An in vitro model demonstrating homeostatic interactions between reconstructed human gingiva and a saliva-derived multispecies biofilm

Abstract

Background: In the oral cavity, host-microbe interactions (HMI) continuously occur and greatly impact oral health. In contrast to the well-studied disease-associated HMI during, for example, periodontitis, HMI that are essential in maintaining oral health have been rarely investigated, especially in a human-relevant context. The aim of this study was to extensively characterize homeostatic HMI between saliva-derived biofilms and a reconstructed human gingiva (RHG). RHG was reconstructed following the structure of native gingiva, composed of a multilayered epithelium formed by keratinocytes and a fibroblast-populated compartment. To mimic the oral environment, RHG were inoculated with pooled human saliva resuspended in different saliva substitute media and incubated for 2 or 4 days. The co-cultured biofilms were retrieved and characterized by viable bacterial counting and compositional profiling (16S rRNA gene sequencing). RHG was investigated for metabolic activity (MTT assay), tissue histology (hematoxylin and eosin staining), epithelial proliferation (Ki67 staining), antimicrobial peptide expression, and cytokine secretion.

Results: Viable biofilms were detected up to day 4 of co-culturing. Bacterial counts indicated biofilm growth from the inoculation to day 2 and maintained thereafter at a similar level until day 4. All biofilms shared similar composition throughout 4 days, independent of co-culture time and different saliva substitute media used during inoculation. Biofilms were diverse with Streptococcus, Haemophilus, and Neisseria being the dominating genera. While supporting biofilm development, RHG displayed no significant changes in metabolic activity, tissue histology, or epithelial proliferation. However, in the presence of biofilms, the antimicrobial peptides elafin and human β-defensin-2 were upregulated, and the secretion of cytokines IL-6, CXCL1, CXCL8, CCL5, and CCL20 increased.

Conclusion: This model mimicked homeostatic HMI where a healthy gingiva supported a viable, diverse, and stable microbial community, incorporating bacterial genera found on native gingiva. The gingiva model maintained its tissue integrity and exerted protective responses in the presence of biofilms over time. This study adds to the evidence that shows the important role of the host in maintaining homeostatic HMI that are essential for oral health. Video Abstract.

Keywords: 16S rRNA gene sequencing; Biofilms; Gingiva; Host-microbe interactions; Organotypic model.

Fig. 1

Comparison of three in vitro biofilm-culturing models. a Setup of the three in vitro biofilm-culturing models. b Viable counts of bacteria derived from the same saliva inoculum after 2 or 4 days in each model. Counts of the AAA model are marked with a gray background as HMI and agar models were inoculated with similar bacterial amount (approximately 10⁴), while AAA model was inoculated using a different procedure (details are described in the “Methods” and in [42, 43]). Each symbol represents the mean of two replicates from one independent experiment. The lines represent the average of at least three independent experiments per condition. AAA, Amsterdam Active Attachment model; MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium; RHG_M, RHG differentiation medium without penicillin-streptomycin and hydrocortisone

Fig. 2

Metabolic activity, tissue histology, and proliferation of RHG in the HMI model. a Metabolic activity of RHG on day 2 and day 4. All values are calculated relatively to the unexposed condition, shown as the first white bar (-) in both graphs. The rest of the white bars in each graph, labelled as biofilm (−), were conditions only exposed to agar supplemented with corresponding media. Data are shown as the mean ± SEM from four independent experiments, each performed in duplicate. b Hematoxylin and eosin (H&E) and Ki67 staining of RHG in the HMI model for 2 or 4 days. The photos represent the average of at least three independent experiments per condition. (-), unexposed; MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium

Fig. 3

Characterization of the HMI biofilms. a Representative FISH staining shows the presence of biofilms on RHG on day 2 (16S probe for all bacteria in green, DAPI in blue). b Viable bacterial counting of saliva inocula (Ino) and HMI biofilms on day 2 and day 4. Each symbol represents the count of inocula or the mean of two replicates of HMI biofilms from one independent experiment. c Relative abundance of major bacterial genera or higher taxa in the pooled data of saliva inocula (Ino) and HMI biofilms (day 2 and day 4). Eleven genera were identified at a relative abundance higher than 1% in any of the samples. The remaining 101 low abundance taxa are shown as one group (others). MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium

Fig. 4

The influence of different conditions on the HMI biofilm profiles. a Number of observed OTUs and Shannon index in all conditions. b Principal component analysis plots of microbial samples including saliva inocula (left) or excluding saliva inocula (right, only HMI biofilms). c OTUs that differentiated the most among all microbial samples ranked by LEfSe. Data are collected from four independent experiments, each performed in duplicate per condition. *Represents statistically significant difference between Ino and HMI biofilms. **P < 0.01, ***P < 0.001, ****P < 0.0001. Ino, saliva inocula; MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium

Fig. 5

Immunohistochemical staining of RHG in the HMI model. Expression of elafin and hBD2 in RHG co-cultured with (+) or without (−) biofilms for 2 or 4 days. Representative staining pictures are shown, from four independent experiments, each with an intra-experimental replicate. Biofilm (−), biofilm-negative condition; biofilm (+), biofilm-positive condition; MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium

Fig. 6

Cytokine secretion of RHG in the HMI model. After exposing RHG to different biofilm conditions, cultures were refreshed with new medium every 24 h, and culture supernatants were collected and used to assess secretion of IL-6, CXCL1, CXCL8, CCL5, and CCL20 by means of ELISA. The unexposed condition is shown as the first white bar (-) in all graphs. The rest of the white bars in each graph, labelled as biofilm (−), were conditions only exposed to agar supplemented with corresponding media. Data represent the mean ± SEM from four independent experiments, each performed in duplicate. Differences were considered significant when P < 0.05. *Represents statistically significant difference between one biofilm-positive condition and its corresponding biofilm-negative condition with the same saliva substitute. *P < 0.05; **P < 0.01; ***P < 0.001. Biofilm (−), biofilm-negative conditions; biofilm (+), biofilm positive conditions; (-), unexposed; MB, McBain medium; AS, artificial saliva medium; FSM, filtered saliva plus mucin medium