A Long-Term Bioreactor-based In Vitro Oral Cavity Model to Study Host-Microbiome Interactions

Oral Cancer Awareness Month

The oral cavity is home to a diverse community of microorganisms—bacteria, fungi, and viruses—collectively known as the oral microbiome. When host-microbiome interactions function properly, they support both oral and overall health; however, imbalances can contribute to conditions like periodontitis (gum disease) and may even increase the risk of oral cancer.

Oral Cancer Awareness Month is an opportunity to highlight the importance of maintaining a healthy oral microbiome. Good oral hygiene not only protects against common dental issues but also helps support systemic health and may reduce the risk of oral cancer.

Despite the critical role of host-microbiome interactions in oral health, our understanding of this complex relationship remains limited. This is largely due to the lack of experimental in vitro models that mimic the structural, physical, and metabolic conditions present in the oral cavity to support the long-term investigation of host–pathogen interactions. In our featured publication, researchers introduce a novel humanized three-dimensional gingival model designed to address these challenges.

Development of a Physiologically Relevant 3D Gingival Tissue Model

Adelfio et al. previously developed a three-dimensional (3D) humanized gingival tissue model using natural proteins to replicate the anatomical structure of adult gingiva. This model sustained interactions between human gingival cells and patient-derived microbiomes for 24 hours while preserving microbial diversity. In this study, the authors extended the model’s capabilities for long-term studies (>24 hours) by integrating it into a bioreactor system that mimics salivary flow, shear stress, and buffering action, with oral hygiene simulated by rinsing with a commercial mouthwash. This oral tissue model (OTM) provides an in vivo-like environment for the investigation of long-term interactions between patient-derived gingival tissue and the oral microbiome.

OTM model establishment

The oral tissue model (OTM) was developed using a biomimetic silk-based scaffold seeded with primary gingival stromal and keratinocyte cells from Lifeline Cell Technologies to replicate the anatomical and functional characteristics of native gingival tissue. Patient-derived subgingival microbiomes were inoculated into periodontal pockets and cultured in a controlled bioreactor system that simulates salivary flow and mechanical stimulation to maintain long-term host-microbiome interactions. Oral hygiene was mimicked on days 3 and 5 with commercial mouthwash rinses.

Host–microbiome interactions within the OTM

This oral tissue model (OTM) shows pro-inflammatory responses to microbial challenge (day 1 post-inoculation), sustained physiological anti-microbial activity, cytokine responses similar to pre-clinical in vivo features, and the maintenance of a viable and eubiotic microbiome.

Early host microbiome responses: At 72 hours, the host tissue maintained viability (LDH) and barrier integrity, and the OTM supported a proliferative (DAPI+/Ki67+) and differentiated (DAPI+/Ki67−) epithelium, suggesting epithelium robustness to microbial challenge (DAPI) and self-renewal capability (Ki67).

Long-term host microbiome responses: Following the early eubiotic response and sustained epithelial barrier function, the researchers examined longer-term (seven-day) host-microbe interactions. Epithelium stratification remained intact, with viability preserved throughout the culture period. An initial spike in cytotoxicity (LDH) stabilized over time, while barrier integrity (as shown by E-Cadherin staining) was maintained. The model exhibited antimicrobial peptide (hBD2) secretion in response to microbial inoculation, alongside distinct cytokine expression patterns over time. Hierarchical clustering revealed cytokines with transient or sustained increases post-inoculation, while others declined. These findings confirm the OTM’s ability to maintain oral epithelial function and host-microbiome interactions for at least seven days.

Subgingival plaque microbiome viability and distribution

Microbial viability within the OTM was assessed using flow cytometry, CLSM, and SEM. Flow cytometry showed a stable viable microbial population from inoculation (79.1%) through day 3 (66.6%) and day 7 (77.8%), indicating that salivary flow stimulation and oral hygiene procedures did not negatively impact viability. CLSM confirmed microbial distribution within the scaffold, while SEM revealed epithelium formation and a mature biofilm with distinct bacterial populations (cocci in upper regions, rods in lower regions). These findings demonstrate that the OTM supports microbial viability, spatial organization, and diversity under physiologically relevant conditions.

On day 7, microbial communities from three OTM regions were sequenced and compared to the initial inoculum to assess microbiome stability. Relative abundance analysis confirmed the presence of key oral genera, including Peptostreptococcus, Streptococcus, Veillonella, Neisseria, Campylobacter, Fusobacterium, Capnocytophaga, and Prevotella, indicating the model’s ability to sustain a diverse and representative oral microbiome over time.

This new in vitro model successfully establishes a symbiotic relationship between the host and microbiome. In the future, this in vitro system can be used explore the link between dysbiosis and inflammation and to test strategies for restoring oral homeostasis in periodontal diseases and beyond.

Lifeline Cell Technology Oral Cells and Media

Lifeline Cell Technology offers a comprehensive portfolio of primary human oral cells and specialized media to support research in oral biology, tissue engineering, and disease modeling including:

• Human Oral Keratinocytes (Gingiva)
• Human Gingival Fibroblasts
• DermaLife K Keratinocyte Medium Complete Kit
• FibroLife S2 Fibroblast Medium Complete Kit
• FibroLife Fibroblast Serum Free Medium Complete Kit

Explore our blog to see how our cells and culture media are advancing biomedical research worldwide. If you have used our products in your publication, we’d love to feature your work here!