Non-invasive in vivo imaging by confocal laser scanning microscopy of gingival tissues following natural plaque deposition.

AIM: Imaging with Confocal Laser Scanning Microscopy (CLSM) generates high-resolution images and may be well suited for basic research in Periodontology and Implant Dentistry. The present study was aimed to explore the in vivo application of CLSM in experimentally induced gingivitis. MATERIALS AND METHODS: Ten subjects were recruited and were advised to stop any oral hygiene of the upper front teeth for 7 days. The gingival tissues were observed using a Heidelberg Retina Tomograph combined with a Rostock Cornea Module at baseline and day 7. The system used a laser of 670 nm and the contrast was given by backscattering from different tissues. Each examination created 800-1200 images that were descriptively analysed. RESULTS: After 7 days of abandoned oral hygiene, plaque scores and bleeding frequencies increased. By using CLSM images tooth hard substances, cells and plaque deposits were distinguishable. Increased epithelial cell irregularities, the apical migration of the sulcular epithelium, cellular infiltrates within the sulcus and plaque deposits were observed at day 7. CONCLUSIONS: The present study showed for the first time that CLSM is suitable for in vivo imaging of the gingival sulcus and adjacent tissues.

Antibacterial Effect of Cupral® on Oral Biofilms - An In-Vitro Study.

Objective: This study aimed to assess the efficacy of Cupral®, a Ca(OH)2 and Cu2+ based materials used in endodontics, against biofilms of the oral species Streptococcus oralis, Streptococcus gordonii and Aggregatibacter actinomycetemcomitans at different maturation stages. Methods: Biofilms of the bacterial target species were grown in brain heart infusion (BHI) medium for 1 and 5 days on titanium disks (titanium, grade 4) to collect microbial communities at different stages of biofilm maturation. Biofilms were subjected to different Cupral® concentrations (4-, 15- and 50-fold dilution) to assess the antimicrobial- and biofilm dissolving effect. 0.2% chlorhexidine gluconate (CHX) solution was used as a positive control. Biovolume and antibacterial efficacy were analyzed by live/dead staining in combination with confocal laser scanning microscopy (CLSM) to quantify biofilm detachment and antibacterial efficacy. Results: All tested Cupral® concentration showed a strong antibacterial effect on tested bacterial species at all biofilm maturation stages. Efficacy of biofilms detachment was concentration dependent, i.e. higher Cupral® concentrations generally led to increased biofilm detachment. The antibacterial efficacy of tested Cupral® concentration was at least equal to CHX treatment (P=0.03). Conclusion: Cupral® shows a strong anti-biofilm efficacy and may be applied for oral biofilm treatment and control in dental disciplines other than endodontics.

In Vitro Antibacterial Effectiveness of a Naturopathic Oral Care Product on Oral Pathogens.

PURPOSE: Currently, the prevention of periodontal diseases focuses on mechanical removal of pathogenic biofilms combined with oral antiseptics as supportive chemical antibacterial control. Due to the risk of resistance development and side effects of existing antiseptics, the interest in alternative medicine with naturopathic treatment modalities is growing in dentistry. In the present study, the antibacterial effect of the naturopathic oral care product Repha OS and some of its derivatives, based on medicinal plant extracts and essential oils, with a specific focus on added sweeteners, was investigated on periodontal pathogenic and halitosis-associated bacteria. MATERIALS AND METHODS: The antibacterial efficacy was investigated by agar dilution assay. The minimum inhibitory concentration (MIC) for the bacterial species Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia and Solobacterium moorei was determined. RESULTS: A concentration-dependent antibacterial effect on oral bacterial species by Repha OS and its derivatives was demonstrated. For the original product, the maximum MIC was 10% of the calculated test solution concentration in agar for all examined bacterial species. The removal of essential oils reduced the antibacterial efficacy, whereas the displacement or replacement of sweeteners had almost no effect. CONCLUSION: In addition to other individual effects of the ingredients, the results of this study show that an antibacterial effect of the naturopathic oral care product on the tested oral bacterial species was achieved in vitro. In vivo, the combination of this antibacterial effect with other properties of the various ingredients may be interesting for a holistic approach in preventive dentistry.

Non-Invasive Luciferase Imaging of Type I Interferon Induction in a Transgenic Mouse Model of Biomaterial Associated Bacterial Infections: Microbial Specificity and Inter-Bacterial Species Interactions.

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-ß) 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-ß expression increased upon infections depending on the type of pathogen and was prolonged by the presence of the implant. IFN-ß 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.

Liquid-Infused Structured Titanium Surfaces: Antiadhesive Mechanism to Repel Streptococcus oralis Biofilms.

To combat implant-associated infections, there is a need for novel materials which effectively inhibit bacterial biofilm formation. In the present study, the antiadhesive properties of titanium surface functionalization based on the "slippery liquid-infused porous surfaces" (SLIPS) principle were demonstrated and the underlying mechanism was analyzed. The immobilized liquid layer was stable over 13 days of continuous flow in an oral flow chamber system. With increasing flow rates, the surface exhibited a significant reduction in attached biofilm of both the oral initial colonizer  Streptococcus oralis and an oral multispecies biofilm composed of S. oralis, Actinomyces naeslundii, Veillonella dispar, and Porphyromonas gingivalis. Using single cell force spectroscopy, reduced S. oralis adhesion forces on the lubricant layer could be measured. Gene expression patterns in biofilms on SLIPS, on control surfaces, and expression patterns of planktonic cultures were also compared. For this purpose, the genome of S. oralis strain ATCC 9811 was sequenced using PacBio Sequel technology. Even though biofilm cells showed clear changes in gene expression compared to planktonic cells, no differences could be detected between bacteria on SLIPS and on control surfaces. Therefore, it can be concluded that the ability of liquid-infused titanium to repel S. oralis biofilms is mainly due to weakened bacterial adhesion to the underlying liquid interface.

Development and characterization of an oral multispecies biofilm implant flow chamber model.

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.

Biofilm formation by the oral pioneer colonizer Streptococcus gordonii: an experimental and numerical study.

For decades, extensive research efforts have been conducted to improve the functionality and stability of implants. Especially in dentistry, implant treatment has become a standard medical practice. The treatment restores full dental functionality, helping patients to maintain high quality of life. However, about 10% of the patients suffer from early and late device failure due to peri-implantitis, an inflammatory disease of the tissues surrounding the implant. Peri-implantitis is caused by progressive microbial colonization of the device surface and the formation of microbial communities, so-called biofilms. This infection can ultimately lead to implant failure. The causative agents for the inflammatory disease, periodontal pathogenic biofilms, have already been extensively studied, but are still not completely understood. As numerical simulations will have the potential to predict oral biofilm formation precisely in the future, for the first time, this study aimed to analyze Streptococcus gordonii biofilms by combining experimental studies and numerical simulation. The study demonstrated that numerical simulation was able to precisely model the influence of different nutrient concentration and spatial distribution of active and inactive biomass of the biofilm in comparison with the experimental data. This model may provide a less time-consuming method for the future investigation of any bacterial biofilm.

Health- and disease-associated species clusters in complex natural biofilms determine the innate immune response in oral epithelial cells during biofilm maturation.

The aim of the present study was to verify our hypothesis concerning the differential induction of various antimicrobial and immunomodulatory responses in oral epithelial cells by diverse bacterial species clusters. For this purpose, oral biofilms between 1 and 14 days of maturation (36 volunteers) were co-incubated with gingival epithelial cells. Subsequently, human ß-defensin (hBD)-2, hBD-3, LL-37, interleukin (IL)-1ß, IL-6, IL-8 and IL-10 mRNA expression profiles were quantified by quantitative reverse transcription PCR. The correlation between bacterial species and the host innate immune response was determined by relating these results to existing 16S rRNA phylogenetic analysis by amplicon sequencing (Langfeldt et al. 2014. PLoS One 9: e87449). Data were analysed by multiple factor analysis. Transcription of hBD-2 and hBD-3 was significantly associated with the abundance of species of the Prevotella cluster and the absence of species of the Streptococcus cluster. IL-1ß, -6, -8 and -10 mRNA syntheses were significant correlated with Leptotrichia species [Leptotrichia 302H02 (0.448, P < 0.0001), Leptotrichia nbw822e09c1 (0.214, P = 0.008) and Leptotrichia wadei (0.218, P = 0.007)] of the Prevotella cluster. In the third dimension IL-10 and members of the Prevotella cluster were negatively correlated, whereas hBD-3 and IL-1ß, IL-6 and IL-8 were positive correlated to axis 3, like members of the Proteobacteria cluster. In conclusion, distinct species of health- and disease-associated bacterial clusters induce antibacterial or immunomodulatory reactions in oral epithelial cells during early stages of bacteria-host interactions.

Bacterial growth on cochlear implants as a potential origin of complications.

OBJECTIVE: To investigate the presence and spectrum of bacterial colonization in relation to cochlear implant infections by introducing molecular biologic methods. METHOD: In a pilot test, a virgin device served as a control to validate the subsequent sampling and analysis process via PCR and SSCP. Subsequently, analysis was performed on specimens of 15 cochlear implants explanted from 15 patients because of device failures, infections, or malinsertions. RESULTS: Positive SSCP results could be verified on 8 of the 15 explanted CI, in detail, 3 of 4 infected cochlear implants and 5 of 9 cochlear implants explanted because of device failure. The germ spectrum shows common germs such as Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae, as well as germs from the dental cavity, with a peak of P. aeruginosa in infections. CONCLUSION: Detection and identification of microbial colonization on cochlear implants can be successfully conducted using the proposed approach of smear test subsequent genetic analysis. The prevalence of P. aeruginosa and germs from the dental cavity may demand an adaption of antibiosis with respect to these germs. Further investigations of the path of infection are needed, and patients might require a prevention by preoperative dental treatment.

Experimental gingivitis induces systemic inflammatory markers in young healthy individuals: a single-subject interventional study.

OBJECTIVES: We here investigated whether experimental gingivitis enhances systemic markers of inflammation which are also known as surrogate markers of atherosclerotic plaque development. BACKGROUND: Gingivitis is a low-level oral infection induced by bacterial deposits with a high prevalence within Western populations. A potential link between the more severe oral disease periodontitis and cardiovascular disease has already been shown. METHODS: 37 non-smoking young volunteers with no inflammatory disease or any cardiovascular risk factors participated in this single-subject interventional study with an intra-individual control. Intentionally experimental oral inflammation was induced by the interruption of oral hygiene for 21 days, followed by a 21-days resolving phase after reinitiation of oral hygiene. Primary outcome measures at baseline, day 21 and 42 were concentrations of hsCRP, IL-6, and MCP-1, as well as adhesion capacity and oxLDL uptake of isolated blood monocytes. RESULTS: The partial cessation of oral hygiene procedures was followed by the significant increase of gingival bleeding (34.0%, P<0.0001). This local inflammation was associated with a systemic increase in hsCRP (0.24 mg/L, P = 0.038), IL-6 (12.52 ng/L, P = 0.0002) and MCP-1 (9.10 ng/l, P = 0.124) in peripheral blood samples between baseline and day 21, which decreased at day 42. Monocytes showed an enhanced adherence to endothelial cells and increased foam cell formation after oxLDL uptake (P<0.050) at day 21 of gingivitis. CONCLUSIONS: Bacterial-induced gingival low-level inflammation induced a systemic increase in inflammatory markers. Dental hygiene almost completely reversed this experimental inflammatory process, suggesting that appropriate dental prophylaxis may also limit systemic markers of inflammation in subjects with natural gingivitis. International Clinical Trials Register Platform of the World Health Organization, registry number: DRKS00003366, URL: http://apps.who.int/trialsearch/Default.aspx.