Matthias Hannig

Background: Dental unit waterlines (DUWLs) often provide optimal conditions for pathogens, including non-tuberculous mycobacteria, causing various infections in patients and practitioners. Preventive measures for non-tuberculous mycobacteria (NTM) remain unclear. Dental simulation units, which are used for student training, are very similar to classic dental units. While the water quality of dental units has been the subject of numerous studies, dental simulation units have received little attention to date. In particular, there are very few studies on the role of NTM in dental simulation units and on influencing factors such as water stagnation, structural conditions, and the effectiveness of a disinfection program. Methods: In this study, 24 dental simulation units from two rooms in two different buildings of the Department for Operative Dentistry, Periodontology and Preventive Dentistry were examined for NTM. Water samples were analyzed from all simulation units, and biofilm swabs were additionally examined from room 2. All samples were incubated on solid and liquid culture media for 8 weeks. The species of NTM were identified using GenoType line probe assays. Results: NTM were detected in 43 of 44 samples. Three different species were identified: M. gordonae was the most common, with 24 detections, followed by M. chelonae with 17 detections and M. chimaera with 3 detections. Conclusion: The high contamination rate of dental simulation units highlights the need for further research to ensure a safe environment. No significant differences in terms of structural conditions could be found, while the influence of water stagnation and disinfection protocols warrants further investigation.

The setup of experimental designs in basic bone research begins with selecting appropriate osteogenic supplements to induce differentiation. Many studies describe the use of the supplements ascorbic acid, dexamethasone, and β-glycerophosphate in varying combinations and concentrations. To clarify their individual and combined effects on MC3T3-E1 cells, we conducted a detailed study focusing on cell morphology, mineralization, and gene expression of key osteogenic markers at four time points. Cell morphology was analyzed using light and scanning electron microscopy. Mineralization was assessed via Alizarin Red staining for calcium and von Kossa staining for phosphate. Additionally, energy-dispersive X-ray analysis was used to detect calcium and phosphorus in the cell layer. Sirius Red staining was applied to characterize the fiber formation observed under electron microscopy. To evaluate the influence of the supplements on gene expression, real-time quantitative PCR was performed for the osteogenic markers alkaline phosphatase, bone sialoprotein, bone morphogenetic protein 2, collagen 1a2, osteocalcin, osterix, and runt-related transcription factor 2. Osteogenic differentiation of MC3T3-E1 cells could be induced by application of ascorbic acid and β-glycerophosphate leading to upregulation of osteogenic markers, changes in cellular morphology, and mineralizing activity. Nevertheless, this effect could be further increased by additional use of dexamethasone. While most osteogenic marker genes were already upregulated on day 7, we did not see the first signs of mineralization until day 14. We recommend using ascorbic acid, β-glycerophosphate, and dexamethasone in order to get an osteogenic phenotype of MC3T3-E1 on day 14 at the earliest.

Foods rich in polyphenols are known to promote oral health by modifying the enamel pellicle. In doing so, they reduce bacterial adhesion, biofilm maturation, and erosion. The goal of this study was to screen local herbal drugs available in Central Europe for their potential suitability as part of a diet promoting oral health by targeting the initial stages of biofilm formation. To achieve this, an in situ study was conducted to evaluate the effects of the four polyphenol-rich herbal extracts of blackcurrant leaves, oak bark, horse chestnut leaves, and sweet chestnut leaves on early bacterial adhesion and biofilm formation on tooth enamel over an 8 h period. This research aimed to identify natural remedies that could support oral hygiene by targeting the initial stages of biofilm formation. Study Design and Experimental Procedures: Aqueous extracts were prepared by ultrasonic extraction. Eight human subjects wore bovine enamel slabs intraorally for 8 h. After 1 min of pellicle formation, the subjects rinsed with 8 mL of the extracts for 10 min, followed by intraoral exposure without food. An 8 h-exposure without rinse served as the negative control; 0.2% chlorhexidine gluconate (CHX) served as the positive control. After 8 h, bacterial adhesion and biofilm matrix formation on the enamel slabs were quantified ex vivo using DAPI/Concanavalin A staining and fluorescence microscopy. The LIVE/DEAD™ BacLight™ assay was used to assess bacterial viability. Statistical analysis was performed by the Mann–Whitney U test and Kruskal–Wallis test (p < 0.05), as well as the Bonferroni–Holm correction (p < 0.01). Results and Conclusions: The screened herbal drugs did not demonstrate a statistically significant impact on the number of adherent bacteria, suggesting that their mode of action may not directly interfere with bacterial adhesion mechanisms. However, all four extracts exhibited consistent trends toward reduced glucan formation and decreased bacterial viability. The observed inhibition of glucan formation indicates that these drugs may potentially target the enzymatic pathways responsible for polysaccharide synthesis. By disrupting glucan production, the structural integrity of the biofilm matrix might be compromised, which indirectly affects bacterial survival within the biofilm environment.

In contact with saliva, tooth enamel is covered by biomolecules forming an initial biofilm. Microorganisms attach to the initial biofilm and form the bacterial biofilm, which can provoke diseases. Therefore, dental biofilms are the focus of preventive research. Enamel consists mainly of hydroxyapatite (HAP). Yet, the composition of dental apatite differs between individuals and influences enamel properties. Standardized surfaces might therefore be useful for biofilm research. Synthetic enamel-like HAP pellets perfectly meet the criteria for such well-defined samples. However, systematic investigations of synthetic HAP on oral biofilm formation have never been performed, especially not in comparison to enamel. Therefore, we systematically compared the in situ biofilm formation on synthetic HAP and enamel to investigate the suitability of HAP as a substitute for natural enamel in biofilm formation studies. We observed no differences in formation kinetics, microstructure and subject-specificity of the initial biofilm on both materials. Furthermore, at the proteome level the development of the biofilm on HAP follows the formation patterns observed for enamel. Formation kinetics and morphology of the bacterial biofilm were also subject-dependent and not distinguishable between the two materials. However, the bacterial viability on HAP was higher than on enamel. For bacterial biofilm viability studies, synthetic HAP may therefore even be the preferred substrate as it is more beneficial for identifying antimicrobial agents. In summary, the results prove synthetic HAP as perfect substrate for dental biofilm studies.

Background: The human microbiome holds promise for identifying biomarkers and therapeutic targets. In obesity, interactions between oral and gut communities are increasingly implicated and end in organ injury. Methods: From the IMAGINE study, we analyzed 418 shotgun metagenomes from three specimen types (dental plaque (n = 143; 65 non-obese, 78 obese), saliva (n = 166; 75 non-obese, 91 obese), and stool (n = 109; 57 non-obese, 52 obese)) to compare site-specific microbial shifts between obese (BMI > 30 kg/m²) and non-obese individuals. Differential abundance was assessed with ANCOM-BC; effect sizes were summarized as Cohen’s d. Results: Across all samples, we detected 240 bacterial species in plaque, 229 in saliva, and 231 in stool, with 46 species present across all three sites. Absolute effect sizes were significantly larger in plaque (mean |d| = 0.26) and saliva (0.25) than in stool (0.21; p = 9 × 10⁻³). Several taxa showed an opposite directionality between oral and gut sites, including Streptococcus salivarius and Bifidobacterium longum, indicating site-specific associations. Notably, Actinomyces sp. and Streptococcus sp. exhibited promising effect sizes as diagnostic markers. Conclusions: The oral and gut microbiomes capture complementary obesity-related signals, with stronger shifts observed in oral sites. We suggest that integrating oral and gut profiling could enhance diagnostic and therapeutic strategies in obesity.

The dental pellicle is a continuously forming layer present at the interface between oral surfaces and saliva. It protects the dental surfaces by shielding against chemical and mechanical damages. The pellicle represents the basis for further biofilm development, and its formation starts after oral hygiene through the adsorption of mostly salivary proteins to all exposed oral surfaces. In spite of its important physiologic role, its formation process and composition are not yet revealed in all details. The objective of the current study was 1) to visualize and elucidate the individual proteomic composition of the very early (only a few seconds) formed in situ pellicle, named seconds -pellicle, and 2) to analyze changes in its ultrastructure and proteomic composition over time. Transmission electron microscopic analyses demonstrated the seconds -pellicle to consist of an already continuous electron-dense layer without a detectable increase in thickness within the first minutes of formation. Proteomic analyses showed it to consist of up to 841 proteins on an individual level, without elevating protein content or diversity over the first minutes. Analyses of several pellicle formation times enabled the detection of changes over time and the first direct verification of protein desorptions occurring during pellicle formation. Comparisons between the salivary and pellicle proteomes at the different formation times revealed insights in the amount of substance of single pellicle proteins over time. The present study provides first evidence that the pellicle forms instantaneously and dynamically on solid surfaces in the oral cavity. Furthermore, the data suggest these first proteins to be replaced by proteins adsorbing with higher selectivity to stabilize the pellicle within the first minutes of its formation. These insights represent the basis for selective modifications of the pellicle layer to control the originating biofilm and develop preventive strategies for oral biofilm management.

Cultivating cells in 3D is considered a significant advancement in cell culture models, as it better reflects natural cellular environments compared to 2D cultures. However, analytical methods like standard light microscopy are less effective for 3D cultures. In this study, 3D cell cultures were generated using the liquid overlay technique with 10,000, 50,000, 100,000 and 200,000 Normal Human Dermal Fibroblasts, analyzed on days 1, 2, and 3 post-seeding. We quantified the influence of fixation with paraformaldehyde or glutardialdehyde/dehydration on their morphology compared to living 3D cell cultures. They were analyzed by light microscopy, scanning electron microscopy as well as by digital light microscopy (height profile measurement). Over time, the cultures decreased in size, likely due to cell shrinkage and structural reorganization. The size reduction could be mathematically described by an exponential decay function. The proportion of round spheroids versus indented aggregates depended on cell number, culture age, and fixation method. On day 1, cultures seeded with 10,000 cells formed nearly 100% round spheroids, regardless of fixation. Higher cell numbers led to fewer round spheroids, and fixation further reduced their number. This suggests that large cell quantities sediment in layers due to steric hindrance, forming indentations. Since aldehydes are responsible for cross-linking proteins, we hypothesize that this chemical reaction, combined with low stability of the 3D cell cultures, leads to the increased formation of the indented 3D cell aggregates. This is consistent with an overall increase in the number of round spheroids and a decrease of the negative influence of fixation over time. In summary, it is important to consider the number of seeded cells, the incubation time, as well as the possible fixation effects when generating stable spheroids using the liquid overlay technique for down-stream experiments.

Background: Stannous ions and polyphenols are effective substances in preventive dentistry. The present study’s aim was to investigate whether a combination of these substance groups can achieve increased efficacy. Methods: Initial biofilm formation was performed on bovine enamel slabs, carried by 10 subjects intraorally. The subjects rinsed with tannic acid, SnCl₂, SnF₂, a combination (50:50) of tannic acid and SnCl₂, or a combination of tannic acid and SnF₂, with no rinsing in the negative control. Bacterial adherence, glucan formation (8 h, 48 h oral exposition,) and calcium release kinetics were measured (pH 2; 2.3; 3). Statistics were performed with the Kruskal–Wallis test (p < 0.05), Mann–Whitney U test (p < 0.05), and Bonferroni–Holm correction. Results: All rinsing solutions reduced bacterial adherence by more than 50%. Initial bacterial colonization and glucan formation was significantly reduced by SnF₂ and SnCl₂ as well as their combinations with tannic acid. The most significant reductions in calcium release at pH 2; 2.3; and 3 were obtained by SnF₂ and the combination of SnF₂ and tannic acid. At the acidic pH 2.0, SnF₂, SnCl₂, and tannic acid and SnF₂ showed significant protection compared to the control (p ≤ 0.01). TEM micrographs indicated that rinsing with SnF₂ and tannic acid leads to pronounced electron dense, thick pellicle layers. Conclusions: SnCl₂ and SnF₂, as well as their combinations with tannic acid, led to a reduction in initial bacterial colonization and glucan formation, showing an erosion-protective effect. These findings confirm the clinical applicability hitherto suspected by in vitro findings.

Objective: To evaluate the effects of cold atmospheric plasma (CAP) on adhesive bonding in Class II composite restorations in vitro. Methods: Forty-eight standardized Class II cavities were assigned to six groups (n = 8), varying in phosphoric acid conditioning, CAP treatment (1.5 W or 3 W), composite filling, and thermo-mechanical loading (TML). Evaluations included dye penetration, adhesive layer morphology, resin tag length, and hybrid layer thickness. Results: CAP combined with phosphoric acid (H₃PO₄) significantly increased hybrid layer thickness and resin tag length (p < 0.006). The lowest dye penetration was observed in Groups 1 and 4. Conclusions: CAP in combination with phosphoric acid improved the adhesive interface in Class II cavities. CAP alone showed limited benefits, and higher power levels may negatively affect bonding.