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PubMed1976

Calcification of alpha-elastin coacervates: a bulk property of elastin.

Scanning electron microscopy and electron probe microanalysis studies are reported on thin sections of calcified coacervates of alpha-elastin. It is found that the capacity of elastin coacervates to initiate calcification is a bulk property of the coacervate and not limited to the serum-coacervate interface, that the calcium phosphate deposits act to bind the protein units together and slow the dissolution and spreading of the coacervate as it floats on an airwater interface, and that, within the limits of detectability, there is no involvement of sulfur. As the charged groups of alpha-elastin had been blocked, the initiation of deposition is due to neutral sites in the protein which are tightly bound to the calcium phosphate deposits.

Animals↗
PubMed1990

Scanning and transmission electron microscopy, and electron probe analysis of the interface between implants and host bone. Osseo-coalescence versus osseo-integration.

Bioinert materials (e.g., alumina implants) and bioactive ceramics (e.g., calcium phosphate ceramics, glass-ceramics) are now extensively used in dentistry. However, the physico-chemical interactions at the interfaces between the implant and the host bone are poorly understood. The purpose of this study was to define the interactions at these interfaces using a combination of analytical techniques: light microscopy, scanning and transmission electron microscopy, electron probe microanalysis, X-ray microradiography, X-ray diffraction, and infrared spectroscopy. Bioinert (pure titanium) and bioactive materials (hydroxyapatite, beta-tricalcium phosphate and biphasic calcium phosphate) were implanted in dogs, and the implants, recovered after various periods of implantation, were analyzed. The results demonstrated the following: the bioactive materials interact with the biological fluid and the living tissues in a specific manner. This process includes biodissolution/biodegradation, apatite crystal precipitation, and bone formation on the implant surface at the expense of the material. The results are discussed according to the limitations of the analytical techniques used. The medical and chemical word coalescence is suggested to describe the specific interactions of bioactive materials and interaction for the phenomenon of physical contact of the bioinert materials with the host bone.

Animals↗
PubMed1996

Bone-bonding behavior of plasma-sprayed coatings of BioglassR, AW-glass ceramic, and tricalcium phosphate on titanium alloy.

The bone-bonding behavior of three kinds of bioactive ceramics coated on titanium alloy by the plasma-spray technique was investigated. Titanium alloy (Ti-6A1-4V) coated with BioglassR (45S5), apatite-wollastonite containing glass ceramic (AW), or beta-tricalcium phosphate (TCP) was prepared, and rectangular specimens were implanted into the tibial bones of mature male rabbits, which were sacrificed 8 or 24 weeks after implantation. The tibiae containing the implants were dissected out and subjected to detachment tests to measure the failure load. The bone-implant interface was investigated by Giemsa surface staining, contact microradiography, and scanning electron microscopy-electron probe microanalysis (SEM-EPMA). Eight weeks after implantation, the failure loads for implants coated with BioglassR, AW, and TCP were 1.04 +/- 0.94, 2.03 +/- 1.17, and 3.91 +/- 1.51 kg, respectively, and 24 weeks after implantation, the respective failure loads were 2.72 +/- 1.33, 2.39 +/- 1.30, and 4.23 +/- 1.34 kg. Failure loads of AW- and TCP-coated implants did not increase significantly with time. After the detachment test, breakage of the coating layer was observed. Bioactive ceramics can act as stimulants that induce bonding between bone and metal implants. However, failure load of metal implants coated with the bioactive ceramics was lower than that of bulk AW or TCP. It appears impossible to obtain a higher failure load using a bioactive-ceramic coating on titanium alloy. Histologically, the coating layer was found to become detached from the metal implant and the bone tissue bonded to the coating layer. SEM-EPMA observation revealed breakage of the coating layer, although bonding between bone and the coating layer was evident. A Ca-P-rich layer was observed at the interface between bone and the AW coating, and a Ca-P-rich and a Si-rich layer were observed at the interface between bone and the BioglassR coating. For clinical application, it would seem better to use coated metal implants for short-term implantation. However, there is a possibility of breakage of the coating layer because of both dissolution of the bioactive ceramic and mechanical weakness at the interface between the coating layer and the metal implant.

Alloys↗
PubMed1985

Ultrastructure and chemical composition of calcite urinary calculi in Chinese swamp buffalo.

Four urinary calculi, derived from Chinese swamp buffalo, were studied by using qualitative chemical analysis, X-ray diffraction, scanning electron microscopy and qualitative energy dispersive (electron probe) microanalysis. Qualitative chemical analysis showed that the predominant ions were calcium and carbonate with small amounts of magnesium and ammonium. X-ray diffraction confirmed that the calculi were primarily composed of calcium carbonate (calcite). On ultrastructural examination, three apparently distinct structural regions were identified in the calculi: outer large laminations; cavities containing variable numbers of small spheres and rods; and large spheres. There did not appear to be material that acted as a nidus and all regions, on qualitative electron probe analysis, contained primarily calcium with trace amounts of magnesium, phosphorus, potassium and chloride. It was concluded that calcite calculi in Chinese swamp buffalo are probably formed through a process of asynchronous layering and that nidus formation may not be necessary. Moreover, the ultrastructure of the calcite calculi is similar to that reported for siliceous calculi in ruminants and this suggests that similar factors may be involved in their formation.

Animals↗
PubMed1995

Artefacts in electron microscopy: ultrastructural features of chrysiasis.

Eleven cases of chrysiasis have been studied ultrastructurally and by electron probe microanalysis. Tissue samples were examined both with and without osmium and uranyl acetate staining. There was a significant morphological difference in the appearance of the gold deposits within aurosomes between the two groups. The untreated group showed finely granular deposits, often arranged in a linear fashion. Aurosomes from samples treated with osmium and uranyl acetate showed more electron-dense deposits and star-like formations. We propose that treatment of the tissue with osmium and uranyl acetate causes a change in the electron microscopic appearance of aurosomes, including the formation of the characteristic star-like aurosomes.

Antirheumatic Agents↗
PubMed1989

Detection of cationic and non-cationic markers in the rat glomerulus by electron probe analysis.

Acidic glycans (glomerular polyanion substances) in the rat kidney were visualized ultrastructurally by three cationic markers: colloidal iron, ruthenium red, and polyethylenimine-phosphotungstic acid (PEI-PTA). Heavy metal atoms (Fe, Ru and W) were detected in ultrathin sections by energy-dispersive electron probe microanalysis (EPMA). Characteristic peaks of the locally bound elements were obtained in spectra derived from the dense structures seen by transmission electron microscopy (TEM)--i.e. the glycocalyx of podocytes and/or the polyanion sites in the lamina rara externa of the glomerular basement membrane. Weaker signals were emitted by some extraglomerular structures. This finding may reflect a low concentration of glycans in structures lacking apparent density by TEM, and/or incomplete specificity of the markers, partial dislocation of reactive substances or the presence of an endogenous element (Fe). Experimental argyrosis was elicited by the peroral administration of silver nitrate. Dense Ag precipitates were seen chiefly in the lamina densa and characteristic peaks of silver were displayed in this site by EPMA, and was best demonstrated in non-contrasted sections. A single i.v. injection of Ag proteinate failed to produce glomerular pigmentation. The only dense granular product in tubular cells yielded characteristic peaks of Fe (endogenous siderosomes) but EPMA excluded detectable amounts of silver.

Animals↗
PubMed2004

A comparative study of white mineral trioxide aggregate and white Portland cements using X-ray microanalysis.

The purpose of this study was to determine and compare the composition of white mineral trioxide aggregate (WMTA) and two different white Portland cements (WPCs). Samples of WMTA and WPCs were prepared and then imaged in a JEOL JSM6400 scanning electron microscope, equipped with an Oxford Instruments light element energy dispersive spectrometer detector for determining the elemental composition. Electron probe microanalysis (EPMA) results indicated that lime (CaO) and silica (SiO2) were the dominant compounds in each case. The results showed that the trace elements are similar in all of the samples but there was no detectable trace of bismuth oxide (Bi2O3) in WPCs. The range of crystal sizes observed in WMTA was found to be distinctly smaller than those observed in the WPCs. It was concluded that there is no significant difference between the dominant compounds in both WMTA and WPCs except the presence of bismuth oxide in WMTA.

Aluminum Compounds↗
PubMed2004

Long-term effects of Ag-containing alloys on mucous tissue present in biopsy samples.

The aim of this study was to investigate the long-term effects of alloys containing silver (mainly Ag-Sn alloy) on oral mucous tissue. We observed biopsy tissue specimens from patients diagnosed as having amalgam tattoo and/or metal pigmentation by light and electron microscopy and electron-probe microanalysis (EPMA). In most cases, Ag-Sn alloy was present in the tissue but it could not be confirmed if the alloy originated from amalgam. Distributions of both Ag-S and Ag-Sn have typical patterns. Most Ag forms Ag2S and is stably deposited in three patterns along the collagen, basement membrane, and fibrous cells without inducing any host reaction. On the other hand, Sn forms large granules that contain Ag, S, C, N, P, and Ca, and is in soft state in the tissue. Tissue reactions to the alloy become weaker as time passes.

Argyria↗
PubMed1989

A histological study of calcium pyrophosphate dihydrate crystal-deposition disease.

Synovial, meniscal, articular cartilage, and other connective tissue from fifty-seven patients who had calcium pyrophosphate dihydrate crystal-deposition disease was examined by light microscopy, electron microscopy, and electron-probe microanalysis. Safranin O-positive hypertrophic chondrocytes that contained proteoglycans were observed in the tissues of each patient. Microcrystals that were suggestive of early precipitation of crystals were found in the degenerating matrix surrounding hypertrophic chondrocytes. The matrix contained electron-dense amorphous material, including proteoglycans and debris of cellular components. The microcrystals were often seen in contact with degenerating collagen fibers. There was never any histological evidence of formation of crystal in the areas that had no hypertrophic chondrocytes. Chondrocytes of this kind, surrounded by characteristic degenerating matrix, were never observed in the articular tissue from sixty-one patients who had only osteoarthritis. On the basis of our results, we speculate that electron-dense amorphous material containing proteoglycans and debris of cellular components, and the degenerating collagen fibers that were seen around the hypertrophic chondrocytes, may play important roles in the formation of calcium pyrophosphate dihydrate crystals.

Adult↗
PubMed1987

Hydroxyapatite deposition in osteoarthritic articular cartilage of the proximal femoral head.

Hydroxyapatite crystal deposition in the articular cartilage of the proximal femoral head was examined by using light and electron microscopy and electron probe microanalysis. The hydroxyapatite deposition was significantly more frequent in patients with osteoarthritis (40.7%, 22 of 54) than in control subjects (6.0%, 3 of 50) (P less than 0.001). Initial deposition of needle-like crystals was always seen on or within the electron-dense, amorphous material around the degenerating hypertrophic chondrocytes. Matrix vesicles were rarely seen in the calcifying areas.

Cartilage, Articular↗
PubMed1991

Secretory granule calcium loss after isolation of rat alveolar type II cells.

Morphological change and lamellar body loss suggests that alveolar type II cells rapidly de- or redifferentiate after several days of primary culture. To determine whether type II cells or lamellar body compositional changes precede these obvious morphological changes, we examined the in situ elemental composition of lamellar bodies and type II cells from intact lung and at different times after isolation using electron probe microanalysis (EPMA). Isolated cells were prepared by standard methods and plated on either tissue culture plastic or kept in suspension with stirrer flasks. Cell pellets obtained at 0, 3, 24, and 48 h after isolation were rapidly frozen, and thin freeze-dried cryosections were prepared and examined cold in a transmission electron microscope equipped for EPMA. Eight to ten type II cells from each of three to four different preparations for each time period were analyzed. A rapid, progressive, and sustained fall in lamellar body calcium and sulfur content occurred by 48 h of primary culture, suggesting rapid alteration in calcium and protein metabolism by type II cells and/or lamellar bodies after isolation. Also, marked changes in type II cell cytoplasmic Na and K occurred in freshly isolated cells, with incomplete normalization by 48 h. Culture on laminin-enriched Matrigel for 1 wk increased both lamellar body calcium or sulfur content, but 100 nM dexamethasone had no effect. Lamellar body calcium accumulation appears to be a very sensitive index of differentiated type II cell function.

Animals↗
PubMed2003

Calcification of degenerating tissues in the periodontal ligament during tooth movement.

OBJECTIVE: Calcification of degenerating tissues in the periodontal ligament (PDL) during tooth movement was investigated longitudinally. MATERIALS AND METHODS: Upper first molars of male Wistar rats were moved lingually for 1, 7 and 21 d, following which unfixed undecalcified sections of the lingual PDL (in the pressure zone) were examined histologically, histochemically (autoradiography and electron probe microanalysis). RESULTS: On d 1 of tooth movement, degenerating tissues, together with some calcified particles, were visible in the pressure zone of the lingual PDL. On d 7, substantial calcified aggregations were seen in the degenerating tissues, predominantly situated between the bone and root. This was confirmed by the 45Ca autoradiography. On d 21 of tooth movement, large calcified aggregations were still clearly evident between the bone and root. CONCLUSIONS: This calcification of the degenerating tissues is a self-defense response of the living body to prevent direct contact between alveolar bone and the tooth root during compression of the PDL, so preventing friction between them and the development of ankylosis.

Alveolar Process↗
PubMed1980

Evaluation of a preparative method for x-ray microanalysis of soft tissues.

We have adapted the cryopreparative methods designed for the radioautography of diffusible substances (Stumpf and Roth, 1966, 1967) to produce freeze-fried sections of soft tissues for electron probe microanalysis. This report concerns the evaluation of these methods for use with X-ray microanalysis with particular reference to: (1) the preservation of cellular morphology, (2) the introduction of structural artefact by ice crystal formation, (3) the preservation of natural elemental distributions under experimental conditions where known quantities of diffusible elements are present, (4) the effects of ice crystal formation upon possible artefactual elemental redistributions, (5) the effect of section thickness on elemental quantitation, and (6) the effect of tissue excision on element translocation. Freeze-dried sections were prepared from mouse pancreas and from 20% (w/v) solutions of bovine serum albumin (BSA) and gelatin containing known amounts of inorganic salts and were analysed in a scanning electron microscope fitted with energy dispersive X-ray detecting equipment. With the preparative methods used, the morphology of pancreatic acinar cells was well preserved. Acinar cell boundaries, nuclear boundaries, chromatin, nucleoli, ergastoplasm and zymogen granules were readily discernible. Ice crystals were present within sections of BSA, but 70% had a cut surface area < 1 micrometer2 and over 90% had a cut surface area of < 2 micrometer2. Characteristic peak-to-continuum ratios of elements in the BSA sections remained constant over an order of magnitude change in magnification. Elemental redistributions were not detected until the magnification was such that the analysed area fell totally within the confines of a single ice crystal. No differences in elemental peak-to-continuum values were obtained between 2 and 4 micrometer sections of the gelatin-salt solution. Tissue excision did not cause element translocations when compared to tissues frozen in situ. We conclude that this method is valid for preparing tissues for microanalysis under our conditions (analysis of nuclear, cytoplasmic, and secretory compartments) and is limiting only when analyses are conducted at very high magnifications.

Animals↗
PubMed1988

Sn in the Ag-Hg phase of dental amalgam.

In a recent study, Sarkar and Eyer (1986) studied an amalgam which was formed using a unique procedure. Based on their results, they concluded that the solubility of Sn in the Ag-Hg (gamma 1) phase of dental amalgam was virtually nil (less than 0.25 wt%). This finding is contrary to the published results of other investigations. The purpose of the present study was to clarify these disparate findings. A low-copper dental amalgam was examined by electron probe microanalysis. This amalgam was selected because it exhibits unusually large gamma 1 grains (10 microns). Because the influence of the electron-analyzing beam can be completely contained within these large grains, grain boundaries or peripheral phases would not be excited, and a valid analysis of the gamma 1 grains themselves could be made. The analyses showed significant Sn contents of 2.0-3.0 wt%. Analyses of areas which contained grain boundaries showed slight or no differences in Sn content compared with that for the grains alone. Furthermore, secondary electron scans of this dental amalgam failed to reveal the intergranular precipitates of Sn-Hg observed by Sarkar and Eyer (1986) in their amalgam. These different results can be explained by the fact that the unique amalgam investigated by Sarkar and Eyer does not exhibit the same diffusional patterns as does dental amalgam, and that the composition of the gamma 1 in the two amalgams is not the same. The final conclusion is that the Ag-Hg (gamma 1) phase in dental amalgam does indeed contain a significant amount of Sn.

Dental Amalgam↗
PubMed1994

Synthesis of bioactive coatings on Ti substrates using glass enamel.

Bioactive coatings on titanium can be prepared by dispersing hydroxyapatite particles into a borosilicate glass enamel doped with TiO2. Adhesion of the coating is obtained when the titanium substrate has been preoxidized before enameling in such a way as to form a continuous TiO2-layer. Adhesion results from the diffusion of this TiO2-layer into the liquid glass during enameling. The evolution of the diffusion zone was followed by scanning electron microscopy and electron probe microanalysis. Adhesion disappears after a critical time of firing which corresponds to the completion of the dissolution of the TiO2-layer by the glass. The borosilicate glass wets the hydroxyapatite particles and adheres strongly to them after cooling.

Bone Substitutes↗
PubMed1986

[Sites of aluminum accumulation in bone marrow: study using electron microscopy, ionic microscopy and X-ray microanalysis].

Two methods of analytical microscopy have been used to study the distribution of aluminum in bone marrow of rats intoxicated by aluminum gluconate. Images of the distribution of aluminum in a field of 250 microns in diameter were obtained by analytical ion microscopy. They show that this element was concentrated in spots, associated with iron or alone, in the cytoplasm of some cells. Electron Probe Microanalysis (EPMA) has shown that aluminum concentration occurred in cells of the reticulo-endothelial system, principally in the reticular cells of erythroblastic islets. In cells of the reticuloendothelial system, aluminum was observed in intracytoplasmic organelles having ultrastructural characteristics of lysosomes or phagolysosomes. In these organelles, aluminum is always associated with phosphorus and sometimes with iron. No cytoplasmic or nuclear aluminum accumulation was detected in any other variety of bone marrow cells. The consequences of the selective accumulation of aluminum in the cytoplasm of reticular cells of erythroblastic islets for the maturation of erythrocytes are discussed.

Aluminum↗
PubMed1999

A bumetanide-sensitive, apically localized Na+2Cl-K+ cotransport in the rat inner medullary collecting duct.

To better characterize loop diuretic-sensitive ion fluxes in the inner medullary collecting duct (IMCD) we examined them in IMCD cells grown as a primary culture on permeable supports. A polarization of the cells with their basolateral side to the support was confirmed morphologically by electron microscopy and functionally by flux studies with ouabain. Within 7 days cells developed a transepithelial resistance of 974+/-52 omega per cm2 and a low transepithelial potential difference (-0.7+/-0.8 mV). Measurements of intracellular ion content by electron probe microanalysis in IMCD depleted of intracellular ions by preincubation in a Na(+)-K(+)-Cl(-)-free medium revealed, compared to the control receiving solvent, significant reductions in intracellular Na+ content (-17.6% within 10 min) and intracellular Cl- content (-43.8% within 30 min) by the addition of bumetanide (10(-4) mol/l) to the apical but not basolateral incubation medium. In 22Na+ and 86Rb+ isotope uptake studies, fluxes from the apical side were significantly inhibited at bumetanide concentrations of 100 micromol/l by 0.27+/-0.10 and 0.21+/-0.04 nmol/cm2 in 10 min, respectively, whereas basolateral fluxes of 86Rb+ but not 22Na+ were significantly reduced by this substance. Removal of Cl- had a similar but not additional effect. mRNA encoding the apical isoform of the Na+2Cl(-)K+ cotransporter could be specifically amplified by reverse transcriptase polymerase chain reaction from the inner medulla and highly purified IMCD cells. Northern blot of mRNA isolated from the inner medulla with a riboprobe of the apical isoform revealed a transcript of approximately 4.9 kb. This probe localized under "low-stringency" conditions to the IMCD in in situ hybridization studies. These results suggest the presence of an apically localized isoform of bumetanide-sensitive Na+2Cl(-)K+ cotransport in at least a subfraction of IMCD cells. This transport may be involved in the ultimate adjustment of urinary electrolyte concentration by this final segment of the tubular system.

Animals↗
PubMed1989

Cellular mechanisms of toxicity and tolerance in the copper-loaded rat. III. Ultrastructural changes and copper localization in the kidney.

The distribution of copper and related changes have been studied in copper-loaded rat kidneys at the ultrastructural level by X-ray electron probe microanalysis, in order to clarify the pathogenesis of copper-induced damage and subsequent recovery in this organ. Male rats fed a high copper diet (1500 ppm) for 16 weeks were killed at intervals; their kidneys were removed and portions of kidney cortex fixed in 4% paraformaldehyde and 2% glutaraldehyde for electron microscopy: other samples were analysed for copper by AA spectrophotometry. Increasing copper accumulation was associated with progressive PCT cell disarray and characterized by irreversible nuclear damage coincident with the intranuclear accumulation of Cu, S, P, and Ca. Copper was also identified within structurally intact lysosomes associated with Zn and Fe (Type I lysosomes) or P and S (Type II lysosomes, putative Cu-MT). Subsequent copper decline and tubular recovery was associated with the facilitated lysosomal sequestration of copper and excretion of copper-containing cell products into the tubule lumina, Cu-MT and alpha-2 urinary protein-copper. The cytotoxicity of copper in the kidney, as well as the liver, is associated primarily with irreversible nuclear damage, whereas lysosomal copper sequestration protects the cell from injury.

Animals↗