Crystal orientation in the shell of the domestic fowl: an electron diffraction study

The eggshell of the domestic fowl has been studied by transmission electron microscopy and diffraction. Thin sections of shell were prepared by chemical and ion-beam thinning techniques. Each calcite column of the palisade layer consisted of crystallites of diameter 20 to 30 micrometer with some tendency for crystallite alignment within a single column. Evidence indicates that there was no significant preferred orientation in the palisade layer as a whole. Only in the surface layer was any preferred orientation detected, and here (1014) planes tended to lie parallel to the surface. The results are compared with previously published data, and calcite nucleation and growth are discussed.     

X-ray diffraction and electron microscope study of osteons during calcification

Summary To obtain information on the changes in the inorganic bone fraction during calcification, low- and wide-angle X-ray diffraction techniques and electron microscopy have been applied to single osteon samples. The samples were cylindrically shaped and their axes corresponded to the axes of the Haversian canals. The selection was made according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteon were selected, that is, longitudinally structured osteons, transversely structured osteons, and alternately structured osteons. The results indicate that in osteonic lamellar bone there are two types of inorganic particles: (1) granules arranged in linear or needle-shaped entities with maximum width 40–45 Å, which are regularly distributed at the level of the main band of the collagen fibrils where their maximum length reaches the length of the main band itself; that is, about 400 Å; and (2) very long crystallites, with a diameter of 40–45 Å, which grow with their crystallographicc-axis parallel to the collagen fibrils and cover much more than a major collagen period.     

The effect of grinding of human dentine examined by X-ray diffraction, infrared spectroscopy, and electron microscopy

Analysis of human dentine by infrared spectrophotometry suggests that ball-grinding may result in damage of the apatite crystallites. The present study includes further assessments of this effect by X-ray diffraction, infrared spectroscopy, and electron microscopy. Each of three coarse-ground dentine samples (Group I) was divided into three portions of 30 mg. One of these portions was ball-ground for approximately 1 min (Group II), the second portion for 6 min (Group III), and the third portion for 23 min (Group IV). The 002 reflection showed line broadening, most marked from Group II to III. Electron microscopy showed a gradual change in crystallite appearance with increased grinding, most pronounced from Group II to III. These observations indicate that by prolonged grinding a limit is approached where no further changes in the crystallites occur. Electron microscopy also indicated that fracture of the crystallites might have occurred. This was probably accompanied by strains in the lattice. The infrared spectra indicated that no breakdown of the apatite structure had occurred during the entire grinding.     

Effects of fluoride on human enamel and selachian enameloid in vitro: A high-resolution TEM and electron diffraction study

Summary The effects of fluoride on human enamel and selachian enameloid in vitro were visualized in TEM and analyzed with electron diffraction. It is demonstrated that under precise pH conditions, inducing concentration balance between F⁻ ions and apatite, calcium fluoride is no longer formed, and crystalline changes occur instead. A secondary growing process, inducing a twofold increase in crystal size, involves all crystal faces, altering the hexagonal symmetry. It is suggested that the mechanism involved is not a dissolution/precipitation process but rather a secondary growth of residual crystallites induced by apatite dissolution.     

An electron microscopic study of the changes observed in osteocytes under ischemic conditions

The purpose of this study was to observe the process of ischemia in osteocytes using light and electron microscopy and to compare the changes in these ischemic osteocytes with those in other types of osteocytes (i.e., degenerative osteocytes in physiological states, steroid-induced lipid-accumulating osteocytes) that have been previously reported. Five female Japanese white rabbits were used in this study. Osteochondral chips were taken from one side of the femoral condyle, covered with Millipore filters, and then inserted into the other side of the knee joint. These tissues were examined after 12 h and after 2, 5, 8, and 14 days of ischemia under both light and electron microscopy. Under light microscopy, osteocytes and lacunae were classified into four types: normal osteocyte, pyknotic osteocyte, pale osteocyte, and empty osteocyte lacuna. The number of each type of osteocyte (or lacuna) in a settled area was counted. The ratio of normal osteocytes decreased significantly (p less than 0.001) after the second day of ischemia. Pyknotic osteocytes increased at 12 h (p less than 0.01) and 2 days (p less than 0.001) of ischemia. On the fifth day of ischemia, the percentage of pale osteocytes reached a peak. This was followed by a gradual increase in the number of empty lacunae. On the fourteenth day of ischemia, empty lacunae constituted greater than 40% of the cell types. When viewed by electron microscopy, these necrotic osteocytes were similar to the degenerative osteocytes that have been observed in physiological states and apparently different from lipid-accumulating osteocytes. The results suggested that there could be at least two types of necrotic processes in osteocytes that eventually lead to cell death.     

An electron microscope study of the formation of the nacreous layer in the shell of certain bivalve molluscs

An electron-microscopic study was made of nacreous shell growth in several species of marine molluscs. Studies of sections of mantle-shell preparations show that the first step in crystal formation is the polymerization of part of the pallial fluid to form lamellae parallel to the surface of the epithelium. These lamellae form compartments enclosing a modified apallial fluid. Initiation of crystals occurs in these compartments in contact with a crystal in an adjacent layer. During crystal growth the organic matrix present in the compartment is displaced by the growing surface of the crystal. When growth is complete the crystal is entirely enveloped by a delicate organic sheath. These studies show that the pallial fluid with its organic constituents is responsible for supplying a matrix or substrate for crystal initiation and growth. It serves as a regulatory device for guiding the orderly growth and arrangement of crystals and, further, it may participate in the induction of new crystals. The formation of compartments during shell growth accounts for the uniform thickness, preferred exhibited orientation and mineralogy of the crystals as well as other features exhibited by the mature nacre.This investigation was aided (in part) by Grant DE-01825, N.I.D.R., U.S.P.H Service.     

A study of the ultrastructure of urinary calculi by scanning electron microscopy

Summary A study of urinary stones obtained from patients after surgery in the Medical College Hospital, Trivandrum, under the scanning electron microscope showed the presence of calcium oxalate and calcium biphosphate crystals as the main constituents. However, the pattern of the different phases of crystal growth was not uniform. Within the crystal lattice, fibrous structures, possibly of protein matrix, were invariably observed. Electron microscopy may be usefully adapted as a particularly suitable method for ultramicroscopic investigation of the fine structure of urinary stones including single crystal surface structure, section of urinary calculi and for possible presence of hitherto unknown components within the calculus.     

Scanning electron microscopy and X-ray diffraction studies of human bone oxalosis

Summary Postmortem scanning electron microscopy of human phalanges in a chronic uremic hemodialysis patient with hyperparathyroidism showed the presence of confluent abnormal rounded formations with a radial rosette-like crystalline pattern in the diaphysis as well as in the epiphyseal part of the bones. These fan-shaped configurations were found either as individual formations within bone trabeculae or as numerous aggregated crystalline deposits replacing large parts of the bone structure. The microdissected content of such large areas submitted to X-ray diffraction analysis revealed the predominant presence of calcium oxalate monohydrate or whewellite with some traces of hydroxyapatite. Oxalate titration analysis indicated the presence of 25% of oxalate, corresponding to 45% in weight of whewellite.     

Articular cartilage and intervertebral disc proteoglycans differ in structure: an electron microscopic study

Articular cartilage and the intervertebral disc tissues have different material and biological properties and different patterns of aging and degeneration. To determine if the proteoglycans of these tissues differ in structure, we used the electron microscopic monolayer technique to compare baboon articular cartilage proteoglycans with baboon annulus fibrosus, transition zone, and nucleus pulposus proteoglycans. Intervertebral disc and articular cartilage proteoglycans differed significantly. Articular cartilage contained large proteoglycan aggregates formed from hyaluronic acid central filaments, multiple monomers, and large nonaggregated monomers. These molecules were identical to those of nasal cartilage, growth plate cartilage, chondrosarcomas, or menisci. In contrast, the intervertebral disc tissues contained only nonaggregated proteoglycan monomers and clusters of monomers without apparent central filaments. Intervertebral disc nonaggregated monomers were shorter and more variable in length than those from articular cartilage, and nucleus pulposus nonaggregated monomers were even shorter and more variable in length than transition zone and annulus fibrosus monomers. These observations suggest that significant differences in proteoglycan metabolism exist between articular cartilage and intervertebral disc.     

Erythropoietin in haemangioblastoma: Immunohistochemical and electron microscopy studies

Summary Immunohistochemical studies for erythropoietin were carried out in six capillary haemangioblastomas, three of which were also studied by electron microscopy. The immunohistochemical studies showed that positively stained cells were scattered in the vicinity of capillaries, and that neither endothelial cells nor stromal cells were stained. In their morphology and distribution, the positively stained cells were identical to mast cells as observed by electron microscopy. In one case, erythropoietin was demonstrated in the cyst fluid of the tumour. These findings suggest that mast cells with abundant secreting granules in haemangioblastomas are capable of producing erythropoietin.     

A back-scattered electron microscopy (BSEM) study of the tight apposition between bone and hydroxyapatite coating

We performed a back-scattered electron microscopy analysis of the interface between newly formed bone and hydroxyapatite coating, in an experimental rabbit model. Twenty cylinders made of Ti6A14V and coated with hydroxyapatite at different crystallinity were implanted in the distal femural canal and retrieved at 4, 8, 26 an 34 weeks. Crystallinity of the coating varied from 90% to 60% and thickness varied between 50 and 100 μm. Osteocytes were detectable a few micrometers in proximity of the coating. They produced new bone which was so tightly apposed to the coating that high magnification BSEM did not resolve any discontinuity at the interface. This was not observed in uncoated implants. Degradation of the hydroxyapatite coating is not a simple hydrolytic process because newly formed bone is remodelled in areas were a tight apposition with hydroxyapatite is present. The coatint itself is likely to be attacked by the resorptive action of multinucleated giant cells and osteoclasts. In conclusion, response to coated samples is morphologically characterized by tight apposition with bone. The substitution of areas of the coating by newly formed bone is possible. Received: 28 April 2000/Accepted: 2 June 2000     

Age-related changes in articular cartilage proteoglycans: electron microscopic studies

Biochemical and biophysical studies have demonstrated that proteoglycan monomers from immature and adult articular cartilage differ in composition and size. To investigate the structural basis of age-related differences in articular cartilage proteoglycan monomers and aggregates, we isolated and purified high buoyant density proteoglycans from the articular cartilages of 2- to 3-month-old calves and 18-month-old steers. The molecular architecture and dimensions of the proteoglycans were examined using the electron microscope monolayer method. Aggregated and nonaggregated monomers from calf cartilage were longer and less variable in length than the corresponding monomers from steer articular cartilage. Calf monomer lengths had unimodal frequency distributions whereas nonaggregated steer monomer lengths had a bimodal distribution. These observations were confirmed by acrylamide-agarose electrophoresis, which demonstrated that the samples contained only one species of proteoglycan monomer in calf but two species in steer. In addition, calf aggregated monomers had longer thin segments indicating that calf and steer monomers differed in structure as well as in size. Steer proteoglycan aggregates were shorter and had fewer monomers than those from calf. These observations demonstrate the existence of significant age-related structural differences in articular cartilage proteoglycans and form the basis for future study of the mechanisms responsible for these differences.     

Demineralization of bone matrix: Observations from electron microscope and electron-probe analysis

Displacement or removal of mineral during the processing of calcified tissues for electron microscopy is a recognized phenomenon. An electron microscope analysis has been made of artefactual mineral loss during ultramicrotomy of osteogenic tissue. It is concluded from morphological investigation and the use of electron diffraction that this loss of crystalline mineral during sectioning can considerably change the morphology of calcified tissues and may lead to inaccurate interpretation of cell and matrix morphology. Electron probe X-ray microanalysis has been used to demonstrate in a semi-quantitative manner, the artefactual loss of calcium and phosphorus. Problems of specimen preparation for such analytical work are discussed.     

A scanning electron microscopic investigation of in vitro osteogenesis

Summary Chick limb mesenchymal cells differentiate into muscle, cartilage, fibrous, and bone tissue. Previous reports show that when stage 24 limb mesenchymal cells are cultured in vitro, chondrocytes, myocytes, fibrocytes, and osteoblasts can be identified on the basis of morphological and biochemical parameters. The study reported here demonstrates that phenotypic expression in culture seems to be dependent on the initial plating density, Scanning electron microscopic observations indicate that when stage 24 limb mesenchymal cells are initially seeded at high densities (5 × 10⁶ cells per 35 mm culture dish), mounds of cells appear in culture. These mounds represent cartilage nodules composed of a fine fibrous matrix and chondrocytes, surrounded by a loose fibrous connective tissue matrix. Cultures initially plated at intermediate densities (2.0–2.5 × 10⁶ cells/35 mm culture dish) produce a flattened layer of fibrocytes overlying a matrix of collagen fibers and calcium phosphate deposits as determined by electron-microprobe analysis; these observations are indicative of osteoblast expression. Cells seeded at this intermediate density appear larger and possess greater surface area than cells seeded at high density. It is suggested that conditions that permit such increased cell surface area coupled with a relative compaction due to cell crowding may provide conditions permissive for osteogenesis. Based on morphological criteria, it appears that chick limb mesenchymal cell osteogenesis in vitro is not associated with chondrogenesis but represents a separate route of phenotypic expression.     

家兔骨折愈合过程中软骨细胞凋亡的透射电镜观察

目的 观察骨折愈合中软骨细胞在软骨化骨时的死亡方式及软骨细胞的其它可能结局。方法 采用家兔左桡骨标准骨折,在软骨骨痂产生的不同时期、不同部位取材,常规制作透射电镜观察标本。结果 ①软骨细胞生长、消亡过程可分为3个阶段:成软骨细胞、肥大软骨细胞、凋亡软骨细胞。软骨细胞凋亡表现为核固缩和凋亡小体形成,但细胞器不被破坏。最后,软骨细胞崩解,凋亡小体散落于软骨基质中。②软骨化骨过程中只是部分软骨细胞凋亡;另一部分软骨细胞并不出现凋亡,直接转变为骨细胞。结论 ①骨折愈合时发生的软骨化骨,是机体通过激活软骨细胞死亡程序,以细胞凋亡达到新老细胞交替,实现化骨。②由肥大软骨细胞组成的软骨骨痂,能直接转变为编织骨并骨化,支持软骨细胞能直接转变为骨细胞的理论。     

Analysis of microvessels in pancreatic cancer: by light microscopy, confocal laser scan microscopy, and electron microscopy

BACKGROUND/PURPOSE: The microvessel density (MVD) of most malignant tumors is considered to be strongly related to metastasis and prognosis. Weidner's "hot spot method" for determining MVD is in general use, but it is possible that cells other than endothelial cells will also be stained. In our previous study, no correlations were observed between MVD determined by the "hot spot method" and prognosis/metastasis. But, using the "lumen method," we found a correlation with the number of vessel structures only. In the present study, we analyzed the staining of microvessels in pancreatic cancer, using light microscopy, confocal laser scan microscopy (CLSM), and transmission electron microscopy (TEM). METHODS: Microvessel staining of pancreatic cancer with CD34, factor VIII, and CD45 antibodies was examined in consecutive slices by light microscopy. For CLSM, freshly resected specimens were immunostained with factor VIII and fluorescein isothiocynate. For TEM, specimens were fixed with 2.5% glutaraldehyde, treated with 1% osmium tetroxide, and embedded in epoxy resin. RESULTS: Staining of vessels with CD34 and factor VIII antibodies appeared similar under light microscopy. However, CD34-stained consecutive slices were judged not to reveal vessel structures, and some cells stained with CD45 antibody were similar in appearance to CD34-stained cells. Under CLSM, irregular arrangements of neovascularization, consisting of many branches, were observed, but many positively stained cells not identified as vessels were also seen. Microvessels were distinctly identified under TEM, but the types of individual cells could not be determined. CONCLUSIONS: An integrated, reproducible method for the measurement of MVD is vital. For pancreatic cancer, the "lumen method" is recommended.     

X-ray diffraction study of the mineralization of turkey leg tendon

X-ray diffraction studies were conducted on calcified turkey leg tendon to establish the effect of mineralization on some of the structural properties of collagen. The principal finding was that the first equatorial reflection of collagen in freshly-excised calcified tendon had a d-spacing intermediate between the values for dried collagen and fresh unmineralized collagen. Since this spacing is a sensitive monitor of moisture levels in collagen, the data suggest that mineralization reduces the amount of water than can associatein vivo with the collagen component in tissue. It was also found that the presence of mineral appears to increase the resistance of collagen to permanent thermal denaturation.

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Zusammenfassung Es wurden Röntendiffraktionsuntersuchungen an calcifizierten Truthahnbeinsehnen durch geführt, um die Wirkung der Mineralisation auf einige der strukturellen Eigenschaften von Collagen festzustellen. Die wichtigste Beobachtung war, daß die erste äquatoriale Reflexion von Collagen in frish herauspräparierten calcifizierten Sehnen einen d-Wert aufwies, der zwischen den Werten für trockenes Collagen und frisches, nicht mineralisiertes Collagen liegt. Da dieser d-Wert empfindlich auf den Feuchtigkeitsgrad des Collagens reagiert, lassen diese Resultate vermuten, daß die Mineralisation die Wassermenge reduziert, welche sich in vivo mit der Collagenkomponente im Gewebe verbinden kann. Es wurde auch beobachtet, daß die Anwesenheit von Mineral den Widerstand von Collagen gegen bleibende thermale Denaturierung heraufzusetzen scheint.

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Résumé Des études de diffraction aux rayons X sont réalisées au niveau du tendon calcifié de la patte de dinde pour déterminer l'effet de la minéralisation sur certaines propriétés structurales du collagène. Le résultat le plus important indique que la première raie équatoriale du collagène de tendon calcifié, fraichement prélevé, présente une équidistance intermédiaire entre les valeurs de collagène déssèché et du collagène frais non calcifié. Comme cette équidistance parait traduire fidèlement le degré d'humidité du collagène, il semble que la minéralisation réduit la quantité d'eau associéein vivo avec le composant collagènique du tissu. La présence du minéral semble augmenter la résistance du collagène à une dénaturation thermique permanente.

     

Vectorial sequence of mineralization in the turkey leg tendon determined by electron microscopic imaging

Summary Turkey leg tendons were used as a model tissue to study the spatial and temporal relationships of mineral deposition between matrix vesicles and collagen fibrils by various electron microscopic techniques—bright field, selected-area dark field (SADF), and electron spectroscopic imaging (ESI). These latter imaging techniques enabled the direct localization and spatial distributions of both apatite crystals and atomic elements (Ca, P) within matrix vesicles and collagen. In longitudinal planes of section, a consistent vectorial gradient of mineralization was observed which started with the first localization of apatite mineral in matrix vesicles; with further development, the mineral spread from the vesicle to the extravesicular interstices and then into the adjacent collagen fibrils. Once intrafibrillar, the mineral was observed to advance both laterally and axially. The association of vesicle/collagen mineral was examined by ESI analysis of Ca and P elemental maps and appeared as a continuum between the vesicles and the adjacent collagen fibrils. Similarly, an intimate spatial relationship was observed between the mineral of vesicles and collagen in transversely cut sections of tendon. The sequential development of this mineralized matrix is discussed in light of matrix vesicle/collagen interactions.     

Orientation of apatite in single osteon samples as studied by pole figures

Summary The X-ray diffraction method based on pole figures has been applied to single osteon samples in order to obtain information about the texture of the inorganic bone fraction and the way it changes during calcification. The osteon samples were cylindrically shaped, with axes corresponding to those of the haversian canals. Selection was carried out according to the degree of calcification and the orientation of collagen bundles and inorganic particles. Osteons at both the initial and final stages of calcification were chosen. Arrangements of fiber bundles and inorganic particles in successive lamellae characteristic of three types of osteons were selected: longitudinal, alternate, and transversal. The results indicate that in all three types of osteons, the long axis of the sample is apparently the only direction of orientation because the transversally oriented crystallites give an isotropic diffuse scattering as would be expected if all the inorganic particles were irregularly oriented around the osteon axis. The number of longitudinally oriented crystallites increases progressively from transversally oriented osteons to alternately and longitudinally oriented ones. The crystallite orientation in an axial direction increases in fully calcified osteons. This last result is in agreement with the electron microscopic finding that the long needle-shaped crystallites covering much more than a major collagen period and measuring 40–45 ? in width increase in number as calcification proceeds.     

Does penile tourniquet application alter bacterial adhesion to rat urethral cells: an in vitro study

Purpose

To investigate the effects of penile tourniquet (PT) application on bacterial adhesion to urothelium.