Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.

Peri-implant infections are the most common cause of implant failure in modern dental implantology. These are caused by the formation of biofilms on the implant surface and consist of oral commensal and pathogenic bacteria, which harm adjacent soft and hard tissues and may ultimately lead to implant loss. In order to improve the clinical situation, there has to be a better understanding of biofilm formation on abiotic surfaces. Therefore, we successfully developed a system to cultivate an oral multispecies biofilm model in a flow chamber system, optimized for the evaluation of biofilm formation on solid materials by direct microscopic investigation. The model contains four relevant oral bacterial species: Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis in ratios similar to the native situation. The reliability of the developed “Hanoverian Oral Multispecies Biofilm Implant Flow Chamber” (HOBIC) model was verified. Biofilm volume and live/dead distribution within biofilms were determined by fluorescence staining and confocal laser scanning microcopy (CLSM). The individual species distribution was analyzed using quantitative real time PCR with propidium monoazide pretreatment (PMA-qRT-PCR) and by urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH). This in vitro model may be used to analyze biofilm formation on dental implants in more detail and to develop future implant systems with improved material properties.

Background: Excessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS^®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time. Methods: An established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS^® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR. Results: REPHA-OS^® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol. Conclusion: The strong antibacterial effect of REPHA-OS^® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

Periodontitis and peri-implantitis are inflammatory conditions with a high global prevalence. Oral pathogens such as Porphyromonas gingivalis play a crucial role in the development of dysbiotic biofilms associated with both diseases. The aim of our study was to identify plant-derived substances which mainly inhibit the growth of “disease promoting bacteria”, by comparing the effect of Rheum palmatum root extract against P. gingivalis and the commensal species Streptococcus oralis. Antiplanktonic activity was determined by measuring optical density and metabolic activity. Antibiofilm activity was quantified using metabolic activity assays and live/dead fluorescence staining combined with confocal laser scanning microscopy. At concentrations of 3.9 mg/L, R. palmatum root extract selectively inhibited planktonic growth of the oral pathogen P. gingivalis, while not inhibiting growth of S. oralis. Selective effects also occurred in mature biofilms, as P. gingivalis was significantly more stressed and inhibited than S. oralis. Our studies show that low concentrations of R. palmatum root extract specifically inhibit P. gingivalis growth, and offer a promising approach for the development of a potential topical agent to prevent alterations in the microbiome due to overgrowth of pathogenic P. gingivalis.

Periodontitis is a common global disease caused by bacterial dysbiosis leading to tissue destruction, and it is strongly associated with anaerobic bacterial colonization. Therapeutic strategies such as oxygen therapy have been developed to positively influence the dysbiotic microbiota, and the use of oxygen-releasing substances may offer an added benefit of avoiding systemic effects commonly associated with antibiotics taken orally or hyperbaric oxygen therapy. Therefore, the oxygen release of calcium peroxide (CaO₂) was measured using a dissolved oxygen meter, and CaO₂ solutions were prepared by dissolving autoclaved CaO₂ in sterile filtered and deionized water. The effects of CaO₂ on planktonic bacterial growth and metabolic activity, as well as on biofilms of Streptococcus oralis and Porphyromonas gingivalis, were investigated through experiments conducted under anaerobic conditions. The objective of this study was to investigate the potential of CaO₂ as an antimicrobial agent for the treatment of periodontitis. Results showed that CaO₂ selectively inhibited the growth and viability of P. gingivalis (p < 0.001) but had little effect on S. oralis (p < 0.01), indicating that CaO₂ has the potential to selectively affect both planktonic bacteria and mono-species biofilms of P. gingivalis. The results of this study suggest that CaO₂ could be a promising antimicrobial agent with selective activity for the treatment of periodontitis.

The performance of biomaterials is often compromised by bacterial infections and subsequent inflammation. So far, the conventional analysis of inflammatory processes in vivo involves time-consuming histology and biochemical assays. The present study employed a mouse model where interferon beta (IFN-β) is monitored as a marker for non-invasive rapid detection of inflammation in implant-related infections. The mouse model comprises subcutaneous implantation of morphologically modified titanium, followed by experimental infections with four taxonomically diverse oral bacteria: Streptococcus oralis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Treponema denticola (as mono culture or selected mixed-culture). IFN-β expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-β expression kinetics reduced with two mixed species infections when compared with the single species. Histological and confocal microscopy confirmed pathogen-specific infiltration of inflammatory cells at the implant-tissue interface. This was observed mainly in the vicinity of infected implants and was, in contrast to interferon expression, higher in infections with dual species. In summary, this non-invasive mouse model can be used to quantify longitudinally host inflammation in real time and suggests that the polymicrobial character of infection, highly relevant to clinical situations, has complex effects on host immunity.