Oral health depends on a complex interplay between the mucosal tissues, physicochemical and microbial components present in the oral cavity. Maintenance of a stable ecosystem is an essential determinant of oral health. However, as a result of a major change in the ecosystem, the stability can be disturbed leading to an increased risk for development of diseases. Epithelial integrity and barrier function are key aspects to support oral health. The oral cavity consists of stratified squamous epithelium that provides a physical barrier that protects the underlying tissues from mechanical or chemical damage, loss of fluids, and invasion from pathogenic bacteria. In order to maintain barrier function, the oral mucosa undergoes constant renewal and repair. When the epithelial integrity is compromised, prompt re-epithelialization is required to restore homeostasis by re-establishment of cell-cell contacts. This thesis describes the development of a quantitative high-throughput scratch assay to study oral re-epithelialization in vitro as well as the influence of different components of the oral ecosystem (i.e. oral commensal bacteria and salivary metabolites) on gingival re-epithelialization.
In Chapter 2, we describe the development and optimization of an automated high-throughput scratch assay that uses a mathematical model to extract biologically relevant parameters that describe re-epithelialization kinetics. The assay was used to screen 39 lactic acid bacteria (LAB) for their influence in re-epithelialization and therefore, assess their potential to promote gingival epithelial re-epithelialization. The results revealed that exposure of the cells to different Streptococcus salivarius strains leads to enhanced re-epithelialization kinetics. In particular, we showed that S. salivarius MS-oral-D6 is able to stimulate re-epithelialization through a secreted serine protease. Furthermore, in Chapter 3, we developed and implemented the Kinetic Re-Epithelialization Analysis Pipeline (KREAP) into a toolbox in Galaxy, providing an open-source, web-based platform for reproducible image processing and data analysis of high-throughput scratch assays. In addition, Chapter 4 provides a detailed procedure of the assay using cell lines originating from oral mucosa as well as from skin, which expands the application of the assay to other research fields.
In Chapter 5, we explored the role of the salivary metabolome on oral re-epithelialization using our scratch assay and the metabolite profiles of a subset of 63 unstimulated saliva samples collected from a healthy cohort (n = 61) during a two-week experimental gingivitis study. Elastic net regression with stability selection led to the identification of a metabolite signature consisting of 10 metabolites that were related to the re-epithelialization kinetics observed in vitro. Using this signature, we were able to predict the re-epithelialization capacity of the remaining 242 saliva samples collected during the clinical study. Higher concentrations of certain plasmalogens, diacylglycerol, and amino acid derivatives in saliva were associated with enhanced re-epithelialization capacity. Moreover, the predicted re-epithelialization capacity of the saliva samples was positively correlated with the gingival bleeding scores determined for the participants during the experimental gingivitis challenge. These scores are used as a clinical parameter to assess the inflammatory state of the periodontal tissues and thus, we propose that the identified metabolite signature reflects the intensity of the mucosal interactions with the resident microbiota. In addition, we found that individuals who displayed larger variation of the metabolite signature over time were associated with a higher increase in gingival bleeding scores during the experimental gingivitis challenge, implying that unstable host-microbe interactions and higher variability of the associated metabolite signature may be an indicator for stronger responses under a challenge.
In Chapter 6, we employed elastic net regression with stability selection to identify operational taxonomical units (OTUs) that were positively and negatively associated with gingival bleeding scores in the participants of a cross-sectional study (n = 268). Representative species of the identified OTUs were screened for their influence in oral re-epithelialization using the high-throughput scratch assay. The results revealed that the group of bacteria that had a negative association with gingival bleeding significantly increased re-epithelialization kinetics in comparison with the positive associated group. Importantly, most of these associations were confirmed in a different cohort that participated in a two-week experimental gingivitis challenge study. Higher relative abundances of Actinomyces oris/viscosus/naeslundii, Rothia dentocariosa and Veillonella dispar were strongly correlated with lower gingival bleeding scores, whereas higher relative abundances of Porphyromonas catoniae, Selenomonas sputigena, Leptrotichia buccalis and Streptococcus anginosus were associated with increased gingival bleeding. Additionally, we showed that microbial signatures in saliva can potentially be used to assess the risk of an individual to develop oral diseases, such as gingivitis. Chapter 7 explored the differential modulatory mechanisms exerted by Actinomyces oris, Actinomyces viscosus, Veillonella parvula and Fusobacterium nucleatum subsp. animalis on gingival re-epithelialization kinetics. Cell-based assays and transcriptomic analysis of scratched gingival cells revealed that treatment with A. oris, A. viscosus or V. parvula enhanced re-epithelialization kinetics by increasing cell survival through downregulation of the p53 signalling pathway. In contrast, the increased re-epithelialization kinetics observed with F. nucleatum subsp. animalis was characterized by a stronger induction of regulatory genes involved in cell proliferation, apoptosis and innate immunity responses. Lastly, we showed that the stimulatory effects observed on cell migration by A. viscosus and F. nucleatum subsp. animalis depend on direct bacteria-host cell contact, whereas A. oris and V. parvula probably secrete soluble compounds that act as chemoattractants for gingival cells.
Chapter 8, summarizes and discusses the key findings of this thesis and provides new avenues for further research. This thesis provides insight in the complex interplay between the oral mucosa and different components of the oral ecosystem that may lead to the development of innovative strategies that could support oral health through the maintenance of epithelial integrity and barrier function. Such strategies may involve the development of novel diagnostic tools to predict disease risk as well as the generation of next-generation probiotics or bioactive compounds to stimulate re-epithelialization in patients with impaired wound healing.