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Biomedical subjects

A Boyde

Publications and source records attributed to A Boyde.

At least 109 records · Page 6Linked to original sources
PubMed1990

Fluorescence in the tandem scanning microscope.

Our studies have shown that the fluorescence mode can be used to good effect in both tandem scanning microscopes (TSM: direct view confocal microscopes) as well as confocal scanning laser microscopes (CSLM). Applications are presented which show that the two great advantages of TSM are real-time viewing and real colour, which allow faster use and interpretation. CSLM are complementary, not competitive, being currently more sophisticated for low-level fluorescence work. This is equally possible with available TSM, but requires further development using CCD cameras and image-processing systems.

Animals↗
PubMed1990

Measurement of osteoclastic resorption pits with a tandem scanning microscope.

The functional capacity of isolated osteoclasts is measured by determining the volume of bone or dentine which they can resorb in vitro when cultured on flat tissue slices. Previous SEM stereophotogrammetric evaluations showed that the measurement of total resorptive area per sample, number of pits per sample, area per pit, etc., may be insufficient and that the volume to area ratio may be of utmost importance. A new procedure for the 3-D description and measurement of resorption pits using confocal microscopy has been established. Osteoclasts were cultured on slices of dentine which were prepared as for SEM. They were imaged with a Tracor Northern TSM equipped with piezo-electric translators for the objective lens focusing motion, interfaced to a TN8500 computer, using a TV camera for image transfer. The procedure consisted of generating a zeta map image of the focus depth at which maximum brightness was found at each pixel. Constructing binary masks corresponding to (i) the edge of the resorption pit to define the area beneath which the volume is to be calculated, and (ii) a uniform width at a small distance outside the edge of the pit, enabled the average height of the original surface to be measured and the slice above the pit to be subtracted. After subtraction of the volume element which lay above the top of the original specimen surface, the zeta map is a measure of both the area and volume of the pit.

Animals↗
PubMed1990

Study of Golgi-impregnated material using the confocal tandem scanning reflected light microscope.

The tandem scanning reflected-light microscope (TSM) is a real-time, direct-view confocal microscope. Only those points in the specimen situated in the focal plane contribute information to the image. A Tracor Northern TMS with piezo-electric control of the objective lens was used to generate 3-D images from Golgi-impregnated hamster cerebral cortex. Stereoscopic pairs of images were recorded as 35-mm colour film transparencies by photographing while automatically through-focusing along inclined axes. Transferring the image via a TV camera to the computer, stereo-pairs were obtained by oblique through-focusing and summing, displaying maximum intensity data in each line of sight. Pseudocolour topographic displays were generated by assigning the pixel value in a zeta map image as the focal depth at which the back-scattered light signal was maximal. The TSM was also modified so that a conventional transmitted-light image with a large depth of field could be obtained simultaneously as the very shallow depth of field confocal back-scattered-light image seen at any focus level. The conventional image is a silhouette of the impregnated neurons: the top surface of the cell is not visible and the relationships of processes that cross over cell bodies cannot be discerned. TSM gives a high-contrast image. The Golgi precipitate over the neuronal surface is resolved as globular or ovoid, coloured particles. The smaller particles also cover the dendritic spines. All the confocal range (extended focus) image display methods satisfactorily demonstrated the 3-D arrangement of cell bodies and processes in the chosen volume.

Animals↗
PubMed1989

The effect of fluoride on the patterns of adherence of osteoclasts cultured on and resorbing dentine: a 3-D assessment of vinculin-labelled cells using confocal optical microscopy.

Osteoclasts were isolated mechanically from chick long bones and cultured on dentine for three days in MEM/10% FCS with or without 1 mg/l NaF, (near to therapeutic ranges for serum fluoride levels) or 15 mg/l NaF (which has been found to reduce the volume:plan-area ratios of resorption pits in vitro). The distribution of close contacts with the substratum was determined by immunolabelling of vinculin and confocal fluorescence microscopy. Four characteristic patterns of contacts were observed: infilled discs, rings, crescents and patches: these reflect the progress of the cell in the process of pit formation. Cell-substratum adherence was more extensive than previously reported with contacts often extending down the sides of pits and sometimes across the floor. This distribution explains the curved shape of unilocular pits and the kidney-shaped extensions of multilocular pits. The peripheral creeping of the contact region was more marked in the osteoclasts of the 15 mg/l NaF cultures, and these cultures showed a decrease in pit depth and inhibition of osteoclastic movement in the presence of fluoride.

Animals↗
PubMed1989

Orientation of collagen in human tibial and fibular shaft and possible correlation with mechanical properties.

The pattern of collagen orientation in two human tibiae and fibulae was studied. Serial 100 microns plane parallel cross-sections at 1 cm intervals at the same level in tibia and fibula were cut using an annular blade saw. Distribution of transversely oriented collagen was mapped using circularly polarized light and an image analyzing computer. In both the tibia and fibula the pattern of collagen orientation is characteristic, which may be related to the distribution of the bending forces normally operative in these bones.

Biomechanical Phenomena↗
PubMed1989

Osteoclast-like cells form in long-term human bone marrow but not in peripheral blood cultures.

Transplantation studies have suggested that peripheral blood mononuclear cells contain precursors for osteoclasts. Thus we tested the capacity of peripheral blood monocytes to form osteoclasts in long-term culture. We have reported previously that mononuclear cells from feline, baboon, and human marrow form osteoclast-like cells in long term cultures. Further, the formation of these cells is increased in response to bone resorption stimulatory agents such as PTH, interleukin 1, and transforming growth factor alpha. We now report that these cells show characteristic cytoplasmic contraction with calcitonin and form resorption lacunae when cultured on sperm whale dentine. Thus, these bone marrow-derived multinucleated cells fulfill the functional criteria for osteoclasts. Although cultured peripheral blood monocytes can be induced to form multinucleated cells with 1,25-dihydroxyvitamin D3, these cells did not show similar responses to the osteotropic factors as multinucleated cells formed in the bone marrow cultures multinucleated cells. These results indicate that osteoclasts or cells closely related to osteoclasts form in long-term human bone marrow cultures. In contrast, few mononuclear cells in the peripheral blood appear capable of forming osteoclasts under the culture conditions used in these experiments.

Bone Marrow↗
PubMed1989

Alkaline phosphatase cytochemistry in confocal scanning light microscopy for imaging the bone marrow stroma.

This paper reports on the use of alkaline phosphatase cytochemistry and combined conventional and confocal reflection and fluorescence scanning light microscopic modes in the study of human marrow stroma. It was found that the end product of the enzyme reaction using Napthol AS phosphate as substrate and Fast Blue BB as coupler reflected the 633 nm (red) light from a Helium-Neon laser. Serial optical sections suitable for 3-D reconstruction and selectively depicting the marrow reticulum cells could be obtained from thick glycol methacrylate sections reacted for Alkaline phosphatase. Furthermore, the yellow background of uncoupled diazonium salt over cytochemically unreactive structures in the same specimens and fields was used for imaging haemopoietic cell mass by operating the microscope at 488 nm (argon ion laser, blue-green). These methods may offer advantages in the investigation of the bone marrow stroma and its interplay with haemopoiesis and osteogenesis in normal and disease conditions.

Alkaline Phosphatase↗
PubMed1988

Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures.

We developed a mouse bone marrow culture system to examine the process of osteoclast-like multinucleated cell formation from its progenitors. When mouse marrow cells were cultured for 8 days with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3, 10(-10) to 10(-7) M] or human PTH (1-34) (25-100 ng/ml), tartrate-resistant acid phosphatase (TRACP)-positive multinucleated cells formed. No TRACP-positive multinucleated cells appeared in the absence of these hormones. 1 alpha,25-(OH)2D3 and PTH also increased the number of the clusters of TRACP-positive mononuclear cells. Time course studies showed that these TRACP-positive mononuclear cell clusters appeared before the formation of TRACP-positive multinucleated cells, suggesting that the TRACP-positive mononuclear cells are precursors of the multinucleated cells. Salmon calcitonin markedly inhibited the formation of TRACP-positive multinucleated cells but not TRACP-positive mononuclear cell clusters induced by 1 alpha,25-(OH)2D3 or PTH. TRACP-positive mononuclear cells and multinucleated cells were rarely stained for nonspecific esterase, but some mononuclear cells were positively stained for both nonspecific esterase and TRACP. More that 90% of the TRACP-positive mononuclear cell clusters and multinucleated cells were found near colonies of alkaline phosphatase-positive mononuclear cells (possibly osteoblasts). When marrow mononuclear cells were cultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 or PTH, numerous resorption lacunae were formed. These results suggest that 1) TRACP-positive multinucleated cells formed in response to osteotropic hormones in mouse marrow cultures satisfy most of the criteria of osteoclasts, and 2) osteoblasts may play an important role in osteoclast formation.

Acid Phosphatase↗
PubMed1988

Basis of the structure and development of mammalian enamel as seen by scanning electron microscopy.

Mature enamel is the most mineralized of mammalian tissues, contains the least water and therefore does not present problems of shrinkage on preparation for SEM. However, the developing enamel is highly hydrated and presents severe problems in preparation. The structure of enamel is determined by the activity of its individual formative cells and their group behaviour. The peculiar, unequal secretion of matrix at the distal pole of the ameloblast leads to the presence of characteristically shaped pits in the surface of the formative tissue. Crystals grow in a special relationship to this surface. Sharp changes in orientation of the surface are reflected in abrupt changes in orientation of neighbouring crystals beneath it, leading to the formation of structural discontinuities at prism boundaries or junctions. Several different patterns of prism cross section have arisen in mammalian enamel. Inequalities in the rate of production of the tissue lead to the formation of features known as varicosities or cross striations. Exaggerations of this presumed daily incremental rhythm lead to the formation of the more major incremental lines which can also be visualized by scanning electron microscopy. Differences in the course of the ameloblasts throughout their life history, in the nature of a translatory motion over the surface which they are secreting, lead to the development of prism decussation, which shows characteristic patterns in different mammalian groups of probable functional significance. One largely ignored area in the study of comparative histology concerns the enamel-dentine junction. Particularly in the marsupial mammals, dentine tubules cross the junction and are continuous with enamel tubules. Methacrylate casting of these features has given new insights into these structures.

Ameloblasts↗
PubMed1988

Enamel structure in primates: a review of scanning electron microscope studies.

Comparative studies of dental enamel microstructure have involved three main areas of enquiry, with structural features having been investigated in relation to developmental mechanisms, function and/or phylogeny. The phylogenetic, or taxonomic aspect has been emphasized in the majority of studies involving the Order Primates, where efforts have focused upon attempts to recognise structural differences among various hierarchical groups. Studies of primate enamel microstructure by SEM are reviewed here, with emphasis on what has been learned concerning the most suitable preparative techniques that can be employed, and with particular emphasis to the relevance of enamel microstructure in taxonomic analyses of living and fossil primates. No one technique of enamel preparation can be held to be the most suitable for all types of material (e.g., fresh developing, wet mature, dry mature, and fossil enamel) but experience to date allows us to make some recommendations. Two aspects of enamel structure have been shown to possess considerable potential in taxonomic analyses: the enamel prism packing patterns, and the enamel formation rates as documented from prism cross-striation repeat intervals. Although the distribution of enamel prism packing patterns among primates suggests considerable homoplasy of this character, this feature does have considerable taxonomic interest at certain hierarchical levels in Primates. The study of rates of enamel secretion coupled with analyses of enamel thickness has considerable potential in resolving taxonomic and phylogenetic questions.

Animals↗
PubMed1987

Changes in the mineral density distribution in human bone with age: image analysis using backscattered electrons in the SEM.

We report a study to test the feasibility of studying mineral density distributions in bone using the backscattered electron signal in scanning electron microscopy. Samples were human sixth ribs ranging in age from 8 weeks to 59 years, embedded in polymethylmethacrylate (PMMA), cut, polished, and carbon coated. The proportions of pixels falling in a uniform set of gray level slices of the BSE signal were determined using a microcomputer-based image analysis system interfaced directly to the SEM. The amount of high-density bone gradually increased with age at the expense of low-density bone, and there was an associated compression of the range of the mineral density distribution. Age-related differences were noted between the density distributions in the outer and inner rib cortices. The distribution in the inner cortex in neonates was influenced by the inclusion of densely mineralized endochondral bone and cartilage trabeculae formed at the growth cartilage zone. In adults it appeared that greater bone turnover occurred in the outer cortex, perhaps reflecting a differential mechanical loading across the rib. The technique enabled rapid, unbiased discrimination between the bone of neonates, children, and adults.

Adolescent↗
PubMed1987

Morphological observations concerning the pattern of mineralization of the normal and the rachitic chick growth cartilage.

The entire calcified layer of the chick growth cartilage is penetrated by canals that contains blood vessel complexes: some of these canals pass through all the layers of the cartilage from the resorptive zone at the metaphysis, through the mineralizing, hypertrophic, proliferative and resting regions. This study aimed to provide more details of the 3-D microanatomy of this region and to establish whether there are differences in the process and progress of mineralization compared with the established mammalian "model". Proximal tibial heads from 6 to 8 weeks old normal and vitamin D deficient chickens were rapidly frozen and prepared for scanning electron microscopy using freeze-fracture, freeze-drying, plasma ashing, and chemical deproteinization techniques. Cartilage samples were also embedded in PMMA and polished for BSE imaging. Other samples were prepared for light microscopy. Zones of (mineralized) cartilage several cells thick separate adjacent canals. At the mineralizing front, calcification of the matrix is most advanced close to the canals, but the matrix adjacent to the canal lumens does not calcify. Mineralisation of the cartilage matrix is incomplete and small fenestrae of unmineralized matrix connect chondrocyte lacunae. These discontinuities in matrix calcification could serve as a route for diffusion of nutrients, metabolites and dissolved gases. The calcified cartilage is more mineralized than the contiguous developing bone. Osteoblasts surrounded by bone were seen to occupy the lacunae of hypertrophic chondrocytes. We tentatively suggest that some osteoblasts represent a terminal stage in the differentiation of hypertrophic chondrocytes. The rachitic cartilage was disorganised. It was penetrated by irrugular vascular canals and exhibited a greatly expanded hypertrophic zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Monoclonal antibody and immunogold cytochemical localization of amelogenins in bovine secretory amelogenesis.

Amelogenin enamel-protein epitopes in developing incisors were ultrastructurally localized with high specificity resolution. They formed clumps scattered over the enamel organic matrix between the hydroxyapatite crystals, and were also present over islands of stippled or granular material at the forming surface of the enamel matrix demonstrating that this material consists in part of amelogenin enamel protein. The amounts of amelogenin, as judged by labelling density, were not greater in the stippled or the surface crystal-containing matrix as compared to the enamel matrix up to 50 micron deep. Amelogenins were also localized in the rough endoplasmic reticulum. Golgi apparatus and secretory granules of the ameloblasts, which suggests they are merocrine secretions.

Amelogenesis↗
PubMed1987

Micro-biomechanics vs macro-biomechanics in cortical bone. A micromechanical investigation of femurs deformed by bending.

Previous studies have shown that longitudinal and transverse lamellae in compact bone signal the presence of tensile and compressive forces, respectively. On this basis an investigation has been carried out to ascertain the distribution of lamellae in femoral shafts that have been deformed by bending. To do this, a series of undecalcified plane-parallel sections 100 microns thick were prepared, and the proportion of transversely oriented collagen and crystallites in the lamellae was measured, using circularly polarized light as an illuminating source and a Quantimet 720 image-analyzing computer. It has been concluded that the distribution of the two types of lamellae is in accordance with the need to compensate for the deformity produced by the bending of the bones, and that an obvious relationship exists between the macroscopic features of the femurs and their modified microscopic structures.

Biomechanical Phenomena↗
PubMed1987

The effects of inhibitors of cysteine-proteinases and collagenase on the resorptive activity of isolated osteoclasts.

The effects of specific inhibitors of cysteine-proteinases ((Z-Phe-Ala-CHN2: benzyloxycarbonyl-phenyl-alanyl alanyl diazomethane and E-64: trans-epoxy-succinyl-L-leucylamido(4-guanidino)-butane) and collagenase and collagenase ((Cl-1: N-(3-N-benzyloxycarbonyl amino-1-R-carboxypropyl)-L-leucyl-O-methyl-L-tyrosine N-methylamide) have been tested on the osteoclastic resorption of dentine. Chick osteoclasts were cultured in the presence or absence of 12.5 microM Z-Phe-Ala-CHN2, 40 or 60 microM E-64, or 40 or 100 microM Cl-1 for 1 or 2 days. In addition, osteoclasts were cultured on oyster shell calcitostracum with or without 12.5 microM Z-Phe-Ala-CHN2. Specimens were studied by light microscopy to count cells and resorption features and by scanning electron microscopy (SEM) stereophotogrammetry for the measurement of the depths, plan-areas and volumes of resorption pits. The numbers, depths and volumes (but not the plan-areas) of the resorption pits in dentine were significantly reduced by Z-Phe-Ala-CHN2 and E-64. Thus, for a given plan-area, the volumes and the depths of resorption pits were smaller in these experimental groups compared with control dentine specimens. The overall inhibition of resorption was at least 75%. Cl-1 did not have this inhibitory effect on the numbers or sizes of resorption pits in dentine. When the oyster calcitostracum was used as a substrate for the osteoclasts, Z-Phe-Ala-CHN2 did not reduce the numbers or volumes of pits, but increased the plan-areas and prevented the formation of deeper pits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Distribution of lamellae in human femoral shafts deformed by bending with inferences on mechanical properties.

On the basis of previous investigations indicating that the distribution of osteonic and interstitial lamellae in a normal femur depends on its mechanical properties, a procedure has been devised to provide information on the distribution of lamellae in human femora diaphyses deformed by bending. To achieve this, exactly plane parallel cross sections, 100 micron thick, were prepared from the portion of maximum bending, using an annular blade microtome. An index of the distribution of longitudinal lamellae (whose fiber bundles and crystallites have a longitudinal course and withstand loading by tension) and transverse lamellae (whose fiber bundles and crystallites have a transverse course and withstand loading by compression) was determined using circularly polarized light as illuminating source and a Quantimet 720 image analyzing computer. The results show that, even in a pathologically deformed bone, both the microscopic structure of sections at the level of osteonic and interstitial lamellae, and their macroscopic shape may be governed by the distribution of the forces active in bone.

Biomechanical Phenomena↗
PubMed1987

On the structural symmetry of human femurs.

The distribution of transverse and longitudinal lamellae from osteons and interstitial bone of the two femurs from a woman aged between 25 and 30 was investigated. Exactly plane parallel cross-sections (100 micron) were prepared from the upper, middle and lower thirds of the two bones. The distributions of transverse and longitudinal lamellae was determined for both osteonic and interstitial bone, using circularly polarised light. The results show that (a) as in the male femur, the transverse and longitudinal lamellae in these female femurs have a characteristic rotational distribution along the shaft consistent with the distribution of the bending strains normally operative in bone; and (b) the rotational distribution of the two types of lamellae is slightly asymmetrical in the two female bones, probably because of imperfectly symmetrical use of the lower limbs.

Adult↗