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PubMed1999

Ultrastructural properties of laser-irradiated and heat-treated dentin.

Previous studies using scanning electron microscopy and infrared absorption spectroscopy reported that laser irradiation causes compositional changes in enamel. The purpose of this study was to evaluate the ultrastructural and compositional changes in dentin caused by irradiation with a short-pulse laser (Q-switched Nd:YAG). The irradiated and non-irradiated areas of the lased dentin samples were investigated by scanning (SEM) and transmission electron microscopy (TEM), micro-micro electron diffraction, and electron microprobe analysis of dispersive energy (EDX). Heat-treated dentin was similarly investigated. This study demonstrated that laser irradiation resulted in the recrystallization of dentin apatite and in the formation of additional calcium phosphate phases consisting of magnesium-substituted beta-tricalcium phosphate, beta-TCMP, beta-(Ca,Mg)3(PO4)2, and tetracalcium phosphate, TetCP, Ca4(PO4)O. TEM analyses of the modified and unmodified zones of the irradiated areas showed two types of crystal populations: much larger crystals from the modified zone and crystals with size and morphology similar to those of dentin apatite in the unmodified zone. The morphology of crystals in the modified zones in the irradiated dentin resembled those of dentin sintered at 800 or 950 degrees C. In the irradiated areas (modified and unmodified zones), the Ca/P ratio was lower compared with that in the non-irradiated dentin. The Mg/Ca ratio in the modified zones was higher than that in the unmodified zones and in the non-irradiated dentin. In sintered dentin, the Mg/Ca ratio increased as a function of sintering temperature. The ultrastructural and compositional changes observed in laser-irradiated dentin may be attributed to high temperature and high pressure induced by microplasma during laser irradiation. These changes may alter the solubility of the irradiated dentin, making it less susceptible to acid dissolution or to the caries process.

Calcium↗
PubMed2002

[Does arteriosclerotic calcinosis of vessel walls imitate osteogenesis? Pathomorphological studies of arteriosclerotic plaque].

OBJECTIVE: For it is supposed that calcification of arteriosclerotic plaques imitates osteogenesis, it was studied whether the morphology of different stages of calcification supports such an assumption. MATERIAL AND METHODS: Investigations were performed on one operation specimen of a dissecting aneurysm of the aorta and five human autopsy aortas. From different arteriosclerotic plaques, paraffin sections without and after EDTA-decalcification were prepared. Light microscopic, scanning (SEM) and transmission electron microscopic (TEM) investigations as well as X-ray microanalysis were performed. RESULTS: By light microscopy, it became obvious that the intensity of calcification of arteriosclerotic plaques is not reflected by the conventional H&E stain--the real amount of calcium-phosphorus deposits becomes only evident by using a special stain, such as the alizarin red S reaction. In the SEM, arteriosclerotic plaques with granular calcification were characterized by accumulations of isolated calcified globules often reflecting the structure of necrotic cells. TEM exhibited even accumulations of calcified bodies corresponding to the shape of cells; cell remnants were present in the neighborhood. X-ray microanalysis of the granules detected calcium and phosphorus and a relative high content of magnesium, reflecting the composition of calcospherites in granular media calcification (71). By SEM, solid calcifications exhibited associations of calcified bodies resembling those of the isolated granules in early calcification but with one stuck onto the other. X-ray microanalysis revealed that these deposits are characterized by high amounts of calcium and phosphorus and a minor component of magnesium in comparison to the isolated granules. CONCLUSIONS: From the investigations it is concluded that calcification of arteriosclerotic plaques imitates the process of granular media calcification which is assumed as "dystrophic" calcification of fragments of necrotic cells. The hypothesis of a "regulated" calcification as it occurs in cartilage or bone cannot be supported for the development of the calcified plaques; however, it cannot be excluded that proteins that are associated with osteogenesis participate in the transition of calcification to ossification in the vessel wall.

Aortic Dissection↗
PubMed1988

X-ray microanalysis of freeze-dried and frozen-hydrated cryosections.

The elemental composition and the ultrastructure of biological cells were studied by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray microanalysis. The preparation technique involves cryofixation, cryoultramicrotomy, cryotransfer, and freeze-drying of samples. Freeze-dried cryosections 100-nm thick appeared to be appropriate for measuring the distribution of diffusible elements and water in different compartments of the cells. The lateral analytical resolution was less than 50 nm, depending on ice crystal damage and section thickness. The detection limit was in the range of 10 mmol/kg dry weight for all elements with an atomic number higher than 12; for sodium and magnesium the detection limits were about 30 and 20 mmol/kg dry weight, respectively. The darkfield intensity in STEM is linearly related to the mass thickness. Thus, it becomes possible to measure the water content in intracellular compartments by using the darkfield signal of the dry mass remaining after freeze-drying. By combining the X-ray microanalytical data expressed as dry weight concentrations with the measurements of the water content, physiologically more meaningful wet weight concentrations of elements were determined. In comparison to freeze-dried cryosections frozen-hydrated sections showed poor contrast and were very sensitive against radiation damage, resulting in mass loss. The high electron exposure required for recording X-ray spectra made reproducible microanalysis of ultrathin (about 100-nm thick) frozen-hydrated sections impossible. The mass loss could be reduced by carbon coating; however, the improvement achieved thus far is still insufficient for applications in X-ray microanalysis. Therefore, at present only bulk specimens or at least 1-micron thick sections can be used for X-ray microanalysis of frozen-hydrated biological samples.

Animals↗
PubMed1997

The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.

Animals↗
PubMed1996

Absorption of iron by dentin: its role in discoloration.

This study investigated the effect of iron-containing mordants on the discoloration of human dentin. Dentin wafers with intact enamel borders were treated with one of several acid solutions containing iron. After exposure to aqueous sodium sulfide, color change was evaluated colorimetrically. X-ray photoelectron spectroscopy (XPS) and energy dispersive x-ray spectrometry (EDS) were used to probe surfaces for the presence of iron. Changes in surface morphology were evaluated by scanning electron microscopy (SEM). The results indicated that dentin treated by iron-containing solutions always discolored when the sodium-sulfide concentration exceeded 9.7 mM; variability in discoloration occurred below this level. XPS detected iron in enamel but not in dentin, while EDS detected iron in both enamel and dentin. Since XPS probes the upper atomic layers, these results indicate that acid-demineralized dentin absorbs iron; however, a water wash removes iron from the uppermost dentin surface, suggesting that the iron is physically absorbed. When exposed to sodium sulfide, at least some of the iron remaining in the bulk region is able to migrate to the surface to form iron-sulfide compounds. Based on SEM findings, discoloration is associated with the formation of a surface film on dentin.

Absorption↗
PubMed2004

Structural and analytical comparison of gallbladder stones collected from a single patient: studies of five cases.

We observed the gross and fine structure of gallbladder stones collected from five adult patients (cases I-V) by optical photography, radiography, scanning electron microscopy, and backscattered electron microscopy, and then measured the components by energy-dispersive X-ray microanalysis and infrared spectroscopy. From the stones, calcium (Ca) phosphate, Ca bilirubinate, and Ca palmitate or fatty acid Ca were identified. The 3 cholesterol stones (case I) and the 2 brown pigment stones (case II) showed macroscopic homogeneity, respectively. In addition, their fine structure and components were also similar to each other. The black pigment stones (case III) showed macroscopic homogeneity, but they were divided into radiopaque (approximately 30 stones) and radiolucent types (approximately 60 stones). The former had Ca phosphate in the center surrounded with Ca bilirubinate, and the latter was dotted with minute deposits of Ca bilirubinate. The 6 cholesterol stones (case IV) were divided into two types in size. The 5 large stones, of macroscopic homogeneity, had a core region of Ca palmitate and clear concentric rings of Ca phosphate, whereas the smaller stone was almost filled with Ca phosphate deposits in the center. From the different distributions of Ca phosphate, the smaller stone may have been formed later than the 5 large stones. Case V contained 4 stones. The 3 large cholesterol stones, of more or less macroscopic homogeneity, had a core region and concentric rings of Ca phosphate, but 1 smaller stone was dotted with minute deposits mainly containing iron (Fe) and/or silicon elements (rare type). Therefore, the stones of cases III, IV, and V showed considerable heterogeneity, respectively. In many stones, the initial precipitation of Ca salts will have become the nidus, and the concentric rings and dotted deposits of Ca salts may have accelerated cholesterol stone growth. In addition, the dotted deposits of Ca bilirubinate in the black pigment stones and the dotted deposits containing Fe in the rare stone may have become also the nidi.

Adult↗
PubMed2002

Characterization of hard metal dusts from sintering and detonation coating processes and comparative hydroxyl radical production.

Dust samples from sintering and detonation coating hard-metal processes were characterized, compared, and contrasted for morphology, composition, and generation of hydroxyl radicals. Inhalation of respirable hard-metal (sintered carbide) dusts from hard-metal processes is known to cause fibrotic and asthmatic lung disease. Scanning electron microscopy/energy-dispersive X-ray analysis was used for morphology, composition, and elemental distribution. An electron spin resonance (ESR) spin trapping technique was used to detect hydroxyl radical generation. Samples were incubated with air-saturated buffer solutions containing a spin trap and analyzed by ESR for the presence of *OH in solution. Postdetonation coating samples often had surface contamination of Co on the WC particles, as shown by elemental mapping of individual particles; this was not evident in predetonation samples or unsintered materials in this study. ESR measurements show that both detonation-gun materials were capable of generating *OH , while the WC, cobalt, and presintered mixture did not produce detectable amounts of *OH radicals. The DMPO/*OH adduct formation was apparently facilitated by Fe-mediated reactions for predetonation dusts, and by Fe-mediated site-specific reactions for postdetonation dusts. The overspray materials from the detonation-gun process produced 9-fold more *OH radicals than the predetonation coating mixture. Overall, this study indicates there are substantial differences between postdetonation materials and both predetonation and unsintered hard-metal process materials with respect to morphology, elemental distribution, and *OH radical generation reactions and that these differences may be important in the toxic potential of those materials.

Air Pollutants, Occupational↗
PubMedDate not supplied

In vitro osteoclast resorption of bone substitute biomaterials used for implant site augmentation: a pilot study.

PURPOSE: This observational study examined the resorptive behavior of normal neonatal rabbit osteoclasts grown on slices of bovine cortical bone as compared to samples of commercially available bone substitute biomaterials. It also examined the surface characteristics of these materials. MATERIALS AND METHODS: The 11 materials tested fell into 3 groups: (1) bone-derived, including freeze-dried human rib block, human demineralized freeze-dried bone, and deproteinated bovine bone; (2) synthetic hydroxyapatites (HA); and (3) synthetic non-HA, including coated methacrylates and coated silica glass. After 4 days in culture, 1 group of samples of each material underwent scanning electron microscopy (SEM) to evaluate resorptive pitting versus controls, while another group underwent tartrate-resistant acid phosphatase staining and light microscopy to examine osteoclast numbers and morphology. The 2 bovine-derived HA materials also underwent immunohistochemical staining and surface chemistry analysis. RESULTS: While most of these materials supported osteoclast attachment, some spreading, and survival in culture, only the bone-derived materials, with the exception of sintered deproteinated bovine bone, showed large scalloped-edged resorption pits with trails and exposed collagen when examined by SEM, although not to the same extent as unprocessed natural bone material. The HA materials and the sintered deproteinated bovine bone showed evidence of etching with smaller pits but no evidence of resorptive trail formation. The non-HA materials showed no evidence of pit formation or trails. Under immunohistochemical staining, Bio-Oss appeared to be positive for type I collagen after osteoclast activity on its surface, while Osteograf/N showed no positive staining. Surface chemistry analysis revealed nitrogen present in Bio-Oss specimens (0.17% to 0.47%), while there was no nitrogen detected in the Osteograf/N (0.00%); the percent nitrogen observed in normal bovine bone controls was 6.01% to 9.25%. DISCUSSION: The bone-derived materials supported osteoclast activity on the material surface in a way that facilitated formation of the more complex resorption pits in vitro. Assuming the rate of pit formation observed in vitro mimics that observed in vivo, the quantity and type of osteoclastic remodeling seen on non-bone-derived materials--and perhaps sintered bone-derived materials--would be extremely slow to negligible. Physiologic removal of non-bone-derived bone substitutes in vivo may occur by methods other than osteoclast resorption. CONCLUSIONS: Allogenous and xenogenous bone-derived materials that undergo delayed physiologic resorption may be more appropriately used with a staged surgical approach when used in sites intended to support osseointegrated dental implants. The combination of collagen staining and the presence of nitrogen suggest that there may be residual protein in Bio-Oss.

Acid Phosphatase↗
PubMed1993

Quantitative histochemistry of phosphorus in the vestibular gelatinous membrane: an electron probe X-ray microanalytical study.

Electron probe X-ray microanalysis was used to study the phosphorus concentration in the otolithic gelatinous membrane of the saccule and the utricle with scanning electron microscopy. The otolithic membranes were plunge-frozen in liquid N2 and freeze-dried. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 salts dissolved in dextran solutions. The otolithic gelatinous membrane consists of a 25-30 microns-thick layer overlying the cilia of the hair cells. Elements detected in the gelatinous membrane are: Na, P, S, Cl, K and Ca. Although Student's t-test did not show significant differences between saccular and utricular concentrations of phosphorus, the distribution of this element in the two organs was different. Regression analysis established that the concentrations of phosphorus in the saccular and utricular gelatinous membrane were dependent. The regression equation was: y = 18.02x2 + 133.9 (r = 0.83, P < 0.05) where y is the concentration of phosphorus in the utricle, and x2 the concentration of phosphorus in the saccule. The findings obtained in the present study could be related to structural differences in organic phosphate residues of the phosphoproteins associated to collagen, or to different polyphosphoinositide turnover rates in the cell membrane.

Animals↗
PubMed2005

Experimental evaluation of a spherical aberration-corrected TEM and STEM.

We have successfully developed a spherical aberration (Cs)-corrected electron microscope for probe- and image-forming systems using hexapole correctors. The performance of the microscope has been evaluated experimentally. The point resolution attained using the image-forming Cs-corrector is better than 0.12 nm. For scanning transmission electron microscopy, the Ronchigram flat area was >40 mrad in half-angle using the probe-forming Cs-corrector.

Letter↗
PubMed1980

A practical method for the identification of particulate and crystalline material in paraffin--embedded tissue specimens.

The combination of light microscopy, low temperature micro-incineration of paraffin sections, and subsequent scanning electron microscopy (SEM), with X-ray energy spectroscopy (XES) has been shown to allow rapid and reliable identification of inorganic crystalline and particulate material in body tissues. Applications of this technique to hospital practice are illustrated and its potential in diagnosis and research are suggested.

Electron Probe Microanalysis↗
PubMed2004

Opacification of a silicone intraocular lens caused by calcium deposits on the optic.

We describe opacification of a plate-haptic silicone intraocular lens (IOL) caused by calcification in a diabetic patient with asteroid hyalosis. The IOL was explanted 48 months after uneventful phacoemulsification because opacification of the posterior surface was causing significant visual disturbance. Light and scanning electron microscopy and x-ray spectrometry of the explanted IOL showed the opacification consisted mainly of calcium and phosphate, presumably hydroxyapatite, in the form of precipitations on the posterior surface of the optic.

Aged↗
PubMed1985

Morphometric and elemental microanalytical studies of human lung in health and disease.

Current methods for determining the fibrogenicity of substances are based on relatively long term exposures of animals to the substance and the evaluation of morphological changes occurring in the lung. The use of inhalation chambers, which produce a more physiological environment, suffer from the need for particularly long exposure times (1-3 years). The present study describes a technique using scanning electron microscopy, energy dispersive analysis, and a digitiser pad with a computer to evaluate the fibrogenicity of silica in cases of known exposure. Scanning electron micrographs taken from silicotic lungs were evaluated for the degree of thickening (fibrosis) and the same areas were analysed for silicon content. Correlations between silicon content and septal thickening were shown to be significant (p less than 0.0001). The study also describes the concentrations of elements found in normal lungs. The technique for establishing correlation curves between elemental concentrations and septal thickening could be of value in determining the fibrogenicity of pure substances after short exposures in an environmental chamber.

Aluminum↗
PubMed1981

Application of scanning electron microscopy to x-ray analysis of frozen-hydrated sections. I. Specimen handling techniques.

X-ray microanalysis of frozen-hydrated tissue sections permits direct quantitative analysis of diffusible elements in defined cellular compartments. Because the sections are hydrated, elemental concentrations can be defined as wet-weight mass fractions. Use of these techniques should also permit determination of water fraction in cellular compartments. Reliable preparative techniques provide flat, smooth, 0.5 micrometers-thick sections with little elemental and morphological disruption. The specimen support and transfer system described permits hydrated sections to be transferred to the scanning electron microscope cold stage for examination and analysis without contamination or water loss and without introduction of extraneous x-ray radiation.

Animals↗