Gradual Acidification at the Oral Biofilm-Implant Material Interface

The colonization of dental implants by oral biofilms causes inflammatory reactions that can ultimately lead to implant loss. Therefore, safety-integrated implant surfaces are under development that aim to detect bacterial attachment at an early stage and subsequently release antibacterial compounds to prevent their accumulation. Since primary oral colonizers ferment carbohydrates leading to local acidification, pH is considered a promising trigger for these surfaces. As a prerequisite for such systems, the present study aimed at specifically analyzing the pH at the interface between implant material and oral biofilms. For this purpose, in vitro-grown Streptococcus oralis monospecies biofilms and an established multispecies biofilm on titanium discs as well as in situ-grown biofilms from orally exposed titanium-equipped splints were used. Mature biofilm morphology was characterized by live/dead fluorescence staining, revealing improved growth from in vitro to in situ biofilms as well as a general decreasing membrane permeability over time due to the static incubation conditions. For pH analysis, the pH-sensitive dye C-SNARF-4 combined with 3-dimensional imaging by confocal laser-scanning microscopy and digital image analysis were used to detect extracellular pH values in different biofilm layers. All mature biofilms showed a pH gradient, with the lowest values at the material interface. Interestingly, the exact values depicted a time- and nutrient-dependent gradual acidification independently of the biofilm source and for in situ biofilms also independently of the sample donor. After short incubation times, a mild acidification to approximately pH 6.3 could be observed. But when sufficient nutrients were processed for a longer period of time, acidification intensified, leading to approximately pH 5.0. This not only defines the required turning point of pH-triggered implant release systems but also reveals the opportunity for a tailored release at different stages of biofilm formation.