Matthias Hannig

The oral cavity—a key system to understand substratum-dependent bioadhesion on solid surfaces in man

One of the greatest challenges in life sciences and biomaterials research is adhesion of biomolecules and bacteria to solid surfaces in aqueous solutions. An example concerning everybody is biofilm formation in the oral cavity on dental materials and dental hard substances, respectively. The main characteristics typical for any bioadhesion can be observed excellently in the oral cavity. Initially, a proteinaceous layer termed pellicle is formed. It mediates the interactions between solid substrata, oral fluids and microorganisms. Numerous different materials with differing physico-chemical properties and possible impact on the acquired pellicle are present in the oral cavity such as enamel, dentine, restorative materials or dental implants. Despite the fact that in vitro studies demonstrate considerable differences of experimental pellicles formed on these materials, the in situ pellicles seem to be relatively similar and level off the different properties of the underlying substrates. However, the bacterial colonisation of pellicle-coated surfaces under in vivo conditions differs considerably. Long-range forces and detachment of biofilm layers may account for this phenomenon despite the masking effect of the pellicle. Accordingly, low-energy surfaces are desirable for restorative materials exposed to the oral cavity to minimise bacterial adhesion. The oral cavity is an easy accessible in vivo model for understanding bioadhesion and for investigation of protein–surface interactions noninvasively. For evaluation of biofilm formation on dental materials, in situ or in vivo studies are preferable.

Killing of adherent oral microbes by a non-thermal atmospheric plasma jet

Atmospheric plasma jets are being intensively studied with respect to potential applications in medicine. The aim of this in vitro study was to test a microwave-powered non-thermal atmospheric plasma jet for its antimicrobial efficacy against adherent oral micro-organisms. Agar plates and dentin slices were inoculated with 6 log(10) c.f.u. cm(-2) of Lactobacillus casei, Streptococcus mutans and Candida albicans, with Escherichia coli as a control. Areas of 1 cm(2) on the agar plates or the complete dentin slices were irradiated with a helium plasma jet for 0.3, 0.6 or 0.9 s mm(-2), respectively. The agar plates were incubated at 37 degrees C, and dentin slices were vortexed in liquid media and suspensions were placed on agar plates. The killing efficacy of the plasma jet was assessed by counting the number of c.f.u. on the irradiated areas of the agar plates, as well as by determination of the number of c.f.u. recovered from dentin slices. A microbe-killing effect was found on the irradiated parts of the agar plates for L. casei, S. mutans, C. albicans and E. coli. The plasma-jet treatment reduced the c.f.u. by 3-4 log(10) intervals on the dentin slices in comparison to recovery rates from untreated controls. The microbe-killing effect was correlated with increasing irradiation times. Thus, non-thermal atmospheric plasma jets could be used for the disinfection of dental surfaces.

Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy

BACKGROUND Cystic fibrosis (CF) lung disease is characterized by perpetuated neutrophilic inflammation with progressive tissue destruction. Neutrophils represent the major cellular fraction in CF airway fluids and are known to form neutrophil extracellular traps (NETs) upon stimulation. Large amounts of extracellular DNA-NETs are present in CF airway fluids. However, the structural contribution of NETs to the matrix composition of CF airway fluid remains poorly understood. We hypothesized that CF airway fluids consist of distinct DNA-NETs that are associated to subcellular structures. METHODOLOGY/PRINCIPAL FINDINGS We employed atomic force microcopy (AFM) and scanning electron microcopy to ultrastructurally characterize the nature of CF sputum and the role of NETs within the extracellular CF sputum matrix. These studies demonstrate that CF sputum is predominantly composed of a high-density meshwork of NETs and NETosis-derived material. Treatment of CF sputum with different DNases degraded CF NETs and efficiently liquefied the mucous-like structure of CF sputum. Quantitative analysis of AFM results showed the presence of three globular fractions within CF sputum and the larger two ones featured characteristics of neutrophil ectosomes. CONCLUSIONS/SIGNIFICANCE These studies suggest that excessive NET formation represents the major factor underlying the gel-like structure of CF sputum and provide evidence that CF-NETs contain ectosome-like structures that could represent targets for future therapeutic approaches.

Modified implant surfaces show different biofilm compositions under in vivo conditions.

OBJECTIVE Plaque accumulation on implant surfaces can result in peri-implantitis with potential implant loss. The aim of the present study was to examine the influence of zirconium nitride (ZrN) as a potential implant surface on the biofilm composition and diversity in vivo. MATERIAL AND METHODS ZrN- or titanium (Ti)-coated glass specimens and ZrN or roughened Ti discs were used as substrates. Pure glass and polished titanium served as controls. The specimens were mounted on removable intraoral splints in five adults. After 24 h of intraoral exposure, the biofilms were analyzed applying single-strand conformation polymorphism (SSCP analysis) of 16S rRNA genes. Sequence analysis of the dominant bands excised from the SSCP fingerprints allowed to taxonomically describe bacteria derived from biofilm samples. RESULTS The highest number of bands was counted on pure glass and Ti 800. ZrN-coated glass and ZrN-coated titanium discs showed the lowest values for species richness. However, no significant differences were observed regarding the diversity of the identified bacterial species among all the surfaces examined. A total of 46 different bacteria were identified. The dominant bands within the fingerprints indicated bacteria belonging to the Streptococcus group as identified by their 16S rDNA sequence. CONCLUSION A coating of glass surfaces with ZrN significantly reduced the species richness in early bacterial colonization but the diversity was not significantly changed. In consideration of the results obtained by this and former studies a ZrN coating appears to rather modify the quantity of early bacterial adherence than the quality of the microbial community structure.

Fracture strength of temporary fixed partial dentures: CAD/CAM versus directly fabricated restorations

OBJECTIVES This study aimed at investigating the influence of fabrication method, storage condition and material on the fracture strength of temporary 3-unit fixed partial dentures (FPDs). METHODS A CrCo-alloy master model with a 3-unit FPD (abutment teeth 25 and 27) was manufactured. The master model was scanned and the data set transferred to a CAD/CAM unit (Cercon Brain Expert, Degudent, Hanau, Germany). Temporary 3-unit bridges were produced either by milling from pre-fabricated blanks (Trim, Luxatemp AM Plus, Cercon Base PMMA) or by direct fabrication (Trim, Luxatemp AM Plus). 10 FPDs per experimental group were subjected either to water storage at 37 °C for 24h and 3 months, respectively, or thermocycled (TC, 5000×, 5-55 °C, 1 week). Maximum force at fracture (Fmax) was determined in a 3-point bending test at 200 mm/min. Data was analyzed using parametric statistics (α = 5%). RESULTS Fmax values ranged from 138.5 to 1115.5N. FPDs, which were CAD/CAM fabricated, showed a significant higher Fmax compared to the directly fabricated bridges (p < 0.05). TC significantly affected Fmax for Luxatemp (p < 0.05) but not for the PMMA based materials (p > 0.05). CAD/CAM milled FPDs made of Luxatemp showed significantly higher Fmax values compared to Trim and Cercon Base PMMA (p < 0.05). SIGNIFICANCE CAD/CAM fabricated FPDs exhibit a higher mechanical strength compared to directly fabricated FPDs, when manufactured of the same material. Composite based materials seem to offer clear advantages versus PMMA based materials and should, therefore, be considered for CAD/CAM fabricated temporary restorations.

Removing Biofilms from Microstructured Titanium Ex Vivo: A Novel Approach Using Atmospheric Plasma Technology

The removal of biofilms from microstructured titanium used for dental implants is a still unresolved challenge. This experimental study investigated disinfection and removal of in situ formed biofilms from microstructured titanium using cold atmospheric plasma in combination with air/water spray. Titanium discs (roughness (Ra): 1.96 µm) were exposed to human oral cavities for 24 and 72 hours (n = 149 each) to produce biofilms. Biofilm thickness was determined using confocal laser scanning microscopy (n = 5 each). Plasma treatment of biofilms was carried out ex vivo using a microwave-driven pulsed plasma source working at temperatures from 39 to 43°C. Following plasma treatment, one group was air/water spray treated before re-treatment by second plasma pulses. Vital microorganisms on the titanium surfaces were identified by contact culture (Rodac agar plates). Biofilm presence and bacterial viability were quantified by fluorescence microscopy. Morphology of titanium surfaces and attached biofilms was visualized by scanning electron microscopy (SEM). Total protein amounts of biofilms were colorimetrically quantified. Untreated and air/water treated biofilms served as controls. Cold plasma treatment of native biofilms with a mean thickness of 19 µm (24 h) to 91 µm (72 h) covering the microstructure of the titanium surface caused inactivation of biofilm bacteria and significant reduction of protein amounts. Total removal of biofilms, however, required additional application of air/water spray, and a second series of plasma treatment. Importantly, the microstructure of the titanium discs was not altered by plasma treatment. The combination of atmospheric plasma and non-abrasive air/water spray is applicable for complete elimination of oral biofilms from microstructured titanium used for dental implants and may enable new routes for the therapy of periimplant disease.

The protective nature of pellicle towards toothpaste abrasion on enamel and dentine

OBJECTIVES To determine the protective nature of pellicle towards toothpaste abrasion. METHODS The enamel region of human enamel-dentine blocks was indented with a Knoop diamond and the profile across the enamel-dentine junction was measured. Blocks were either exposed to deionised water or placed onto intra-oral appliances and worn in the mouth to produce in situ pellicles. This was followed by a 10-day period of tooth brushing experiments. Each day, specimens were brushed with a slurry of either Toothpaste A (RDA=90) or Toothpaste B (RDA=204) for 25 cycles (10s) on a brushing machine. This was repeated three times per day for a total of 750 brushing cycles. Between brushing cycles specimens were returned to water or in situ. The geometry of the Knoop indents and the enamel-dentine profile were re-measured and the enamel and dentine wear calculated. Specimens were also prepared for TEM analyses. RESULTS The mean enamel wear (microm) for Toothpastes A and B (water) was 0.23 and 0.06, and for Toothpastes A and B (in situ) was 0.03 and 0.08, respectively. The mean dentine wear (microm) for Toothpastes A and B (water) was 5.08 and 6.03, and for Toothpastes A and B (in situ) was 1.94 and 1.70, respectively. For Toothpaste A, the presence of in situ pellicle significantly (p<0.05) reduced enamel and dentine wear compared to water and for Toothpaste B, dentine wear was significantly reduced compared to water. After tooth brushing, residues of the in situ pellicle layer could be detected on enamel and dentine surfaces by TEM analysis. CONCLUSIONS The study has demonstrated for the first time that the presence of pellicle can significantly reduce toothpaste abrasion.

Bacterial colonization of enamel in situ investigated using fluorescence in situ hybridization.

Oral biofilms are one of the greatest challenges in dental research. The present study aimed to investigate initial bacterial colonization of enamel surfaces in situ using fluorescence in situ hybridization (FISH) over a 12 h period. For this purpose, bovine enamel slabs were fixed on buccal sites of individual splints worn by six subjects for 2, 6 and 12 h to allow biofilm formation. Specimens were processed for FISH and evaluated with confocal laser-scanning microscopy, using probes for eubacteria, Streptococcus species, Veillonella species, Fusobacterium nucleatum and Actinomyces naeslundii. The number of adherent bacteria increased with time and all tested bacterial species were detected in the biofilm formed in situ. The general percentage composition of the eubacteria did not change over the investigated period, but the number of streptococci, the most frequently detected species, increased significantly with time (2 h: 17.7+/-13.8 %; 6 h: 20.0+/-16.6 %; 12 h: 24.7+/-16.1 %). However, < or =1 % of the surface was covered with bacteria after 12 h of biofilm formation in situ. In conclusion, FISH is an appropriate method for quantifying initial biofilm formation in situ, and the proportion of streptococci increases during the first 12 h of bacterial adherence.

The effect of acidic beverages on the ultrastructure of the acquired pellicle—An in situ study

AIM The aim of the present in situ study was to investigate ultrastructural alterations as well as protective properties of the pellicle layer during consumption of acidic beverages. METHODS Bovine enamel slabs were fixed on buccal and palatal aspects of individual splints and exposed in the oral cavities of three subjects for 120 min. In the following, the subjects drank orange juice, coke light or sprite light. Half of the specimens were removed afterwards, the others were exposed to the oral fluids for another 120 min. Erosive alterations of the bovine enamel slabs were measured by determination of the Knoop-micro-hardness. In addition, the ultrastructure of the pellicle was evaluated by transmission electron microscopy (TEM). RESULTS Determination of Knoop-micro-hardness yielded only little reduction of the relative Knoop-hardness in situ during consumption of sprite light (-0.053+/-0.019) and coke light (-0.075+/-0.04). With orange juice nearly no change of the hardness was recorded. TEM-pictures showed that the globular outer layers of the pellicle were removed to a different extent according to the localisation of the specimens in the oral cavity, whereas the basal pellicle was not affected by the acidic beverages. On the specimens carried for another 120 min after the erosive attack, lacunae filled with organic structures were observed underneath the basal side of the pellicle. CONCLUSION During fast consumption of acidic beverages in situ, the erosive effects on pellicle coated bovine enamel are moderate and juices seem to be less harmful as compared with low pH soft drinks. Pellicle proteins in eroded lacunae may impact the remineralization process.

Initial bioadhesion on dental materials as a function of contact time, pH, surface wettability, and isoelectric point.

The adsorption of bovine serum albumin (BSA) on surfaces of dental enamel and of dental materials was investigated by scanning force spectroscopy. This method provides adhesion forces which can be measured as a function of contact time between protein and surface, pH, wettability, and isoelectric point of the surface. Whereas the chosen ceramic and composite materials resemble very well the adhesion on natural enamel, a much stronger adhesion was found for the more hydrophobic surfaces, that is, gold, titanium, poly(methyl methacrylate) (PMMA), and poly(tetrafluoroethylene) (PTFE). On hydrophilic surfaces, adhesion is mainly influenced by the electrostatic forces between protein and surface. However, the conformational change of BSA at pH values above pH 8 has to be taken into account. On the very hydrophobic PTFE surface, the special interface structure between PTFE and water plays an important role which governs BSA adhesion.