STEM and HRTEM studies of accumulated deposits on human tooth surface.

The aim of this study was to clarify the fine structure of accumulated deposits on the surface of teeth that are considered to affect the gloss of teeth. The study was carried out using, as specimens, human incisor teeth having gloss, which were extracted from teenage donors and those incapable of showing gloss even by brushing which were extracted from donors in their 50s. Thin longitudinal sections of tooth enamel with accumulated deposits on the surface were prepared by focused ion beam (FIB) milling, and the fine structure was analyzed using a scanning transmission electron microscope (STEM) and a high resolution transmission electron microscope (HRTEM). By FIB, thin longitudinal sections could be prepared from tooth enamel together with organic and inorganic substances accumulated on the surface without artifacts. The accumulated deposits on the surface of teeth having gloss were composed of organic substances. However, it was first revealed by STEM observation that the accumulated solid deposits on the surface of teeth having no gloss had a complicated structure wherein inorganic and organic substances coexisted. It is suggested that the organic substances contain proteins derived from saliva. The inorganic substances were spherical and needle-like hydroxyapatites (HAs). It is considered that amino acids constituting the proteins affected the nucleus formation and the crystal formation of HA. It is considered that the unevenness of the accumulated deposits existing on the surface of tooth enamel having no gloss causes the decrease in gloss of teeth due to diffuse reflection of light.

Evaluation of the smear layer and hybrid layer in noncarious and carious dentin prepared by air abrasion system and diamond tips.

PURPOSE: To analyze the smear layer and the hybrid layer in noncarious and carious dentin prepared by different cutting instruments and restored with composite resin. STUDY DESIGN: Cavities were randomly prepared in 160 specimens (noncarious and artificial carious dentin) by high-speed diamond tips (KG Sorensen 1013), air abrasion system (Prepstart, Danville Engineering), ultrasonic tip (CVDentus 8.3231-1), and ultrasonic tip associated with ultrasonic cavitation by water for 10 s. Half of the cavities in each group were conditioned with 37% phosphoric acid for 15 s. The amount of smear layer and dentinal tubules present were analyzed using scanning electron microscopy and graded from 0 to 3. Cavities were prepared in another 20 noncarious specimens and 20 carious specimens and restored with adhesive composite resin system. The restorations were hemisected longitudinally and analyzed using scanning electron microscopy to evaluate the hybrid layer and resinous prolongation characteristics, using scores ranging from 1 to 6. RESULTS: The data were statistically analyzed using Kruskal-Wallis and Dunn tests at 5% of significance level. There was evidence that the most efficient smear layer removal was the acid etching in the noncarious dentin and the water ultrasonic cavitation in the carious dentin. The hybrid layer formed on the noncarious and carious dentin prepared by the ultrasonic tip was more regular than in the specimens prepared by high-speed diamond tip, with many resinous prolongations. CONCLUSION: The ultrasonic tip seems to be a promising tool for carious dentin cavity preparation.

[Biofilm formation on root canal--review]. / Biofilm w kanalach korzeniowych w swietle pismiennictwa.

It seems likely that one of the reasons for failures in the endodontic treatment is the presence of biofilm in root canals. Biofilm bacteria have a slower metabolism and higher resistance and virulence due to phenotypic changes. The occurrence of biofilms has been reported both inside the canal and on the external root surface. The results of many studies suggest that biofilm may be associated with refractory periapical periodontitis and is often caused by the coronal leakage.

Structural conformation and leaching from in vitro aged and retrieved Invisalign appliances.

The objectives of this study were to investigate the structure of Invisalign appliances (Align Technology, Santa Clara, Calif) after intraoral exposure, and to qualitatively and quantitatively characterize the substances leached from the aligners after accelerated in vitro aging. Samples of Invisalign appliances were randomly selected from 10 patients before intraoral placement and after retrieval, and the prepared specimens were subjected to (1) bright-field optical reflection microscopy to study the surface morphology; (2) Fourier transform infrared microspectroscopy to characterize the in vivo changes in molecular composition induced on appliance surfaces, (3) scanning electron microscopy and energy dispersive X-ray microanalysis to identify the elemental composition of integuments formed on the surface, and (4) Vickers hardness (HV 200) testing. Another set of reference and retrieved appliances was subjected to artificial aging for 2 weeks, and the extracts were subjected to gas chromatography-mass spectroscopy. The retrieved appliances demonstrated substantial morphological variation relative to the as-received specimens involving abrasion at the cusp tips, adsorption of integuments, and localized calcification of the precipitated biofilm at stagnation sites. Buccal segments of retrieved appliances showed an increase in hardness, which might be attributed to mastication-induced cold work; however, the clinical implication of this effect on mechanotherapy is unknown. In vitro aged and retrieved appliances were found to leach no traceable amount of substances in an ethanol aging solution.

A preliminary investigation into the ultrastructure of dental calculus and associated bacteria.

INTRODUCTION: Though dental calculus is generally recognised as comprising mineralised bacteria, areas of non-mineralised bacteria may be present. AIM: To investigate the ultrastructure of non-decalcified young and mature supragingival calculus and subgingival calculus, and the possible presence of internal viable bacteria. MATERIALS AND METHODS: Supragingival calculus was harvested from five patients, 9-10 weeks after scaling and root debridement. Five samples of mature supragingival and subgingival calculus were taken from patients presenting with adult periodontitis. Specimens were fixed and embedded for transmission electron microscopy. RESULTS: The ultrastructure of young and mature supragingival calculus was similar with various large and small crystal types. Non-mineralised channels were observed extending into the calculus, often joining extensive lacunae, both containing intact non-mineralised coccoid and rod-shaped microorganisms. Subgingival calculus possessed more uniform mineralisation without non-mineralised channels and lacunae. CONCLUSION: Supragingival calculus contains non-mineralised areas which contain bacteria and other debris. The viability of the bacteria, and their identification could not be determined in this preliminary investigation. As viable bacteria within these lacunae may provide a source of re-infection, further work needs to be done to identify the bacteria in the lacunae, and to determine their viability.

Assessment of enamel erosion and protective effect of salivary pellicle by surface roughness analysis and scanning electron microscopy.

PURPOSE: To assess dental erosion caused by 0.1% and 1.0% citric acid in vitro and to estimate the protective influence of experimentally formed salivary pellicle. MATERIALS AND METHODS: Bovine enamel slabs (n = 80) were polished and embedded in epoxy resin. For the formation of pellicle layer 40 specimens were immersed for 24 h in pooled human saliva. Erosion was caused by immersion in citric acid solution for 1, 5, 10 and 30 min. Erosive alterations on the pellicle-covered and non-covered enamel specimens were scored as a change (delta) of surface roughness parameters Ra, Rt and RzDIN using contact profilometer and observed in scanning electron microscope. RESULTS: Profilometric analysis of eroded enamel specimens emphasized the aggressiveness of even low concentrated citric acid with a short period of challenge. The change of roughness parameters after 1-min immersion in 0.1% citric acid were 16.4, 182.6 and 132.2 nm for deltaRa, deltaRt and deltaRzDIN, respectively, and 54.8, 516.6 and 258.2 nm after 1-min immersion in 1.0% citric acid. Changes of the surface roughness were dependent on the exposure time and concentration of acidic solution. Pellicle layer significantly reduced the extent of erosive destruction, which was additionally documented on SEM-micrographs. Residual pellicle-like structures were detected after 5 min of immersion in 0.1% citric acid. However, there were no significant differences in pellicle-covered and non-covered enamel slabs measured profilometrically for 1.0% citric acid with 10 min and 30 min exposure time. CONCLUSION: The findings confirm the property of pellicle layer to resist against erosive influence of organic acids, which is, however, limited by duration of acidic treatment and concentration of erosive agent.

Electron-microscopic demonstration of proline-rich proteins, statherin, and histatins in acquired enamel pellicles in vitro.

Proline-rich proteins (PRPs), histatins, and statherin are salivary proteins that exhibit high affinities for hydroxyapatite surfaces. In vitro experiments with parotid submandibular/sublingual or whole saliva have shown these proteins to adsorb selectively to tooth surfaces. This investigation focuses on the histo-morphological identification of PRPs, histatins, and statherin in acquired enamel pellicles. Synthetic hydroxyapatite or bovine enamel were exposed to glandular secretions, and whole saliva and pellicle precursor proteins were identified immunohistologically by electron microscopy. Results obtained by back-scattered scanning electron microscopy showed these proteins to be present in pellicles. Pellicles displayed a distinct structure consisting of a sponge-like meshwork of microglobules. Interconnections between structural elements were identified in submandibular/sublingual and whole saliva pellicles only. Transmission electron microscopy of pellicles formed on bovine enamel surfaces revealed a tendency for preferential localization of precursor proteins within the protein film. Since the data showed the presence of pellicle precursors in pellicles derived both from glandular secretions and from whole saliva, it is likely that PRPs, histatins, and statherin are integral components of acquired enamel pellicles in vivo.

Protective properties of salivary pellicles from two different intraoral sites on enamel erosion.

The purpose of this study was to investigate the protective effect and ultrastructure of salivary pellicles formed in vivo near the orifices of the ducts of parotid and submandibular/sublingual salivary glands. Pellicles were formed by exposing bovine enamel slabs to the oral environment at the buccal aspect of the upper first molars and at the lingual aspect of the lower incisors in 3 subjects over periods of 24 h. Enamel specimens with and without 24-hour pellicles were immersed in citric acid (0.1 and 1%) for periods ranging from 30 s to 5 min, and processed for measurement of surface microhardness (SMH) and transmission electron microscopy (TEM). In comparison to uncovered enamel specimen significantly less decrease in SMH due to acid exposure was observed in pellicle-coated enamel specimens. Pellicles formed at the buccal aspect of the upper molars were less effective in protecting the enamel against acid-induced softening as compared to pellicles formed at the lingual aspect of the lower incisors only after 5 min exposure in 1% citric acid. TEM analysis showed that pellicle layers were dissolved continuously due to acid exposure. However, even after 5 min exposure to 1% citric acid, a residual pellicle layer could be detected on the enamel surface. In conclusion, site-dependent differences of buccally and lingually in vivo formed 24-hour pellicles have minor importance concerning the pellicle-induced protection of the enamel surface against erosive changes.

Transmission electron microscopy of early plaque formation on dental materials in vivo.

This in vivo study describes the ultrastructural pattern of early plaque formation on various dental materials. Test pieces of amalgam, casting alloys, titanium, ceramics, glass polyalkenoate cement, composite resins, unfilled resins, and bovine enamel were attached to the buccal and lingual surfaces of the upper first molars in 3 subjects using removable intraoral splints. Specimens were exposed to the oral environment over a period of 24 h and subsequently processed for transmission electron microscopic evaluation. Only less pronounced variations could be detected in the ultrastructural appearance of the early plaque formed on the different material surfaces. However, electron microscopic observations revealed distinct differences in early biofilm formation between buccally and lingually mounted test pieces. While the bacterial colonization of specimens worn in the lingual position remained limited to the adherence of individual micro-organisms in the area of surface irregularities, a multi-layer adherence of micro-organisms was observed on all specimens carried in buccal areas. It is concluded that early plaque formation on solid surfaces is influenced predominantly by the oral environment rather than by material-dependent parameters. These findings may be ascribed to the presence of the pellicle layer, which apparently masks any difference among materials, with regard to surface properties and biocompatibility.

Ultrastructural investigation of pellicle morphogenesis at two different intraoral sites during a 24-h period.

The purpose of the present in vivo study was to examine salivary pellicle formation on enamel surfaces at two different intraoral sites for periods of 1 min up to 24 h by means of transmission electron microscopy. Bovine enamel specimens were attached to the buccal and lingual surfaces of the upper first molars in three subjects using removable intraoral splints. Specimens were carried over period of 1, 10, 30 and 60 min, 2, 6 and 24 h and were processed for transmission electron microscopy. After 1 min, an electron dense pellicle layer, 10-20 nm thick, was observed on the enamel surfaces. The subsequent adsorption of salivary biopolymers was governed by local influences of the oral cavity. Specimens located on the lingual aspect were covered within 2 h by a 20- to 80-nm-thick, homogeneous, predominantly granular-structured pellicle. The thickness of the surface coatings that were adsorbed on lingually carried specimens increased to 100-200 nm after 24 h. In contrast, on the buccally mounted specimen surfaces, a variably structured pellicle with granular and globular components could be detected after intraoral exposure for 2 h. The thickness of the 2-h buccal pellicles ranged between 200 and 700 nm. After 24 h, the buccally positioned specimens were covered by a dense globular pellicle layer varying in thickness from 1000 to 1300 nm. It is suggested that in vivo pellicle formation is initiated by adsorption of an electron-dense layer of salivary proteins. Further adsorption of salivary biopolymers leads to the formation of an outer loosely arranged pellicle layer. Under oral conditions, the locally available salivary biopolymers and the influence of locally effective shearing forces are of significance for the ultrastructural pattern and extent of pellicle formation.

Transmission electron microscopic study of in vivo pellicle formation on dental restorative materials.

This electron microscopic investigation was performed to examine the ultrastructure of the in vivo formed salivary pellicle on 15 different dental materials. Test pieces of amalgam alloys, casting alloys, titanium, ceramics, resins, composite resins, glass polyalkenoate cement, and bovine enamel were attached to the buccal and lingual surfaces of the upper first molars in three adults using removable intraoral splints. The splints were carried over periods of 2 and 6 h. Pellicle-like structures could be identified on all tested material surfaces by transmission electron microscopic analysis. The pellicle layer showed a high degree of similarity on different kinds of surfaces with regard to the ultrastructural appearance. However, distinct differences could be detected in the ultrastructural pattern and thickness of the pellicle layer formed on buccally and lingually mounted specimens. Pellicles adsorbed on buccally carried test pieces were characterized by a heterogeneous, globular appearance and a thickness ranging from 500 to 1,000 nm after 6 h. In contrast, all lingually mounted test pieces were covered by a granular pellicle of about 100 nm thickness after 6 h. It is concluded that the ultrastructural pattern and extent of salivary pellicle formation are influenced by locally available salivary biopolymers and locally effective shearing forces rather than by material-dependent parameters.

Scanning electron microscopic examination of different cleaners: surface contaminant removal from dentures.

Dentures were examined by scanning electron microscopy to evaluate removal of surface contaminants. Five complete dentures were obtained during patient appointments. The palatal surface of each denture was divided into eight pieces (1 cm2) then each sample cleaned with Corega, Dentipur, Fittydent, sodium hypochloride, Savlon, Ipanol, brushing methods and one sample was also kept as a control. They were prepared for SEM examination and photographed at x500. One photograph of each sample was evaluated in random order by three judges for a total of 120 observations. Photographs were compared with one of a clean denture sample. Statistical analysis of the results showed that soaking dentures in sodium hypochloride and Savlon removed significantly more contaminants than any of the other methods used in this study.

Analytical and ultrastructural studies of pellicle on primary teeth.

The pellicle on permanent enamel has been thoroughly studied. The aims of this study were to compare the chemical composition, rate of formation, and ultrastructural appearance of pellicle formed on deciduous enamel in children with those on permanent teeth. This was done by amino acid analyses, Auger analyses, and transmission electron microscopy. The amino acid composition of 2-h pellicle on deciduous and permanent enamel had an overall similar pattern, but the contents of serine, glycine, and tyrosine were statistically significantly different. An initially slower pellicle formation and a thinner 2-h pellicle without a globular structured second layer was observed on deciduous enamel. The results indicated therefore distinct differences in chemical composition, rate of formation, and ultrastructural appearance between pellicle on primary teeth and that on permanent teeth.

A scanning electron microscope investigation of calcium fluoride-like material deposited during topical fluoride exposure on sound human enamel in vitro.

1. The morphological aspects of globules deposited in vitro when a NaF solution is applied to sound human enamel were studied by scanning electron microscopy. 2. Particles, presumably of calcium fluoride-like material, formed on the surfaces of an unerupted tooth and consisted of subunits showing a "cauliflower" appearance, indicating agglomeration of even smaller particles. The number and size of the crystals increased with time of exposure. 3. These particles represent highly resistant fluoride reservoirs and may be of clinical significance.

A scanning electron microscope investigation of calcium fluoride-like material deposited during topical fluoride exposure on sound human enamel in vitro

1. The morphological aspects of globules deposited in vitro when a NaF solution is applied to sound human enamel were studied by scanning electron microscopy. 2. Particles, presumably of calcium fluoride-like material, formed on the surfaces of an unerupted tooth and consisted of subunits showing a "cauliflower" appearance, idnciating agglomeration of even smaller particles. The number and size of the crystals increased with time of exposure. 3. These particles represent highly resistant fluoride reservoirs and may be of clincial significance

Acid resistance of human enamel by brushing with and without abrasive dentifrice.

Daily brushing with and without abrasive dentifrice pieces of was performed on the ground surfaces of human enamel attached to resin plates which were exposed to the oral cavities of 4 subjects for 8 weeks. The difference in acid resistance between a pair of enamel pieces obtained from the same tooth was examined with atomic absorption spectrometry. After etching with a lactate buffer solution at pH 4.5, the amount of Ca dissolved from the enamel in each pair by brushing without dentifrice was significantly lower than that when brushing with an by abrasive paste. Under SEM, the brushing with paste caused prism structures to appear clearly on the enamel surface, while brushing without dentifrice caused a nonbacterial organic film, i.e., salivary pellicle, to cover the surface. These results indicate that the pellicle is protected by mineral deposits when brushing without dentifrice but brushing with an abrasive paste tears off the unmineralized pellicle. Brushing without dentifrice induces a stronger resistance to acid on the enamel surface.

Effect of two organic phosphonates on protein adsorption in vitro and on pellicle formation in vivo.

Organic phosphonates have been introduced in dentifrices to reduce the formation of dental calculus. They may conceivably act as calcium sequestrants or crystal growth inhibitors, interfering directly with the calcium ions on the hydroxyapatite (HA) and enamel surfaces. The aim of the present study was to examine the effect of two organic phosphonates on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo. The effect on protein adsorption in vitro was studied by adsorption of albumin to either untreated or phosphonate treated HA powder. Ion exchange chromatography was also performed with columns with untreated and phosphonate treated HA as bed materials and with linear gradients of either phosphate or phosphonates. The effect on pellicle formation in vivo was studied by scanning electron microscopy on untreated and phosphonate treated enamel fragments which had been carried in the mouth to acquire pellicle materials. The present study showed that phosphonate-treated HA took up less protein. The adsorbed protein was, furthermore, less firmly bound to phosphonate treated hydroxyapatite. Phosphonate-treated enamel fragments carried in the mouth also exhibited a slower rate of pellicle formation as compared to the untreated enamel fragments.

Pits and fissures: remnant organic debris after acid-etching.

The purpose of this study was to compare the ability of liquid and gel etches to remove remaining debris in occlusal pits and fissures in vitro after cleansing using a conventional technique or air-water slurry method. A suitable stain differentiated between etched enamel, unetched enamel, and pellicle or debris. The results statistically indicate that etching does not remove much of the organic debris, and there was no difference between a liquid or gel etchant. Agitation of the etchant did not aid pellicle removal.

Effect of sodium lauryl sulfate on protein adsorption to hydroxyapatite in vitro and on pellicle formation in vivo.

Sodium lauryl sulfate (SLS) is widely used as a synthetic detergent in dentifrices. It has been shown to have high affinity for hydroxyapatite (HA), and the binding mechanism has been proposed to be electrostatic, involving the negative sulfate terminals of the SLS and the calcium sites on the HA. The binding of SLS to HA may thus well interfere with the protein adsorption to HA. The aim of the present study was to examine the effect of SLS on protein adsorption in vitro and on pellicle formation in vivo. The effect on protein adsorption was studied using ion exchange chromatography. The effect on pellicle formation was studied using enamel fragments carried in the mouth. The study showed that SLS-treated HA adsorbed less protein than untreated HA. Protein adsorbed to SLS-treated HA was more firmly bound to HA as compared to untreated HA. SLS-treated enamel fragments carried in the mouth showed a slower rate of pellicle formation than non-treated enamel.