(E-193) Investigating the efficacy of probiotic treatment in an in vitro gingival tissue model

Introduction:: Probiotic species Lactobacillus and Bifidobacterium are lactic acid-producing bacteria with great potential towards prevention and restoration of oral inflammatory conditions. Specifically, clinical studies identified that the interactions of Lactobacillus and Bifidobacterium with the oral host could be based on 1) supporting homeostasis of the oral microbiome via competitive inhibition of pathogenic species colonizing the oral epithelium; 2) promoting fibroblast growth; and 3) re-establishing damaged epithelial barrier functions by enhancing immune defense. In view of their beneficial effects, probiotics are currently used as treatment (health supplements or cosmetic products) methods for periodontitis, an oral inflammatory disease affecting 47.2% of adults (< 30 y/o). However, the fundamental mechanisms underlying the action of probiotics on oral health are still not well defined, due to the complexity and microbial inter-variability among different patients, as well as the different methodologies employed for processing bacterial strains (whole cells, lysate and filtrate). Therefore, there is a need for an in vitro, standardized model that allows investigations of probiotics’ role on oral health conditions. This work proposes the utilization of a previously validated in vitro anatomical gingival tissue model with humanized and physiologically relevant features (cyto-and anatomical architectures, oxygen and pH gradients, saliva and mechanical cues) to investigate the role of Lactobacillus rhamnosus (LR) in the form of lysate (LR-L) and filtrate (LR-F) on the in vitro oral mucosa.

Materials and Methods:: An anatomical replica of the lower jaw gingiva was manufactured based on silk protein as a sponge scaffold populated with human primary oral stromal and keratinocyte cells and cultured in static condition for fourteen days. To obtain LR-L and LR-F, LR was cultured for 12 hours until it was within the log (exponential growth) phase, then filtered (0.24 µm) and frozen (LR-F) or directly frozen at -80⁰C (LR-L). Scaffolds were chronically treated with 1:4 LR-L (n=3) and LR-F (n=3) in optimized artificial saliva beginning on day 10 of culture until the end of culture (day 14). pH and oxygen assessments were performed prior and throughout the experiments to monitor any changes within the gingival sulcus of the model as a direct effect of the bacteria. TEER was performed to assess the probiotic impact on the epithelial barrier integrity of the in vitro model (IVM), while lactate dehydrogenase (LDH) and PicoGreen were used to assess cellular cytotoxicity and viability, respectively. Statistical analysis was performed using Student’s T-test and Two-way ANOVA repetitive measurements with a Fisher post-hoc test.




Results, Conclusions, and Discussions:: The results of assessing the probiotic bacteria exposure effect on cellular viability of the IVM indicated that LR-L and LR-F did not have significantly different effects on cellular viability and number, or on epithelial barrier integrity. PicoGreen showed a significant decrease in cell number, and LDH levels decreased significantly compared to not treated (NT) groups due to a local effect of an acidic pH of the LR-F/L treatments. Even so, TEER results report no significant difference between the means of any group (LR-L, LR-F, and not treated (NT)), indicating that no significant difference in effect on the epithelial barrier integrity post-exposure. This demonstrates the beneficial effect of LR-L and LR-F on the oral epithelium. Lastly, a range of 17% to 20% oxygen saturation was maintained throughout the culture and within the range of NT. These findings potentiate the IVM as a viable, robust approach to analyzing the effect of probiotics. However, further work is needed to determine specific mechanisms of action that probiotics have in oral health and disease. The anatomical gingival tissue model has previously been proven to recapitulate the gingival tissue architecture and the clinical inflammatory response in periodontal states using lipopolysaccharide (LPS) portion of Porphyromonas Gingivalis (P.G.) as a surrogate for periodontal disease. To thus investigate the role of action of probiotics, a probiotic mouthwash consisting of Lactobacillus rhamnosus and Bifidobacterium longum in phosphate buffered saline will be used to treat the IVM while in disease states (gingivitis and periodontitis) and viability, morphological, and functional tissue assessments will be performed throughout the culture to assess therapeutic effect. Future studies will focus on the inoculation of healthy and diseased human oral microbiome to study host-pathogen interactions and probiotic efficacy in treatment of disease (gingivitis and periodontitis).

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