Microsensor Measurements of Metabolic Activities (O₂, pH) Inside Dental Biofilms

Objectives: Dental biofilms display spatial heterogeneity on the microscale with respect to oxygen (O₂) and pH which allows micro-organisms with different metabolic requirements to coexist. The aim of this study was to investigate metabolic activities in dental biofilms in situ using a microsensor technique before and during sucrose exposure. Methods: An intraoral splint supplied with standardized human enamel slivers was worn for 3d/4d to obtain in vivo generation of dental biofilm. After staining (LIVE/DEAD BacLight Viability Kit) they were investigated by CLSM. Biofilm thickness (BT) and viability (V) were measured. Furthermore, the biofilms were analyzed ex vivo by means of a flow cell setup. Microelectrodes were used to measure O₂-concentration and pH at high spatial resolution within the biofilm (profiles). Dynamic responses (time series) were recorded at the surface (O₂) or at the base (pH) of the biofilm. Measurements were performed in sterile human saliva as medium (control) and with addition of 10% sucrose to stimulate activity. Results: CLSM analysis revealed a BT of 300µm and 65% V. Without external substrate present O₂ penetrated 175-200µm into the biofilm and pH was about 7.1. Upon sucrose addition O₂-penetration was lowered to 100µm and the concentration at the surface decreased within seconds from 35% air saturation to 23%. A strong pH decrease to 4.4 was obtained in a depth of 200µm. 50% of this response occurred within about 2min. Conclusion: O₂-consumption inside the biofilm was high causing limited penetration into deeper layers. Almost half of the biofilm was anoxic. Sucrose addition led to a drastic change of the local environment with an increase of the anoxic areas and a decrease in pH. Thus, the microsensor approach is suitable for real time non destructive in situ analysis of dental biofilms grown in vivo and monitoring changes in the micro-environment.