, the OTM sustained interbacterial dialogues further substantiated the need to utilize the whole microbiome to replicate microbe-microbe and host-microbe networking in the periodontal niche.

Taken together, this data supports the engineering of a physiologically relevant human OTM, demonstrating for the first time the ability of an in vitro model to sustain long-term (7-day) host-microbiome interactions. The OTM represents a breakthrough in the field of oral tissue engineering, as it allows to investigate in a simple, yet relevant, in vitro model the mechanisms and biomarkers that could result in disruption of homeostasis. OTM makes possible long-term in vitro cultivation of complex microbiomes and incorporates oral hygiene practices for the first time without compromising tissue viability. Although this study represents a comprehensive analysis of sustained interactions between host and healthy microbiome, the OTM can offer more. The OTM can be used as a platform to study microbial assembly, metabolite analysis, and host and microbiome responses to perturbation events such as antibiotics, anti-inflammatories, probiotics or microbial species enrichment/depletion. The scientific efforts provided by the OTM can underpin the development of intervention strategies to promote overall human health.