Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4′,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

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Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle

Caries Research ◽

10.1159/000451036 ◽

2016 ◽

Vol 51 (1) ◽

pp. 34-45 ◽

Cited By ~ 13

Author(s):

Susann Hertel ◽

Sandra Pötschke ◽

Sabine Basche ◽

Judith Delius ◽

Wiebke Hoth-Hannig ◽

...

Keyword(s):

Tannic Acid ◽

Ex Vivo ◽

Oral Exposure ◽

Dependent Manner ◽

Pellicle Formation ◽

Protective Properties ◽

Transmission Electron ◽

Bovine Enamel ◽

The Impact

Objectives: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. Methods: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4′,6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). Results: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. Conclusion: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

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Localization of ‘Candidatus Liberibacter solanacearum’ and Evidence for Surface Appendages in the Potato Psyllid Vector

Phytopathology ◽

10.1094/phyto-04-15-0088-r ◽

2016 ◽

Vol 106 (2) ◽

pp. 142-154 ◽

Cited By ~ 20

Author(s):

J. M. Cicero ◽

T. W. Fisher ◽

J. K. Brown

Keyword(s):

Electron Microscopy ◽

Light And Electron Microscopy ◽

Fluorescence Labeling ◽

Potato Psyllid ◽

Bactericera Cockerelli ◽

Visual Identification ◽

Transmission Electron ◽

Candidatus Liberibacter ◽

Identification Of Bacteria

The potato psyllid Bactericera cockerelli is implicated as the vector of the causal agent of zebra chip of potato and vein-greening of tomato diseases. Until now, visual identification of bacteria in the genus ‘Candidatus Liberibacter’ has relied on direct imaging by light and electron microscopy without labeling, or with whole-organ fluorescence labeling only. In this study, aldehyde fixative followed by a coagulant fixative, was used to process adult psyllids for transmission electron microscopy (TEM) colloidal gold in situ hybridization experiments. Results indicated that ‘Ca. Liberibacter solanacearum’ (CLso)-specific DNA probes annealed to a bacterium that formed extensive, monocultural biofilms on gut, salivary gland, and oral region tissues, confirming that it is one morphotype of potentially others, that is rod-shaped, approximately 2.5 µm in diameter and of variable length, and has a rough, granular cytosol. In addition, CLso, prepared from shredded midguts, and negatively stained for TEM, possessed pili- and flagella-like surface appendages. Genes implicating coding capacity for both types of surface structures are encoded in the CLso genome sequence. Neither type was seen for CLso associated with biofilms within or on digestive organs, suggesting that their production is stimulated only in certain environments, putatively, in the gut during adhesion leading to multiplication, and in hemolymph to afford systemic invasion.

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A Study of Membrane Impact on Spatial Resolution of Liquid In Situ Transmission Electron Microscope

Microscopy and Microanalysis ◽

10.1017/s143192761901523x ◽

2020 ◽

Vol 26 (1) ◽

pp. 126-133

Author(s):

Ming Li ◽

Ruth Knibbe

Keyword(s):

Electron Microscope ◽

Spatial Resolution ◽

Transmission Electron Microscope ◽

Signal To Noise Ratio ◽

Chromatic Aberration ◽

Membrane Thickness ◽

Additional Material ◽

Transmission Electron ◽

The Impact

AbstractMicrochip technology with electron transparent membranes is a key component for in situ liquid transmission electron microscope (TEM) characterization. The membranes can significantly influence the TEM imaging spatial resolution, not only due to introducing additional material layers but also due to the associated bulging. The membrane bulging is largely defined by the membrane materials, thickness, and short dimension. The impact of the membrane on the spatial resolution, especially the extent of its bulging, was systematically investigated through the impact on the signal-to-noise ratio, chromatic aberration, and beam broadening. The optimization of the membrane parameters is the key component when designing the in situ TEM liquid cell. The optimal membrane thickness of 50 nm was found which balances the impact of membrane bulging and membrane thickness. Beyond this, the short membrane window dimension and the chip nominal spacing should be minimized. However, these two parameters have practical limitations in regards to chip handling.

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Adhesion of Hydroxyapatite Nanoparticles to Dental Materials under Oral Conditions

Scanning ◽

10.1155/2020/6065739 ◽

2020 ◽

Vol 2020 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Cíntia Mirela Guimarães Nobre ◽

Norbert Pütz ◽

Matthias Hannig

Keyword(s):

Electron Microscopy ◽

Dental Materials ◽

Hydroxyapatite Nanoparticles ◽

Pellicle Formation ◽

Transmission Electron ◽

Different Shapes ◽

Oral Conditions ◽

Dental Applications

Hydroxyapatite nanoparticles (nano-HAP) are receiving considerable attention for dental applications, and their adhesion to enamel is well established. However, there are no reports concerning the effects of HAP on other dental materials, and most of the studies in this field are based on in vitro designs, neglecting the salivary pellicle-apatite interactions. Thus, this in situ pilot study aims to evaluate the effects of three hydroxyapatite-based solutions and their interactions with different dental material surfaces under oral conditions. Hence, two volunteers carried intraoral splints with mounted samples from enamel and from three dental materials: titanium, ceramics, and polymethyl-methacrylate (PMMA). Three HAP watery solutions (5%) were prepared with different shapes and sizes of nano-HAP (HAP I, HAP II, HAP III). After 3 min of pellicle formation, 10 ml rinse was performed during 30 sec. Rinsing with water served as control. Samples were accessed immediately after rinsing, 30 min and 2 h after rinsing. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the particles, and SEM evaluated the pellicle-HAP interactions. SEM and TEM results showed a high variation in the size range of the particles applied. A heterogeneous HAP layer was present after 2 h on enamel, titanium, ceramics, and PMMA surfaces under oral conditions. Bridge-like structures were visible between the nano-HAP and the pellicle formed on enamel, titanium, and PMMA surfaces. In conclusion, nano-HAP can adhere not only to enamel but also to artificial dental surfaces under oral conditions. The experiment showed that the acquired pellicle act as a bridge between the nano-HAP and the materials’ surface.

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Influence of Calcium Phosphate and Apatite Containing Products on Enamel Erosion

The Scientific World JOURNAL ◽

10.1155/2016/7959273 ◽

2016 ◽

Vol 2016 ◽

pp. 1-12 ◽

Cited By ~ 12

Author(s):

A. Kensche ◽

S. Pötschke ◽

C. Hannig ◽

G. Richter ◽

W. Hoth-Hannig ◽

...

Keyword(s):

Oral Cavity ◽

Oral Exposure ◽

Ion Release ◽

Pellicle Formation ◽

Protective Properties ◽

Ph Values ◽

Mineral Components ◽

Tooth Mousse ◽

Bovine Enamel

For the purpose of erosion prevention the present study aimed to compare the efficacy of two biomimetic products and a fluoride solution to optimize the protective properties of the pellicle. After 1 min ofin situpellicle formation on bovine enamel slabs, 8 subjects adopted CPP-ACP (GC Tooth Mousse), a mouthwash with hydroxyapatite microclusters (Biorepair), or a fluoride based mouthwash (elmex Kariesschutz) for 1 min each. Afterwards, samples were exposed in the oral cavity for 28 min. Native enamel slabs and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After oral exposure, slabs were incubated in HCl (pH values 2, 2.3, and 3) for 120 s and kinetics of calcium and phosphate release were measured photometrically; representative samples were evaluated by SEM and TEM. The physiological pellicle reduced demineralization at all pH values; the protective effect was enhanced by fluoride. The biomimetic materials also reduced ion release but their effect was less pronounced. SEM indicated no layer formation after use of the different products. However, TEM confirmed the potential accumulation of mineral components at the pellicle surface. The tested products improve the protective properties of thein situpellicle but not as effectively as fluorides.

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Application of Plant Extracts for the Prevention of Dental Erosion: An in situ/in vitro Study

Caries Research ◽

10.1159/000431294 ◽

2015 ◽

Vol 49 (5) ◽

pp. 477-487 ◽

Cited By ~ 16

Author(s):

Marie-Theres Weber ◽

Matthias Hannig ◽

Sandra Pötschke ◽

Franziska Höhne ◽

Christian Hannig

Keyword(s):

Plant Extracts ◽

Dental Erosion ◽

Oral Exposure ◽

Wild Form ◽

Dependent Manner ◽

Mouth Rinse ◽

Protective Properties ◽

The Impact

Objectives: Antiadherent and antibacterial effects of certain plant extracts have been proven to be beneficial in preventive dentistry. In the present in situ/in vitro crossover study, the impact of plant extracts rich in polyphenols on the erosion-protective properties of the in situ pellicle was evaluated. Methods: Individual splints were prepared for 12 subjects for intraoral exposure of bovine enamel specimens. Following formation of a 1-min pellicle, watery plant extracts (leaves of the wild form of Ribes nigrum, the wild form of Origanum as well as a combination of both) were administered for 10 min in situ. Alternatively, a mouth rinse with fluorides (Elmex Kariesschutz) was performed for 1 min. After further oral exposure for 19/28 min, respectively, slabs were removed and incubated with HCl in vitro over 120 s (pH 2, 2.3, 3). The resulting calcium and phosphate release was quantified photometrically. Slabs with and without a 30-min in situ pellicle served as controls. The modification of pellicle ultrastructure was evaluated by transmission electron microscopy (TEM). Results: Plant extracts modulated the erosion-protective properties of the native in situ pellicle in all test groups in a pH-dependent manner. The combination of R. nigrum leaves and Origanum enhanced the protective properties of the pellicle at all pH values; the administration of this preparation was comparable, yet superior, to the effect of the fluoridated mouth rinse. TEM images indicated that rinsing with R. nigrum leaves/Origanum yielded a distinctly thicker and more electron-dense pellicle. Conclusion: The combination of certain plant extracts offers a novel approach to the complementary prevention of dental erosion.

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Modification of the Lipid Profile of the Initial Oral Biofilm In Situ Using Linseed Oil as Mouthwash

Nutrients ◽

10.3390/nu13030989 ◽

2021 ◽

Vol 13 (3) ◽

pp. 989

Author(s):

Anna Kensche ◽

Marco Reich ◽

Christian Hannig ◽

Klaus Kümmerer ◽

Matthias Hannig

Keyword(s):

Fatty Acid ◽

Linolenic Acid ◽

Impact Ionization ◽

Linseed Oil ◽

Lipid Vesicles ◽

Oral Exposure ◽

Ionization Mass ◽

Pellicle Formation ◽

Fatty Acid Accumulation

Lipids are of interest for the targeted modification of oral bioadhesion processes. Therefore, the sustainable effects of linseed oil on the composition and ultrastructure of the in situ pellicle were investigated. Unlike saliva, linseed oil contains linolenic acid (18:3), which served as a marker for lipid accumulation. Individual splints with bovine enamel slabs were worn by five subjects. After 1 min of pellicle formation, rinses were performed with linseed oil for 10 min, and the slabs’ oral exposure was continued for up to 2 or 8 h. Gas chromatography coupled with electron impact ionization mass spectrometry (GC-EI/MS) was used to characterize the fatty acid composition of the pellicle samples. Transmission electron microscopy was performed to analyze the ultrastructure. Extensive accumulation of linolenic acid was recorded in the samples of all subjects 2 h after the rinse and considerable amounts persisted after 8 h. The ultrastructure of the 2 h pellicle was less electron-dense and contained lipid vesicles when compared with controls. After 8 h, no apparent ultrastructural effects were visible. Linolenic acid is an excellent marker for the investigation of fatty acid accumulation in the pellicle. New preventive strategies could benefit from the accumulation of lipid components in the pellicle.

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Direct imaging of short-range order and its impact on deformation in Ti-6Al

Science Advances ◽

10.1126/sciadv.aax2799 ◽

2019 ◽

Vol 5 (12) ◽

pp. eaax2799 ◽

Cited By ~ 11

Author(s):

Ruopeng Zhang ◽

Shiteng Zhao ◽

Colin Ophus ◽

Yu Deng ◽

Shraddha J. Vachhani ◽

...

Keyword(s):

Direct Observation ◽

Short Range ◽

Short Range Order ◽

Range Order ◽

Ex Situ ◽

In Situ Tem ◽

Dislocation Plasticity ◽

Transmission Electron ◽

The Impact

Chemical short-range order (SRO) within a nominally single-phase solid solution is known to affect the mechanical properties of alloys. While SRO has been indirectly related to deformation, direct observation of the SRO domain structure, and its effects on deformation mechanisms at the nanoscale, has remained elusive. Here, we report the direct observation of SRO in relation to deformation using energy-filtered imaging in a transmission electron microscope (TEM). The diffraction contrast is enhanced by reducing the inelastically scattered electrons, revealing subnanometer SRO-enhanced domains. The destruction of these domains by dislocation planar slip is observed after ex situ and in situ TEM mechanical testing. These results confirm the impact of SRO in Ti-Al alloys on the scale of angstroms. The direct confirmation of SRO in relationship to dislocation plasticity in metals can provide insight into how the mechanical behavior of concentrated solid solutions by the material’s thermal history.

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Investigation of structurally less-ordered areas in the Nb filaments of a heavily cold-rolled Cu-20 wt. % Nb in situ composite

Journal of Materials Research ◽

10.1557/jmr.1995.3050 ◽

1995 ◽

Vol 10 (12) ◽

pp. 3050-3061 ◽

Cited By ~ 10

Author(s):

D. Raabe ◽

U. Hangen

Keyword(s):

Large Strain ◽

High Strength ◽

Structural Regularity ◽

Transmission Electron ◽

Hardening Models ◽

Dislocation Cells ◽

Cold Rolled ◽

Cold Worked ◽

The Impact

A fiber-reinforced in situ metal matrix composite (MMC) consisting of copper (Cu) and 20 mass% niobium (Nb) was produced by large strain cold rolling. The rolled MMC revealed a very high strength combined with good electrical conductivity. The microstructure of single Nb filaments was investigated employing transmission electron microscopy (TFM). In heavily rolled specimens (∊max = 99.4%) randomly arranged dislocations as well as dislocation cells were observed. Furthermore, structurally less-ordered areas were discovered, the size of which frequently extended over the entire filament width. The shrinkage of these zones during heating was directly observed in the TEM. The impact of such structurally less-ordered areas on the strength was assessed. The discovery of the degradation of structural regularity in the Nb filaments of heavily cold-worked Cu-20 wt. % Nb shows that the underlying microstructural mechanisms responsible for the high strengths observed are far from being understood and that the strain-hardening models for Cu-based in situ composites currently discussed do not yet account for all relevant microstructural features.

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Monitoring Electrical Biasing of Pb(Zr0.2Ti0.8)O3 Ferroelectric Thin Films In Situ by DPC-STEM Imaging

Materials ◽

10.3390/ma14164749 ◽

2021 ◽

Vol 14 (16) ◽

pp. 4749

Author(s):

Alexander Vogel ◽

Martin F. Sarott ◽

Marco Campanini ◽

Morgan Trassin ◽

Marta D. Rossell

Keyword(s):

Thin Film ◽

Real Time ◽

Data Storage ◽

Ferroelectric Properties ◽

Polarization State ◽

Ferroelectric Materials ◽

Time Data ◽

Transmission Electron ◽

The Impact

Increased data storage densities are required for the next generation of nonvolatile random access memories and data storage devices based on ferroelectric materials. Yet, with intensified miniaturization, these devices face a loss of their ferroelectric properties. Therefore, a full microscopic understanding of the impact of the nanoscale defects on the ferroelectric switching dynamics is crucial. However, collecting real-time data at the atomic and nanoscale remains very challenging. In this work, we explore the ferroelectric response of a Pb(Zr0.2Ti0.8)O3 thin film ferroelectric capacitor to electrical biasing in situ in the transmission electron microscope. Using a combination of high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and differential phase contrast (DPC)-STEM imaging we unveil the structural and polarization state of the ferroelectric thin film, integrated into a capacitor architecture, before and during biasing. Thus, we can correlate real-time changes in the DPC signal with the presence of misfit dislocations and ferroelastic domains. A reduction in the domain wall velocity of 24% is measured in defective regions of the film when compared to predominantly defect-free regions.

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