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Biomedical subjects

A Boyde

Publications and source records attributed to A Boyde.

At least 163 records · Page 9Linked to original sources
PubMed1984

Microdissection--elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick.

The concentrations of elements in avian growth cartilage were studied by electron probe x-ray emission microanalysis (EDX). The cartilage was prepared for analysis by freezing, freeze-fracturing, freeze-drying, and carbon coating techniques. Cells and matrix fragments were removed from the tissue by microdissection with a tungsten needle in a scanning electron microscope (SEM) equipped with a real-time stereoscopic viewing facility. The samples were analyzed in the same SEM by EDX. Elemental analyses were performed on each fragment at a distance from the tissue sample, and hence background radiation due to the sample was eliminated. An important finding was that the intracellular potassium concentration of chondrocytes in calcified cartilage was similar to the levels in the premineralized zones. This observation supports the view that chondrocytes do not die in the process of, or as a consequence of, mineralization of the surrounding matrix. Calcium peaks were seen in the matrix at all levels and in chondrocytes immediately prior to mineralization. In contrast, phosphorus levels were always high in cells and low or absent from the premineralized matrix. At the mineralization front the appearance of a phosphorus peak in the matrix just preceded the deposition of mineral. We propose that the transfer of phosphorus from cell to matrix is a rate-limiting step in mineralization. Finally, when rachitic and normal cartilage were compared, little difference was seen in the profile of either intracellular or extracellular elements. However, in rickets the mineralized matrix remained soft in consistency. We suggest that this may reflect a phosphorus-related calcification defect that prevents growth and interlocking of the apatite crystallites.

Animals↗
PubMed1984

Incident light microscopy of surfaces of plastic embedded hard tissues.

To eliminate sectioning artefacts, we devised a method for the preparation of surfaces of plastic embedded hard tissues and their examination by incident light fluorescence microscopy. Flat surfaces produced by cutting on a microtome or using a low-speed saw were stained with dyes in aqueous solutions. Best results were obtained in tissues embedded in glycol-methacrylate. This polymer allowed good penetration of the dyes and its optical properties render cutting defects invisible with the present method. The relationship between hard and soft tissues was well preserved and the integrity of the mineralized component maintained. Cellular details and the distinction between osteoid and bone could be clearly/demonstrated. The procedure is simple, rapid and produces good results even in instances when histological sectioning is unsatisfactory.

Animals↗
PubMed1983

A new method of scanning electron microscopy for imaging biological tissues.

The scanning electron microscope (SEM) has proved of little value in the examination of material prepared for light microscopic histology. One of the chief reasons for this is that the secondary electron signal used for image formation in routine scanning microscopy derives from the surface of the specimen. In the case of histological material this surface is one which has been severely distorted by processing and cutting procedures. Light microscopy sections can be usefully studied in te SEM if the signal used to form the image derives from a considerable portion of the thickness of the section. Thus the backscattered electron (BSE) image has been successfully used in studying the distribution of dense material or densely staining components several micrometres deep to the surface of dried sections. Such sections are, however, usually mounted on low density (poorly BSE reflecting) non-transparent substrates such as beryllium or carbon, so that matching light microscopy of the same samples is not possible. We report here a method by which histological sections mounted on glass slides can be imaged in the SEM at a resolution higher than that obtained using conventional light microscopy. The method exploits the facts that the ordinary, cheap light microscope slide is strongly cathodoluminescent, yet the standard histological (7 micrometers) section is of such a mass thickness that it absorbs a significant proportion of electrons which energies (5-20 keV) usually used in biological SEM. Thus the measure of the glass cathodoluminescence signal is the measure of the electron flux passing through the specimen.

Animals↗
PubMed1983

Backscattered electron imaging of dental tissues.

New findings concerning the processes and results of the mineralization of the dental tissues have been made using new technical developments in scanning electron microscopic specimen-preparation and operation of the instrument. Anorganic preparations have been made with oxygen plasma ashing in the dry state, and with deproteinising solutions and very careful washing and drying for wet specimens: these were imaged with backscattered electrons (BSE) to provide charge-free, topographic images. Methacrylate embedded samples were diamond micromilled to provide flat surfaces: in the absence of topography, BSE images could be used to study even small density differences. The technological advances described here have greatly improved the ability to image tenuously linked, microscopic mineral particles in dental tissues. Additionally, we now have a technique providing atomic number contrast of topographically flat, calcified tissues so that minor variations in the level of mineralization can be detected and regions compared. The advances in the interpretation of the structure of the three dental tissues that these methods have yielded are: the variation in mineral density of the enamel prisms coincident with the pattern of cross-striations seen by light microscopy; the identification of a dense centre of mineralization at the initiation point of calcospherites in dentine; and the relative mineralization of the acellular cement compared with dentine.

Dental Cementum↗
PubMed1983

Tetracycline cathodoluminescence in bone, dentine and enamel.

The emission of light under electron bombardment (cathodoluminescence-CL) in an SEM has been demonstrated from tetracycline and tetracycline labelled areas of bone, dentine and secretory and maturation zone enamel. CL images could be compared with backscattered (BSE) and secondary (SE) electron images in (a) fresh, air-dried or freeze-dried samples taken without fixation at a short term after labelling: (b) polished block faces of fixed, plastic embedded tissue and (c) cut section surfaces cut through fixed and unfixed tissue. These results indicate a considerable future potential for the tetracycline-CL-SEM mode in experimental studies on teeth and histopathological and growth studies of bone.

Aging↗
PubMed1983

Cyclical uptake pattern of tetracycline in post-secretory maturation phase enamel demonstrated in rooted teeth.

Uptake of tetracycline by enamel in the short-term was studied at an advanced stage of crown formation and after completion of crown formation in deciduous molars in the cat. Both secretory phase enamel and bands of postsecretory, maturation phase enamel labeled rapidly. The pattern of labeling mimicked that seen in the continuously growing, rootless incisor teeth of the rat, with narrow doublets fusing to form narrow bands with wide unlabeled intervals in the short term. This is a physiological demonstration which indicates that cyclical activity and changes may occur in vivo during the maturation phase of amelogenesis in rooted teeth. It is also noted that dentine did not, and that some circumscribed patches of bone did label in the same animals in the same time interval. Short-term tetracycline labels are lost following conventional histological processing, but are retained after freeze-drying or air-drying.

Ameloblasts↗
PubMed1983

Simple collectors for cathodoluminescence in the SEM made from aluminium foil.

Inexpensive cathodoluminescence collectors for scanning electron microscopes can easily be made from aluminium foil fashioned as tubes which abut against the window of the photomultiplier at one end and shroud the specimen at the other. Their use in the study of fluorescent labelled mineralized tissues is illustrated.

Aluminum↗
PubMed1983

Tandem scanning reflected light microscopy of internal features in whole bone and tooth samples.

An improved version of the tandem-scanning reflected-light microscope (Petran et al., 1968) has been studied with respect to its applicability to the study of mammalian mineralized tissues. It was found to have important advantages compared with other light microscopic methods, since it allowed microscopic structures to be seen at considerable depths within intact specimens. It has a small depth of focus and gives high contrast for features such as osteocyte lacunae and canaliculi in bone, and prism boundaries in dental enamel.

Animals↗
PubMed1983

New methods for cathodoluminescence in the scanning electron microscope.

Experiments using the CL imaging mode to recognise osteoid in the polished, cut surfaces of bone biopsies embedded in PMMA led to the development of a number of new methods for contrast formation in CL images in the SEM. These involve: (1) enhancing or (2) reducing the CL signal by staining the specimen, (3) utilising the cathodoluminescence of glass microscope slides to produce images of histological sections mounted on glass so that features in the section which scatter the electron beam appear dark against a light background, and (4) enhancing the CL signal from PMMA so that features which are less penetrated by the scintillator show up dark against a bright background. Efforts to increase the efficiency of light collection resulted in the development of a new means for manufacturing reflector-cum-light guide CL detectors by wrapping aluminum foil around a wooden former. These detectors enshroud the specimen so that CL light can only escape to the photomultiplier window (or back up the final lens). A variety of such designs have proved more efficient than the conventional plastic light guides used as CL detectors. By enlarging the beam entry aperture, other SE and BSE detectors can be used simultaneously. Examples of the value of the CL mode in mineralised tissue research include the use of enhanced CL plastic embedding media to detect marrow space and of enhanced osteoid CL to detect unmineralised bone matrix; the use of tetracycline as a growth marker in pathological studies of bone and experimental studies with bone, dentine and enamel; the use of bisbenzamid to locate and count nuclei in osteoclasts, the hard tissue resorbtive cells; and the use of superficial stain absorption of auto-CL to locate stained material on tooth surfaces, with the view to monitor the efficiency of periodontal therapy.

Animals↗
PubMed1982

Enamel microstructure determination in hominoid and cercopithecoid primates.

Enamel structure was determined in primate teeth by scanning electron microscopy. It was found that the important organisational features of this tissue can be determined solely from the examination of developing material and that significant differences in internal structure of the mature tissue are reflected in differences in the surface (cell-matrix interface) of the developing tissue. Several very conservative, relatively non-destructive techniques can be used to acquire information from fully formed teeth, whereas the examination of small areas of heavily etched mature tissue samples may be inadequate or provide biased or misleading information. Pattern 3 prism packing occurs predominantly in Hominoidea, Pattern 2 in Cercopithecoidea. Pattern 1 was found in the one callitricid and the one lemur specimen studied.

Animals↗
PubMed1982

In vitro histological and tetracycline staining properties of surface layer rat incisor enamel also reflect the cyclical nature of the maturation process.

Rat incisors cleaned of overlying enamel organ cells stain unevenly with several common histological stains producing two kinds of banding pattern. In one a pattern of widely spaced stained bands run in an oblique to transverse direction from a more apical level on the medial side to a more incisal level on the lateral side of each tooth. The pattern correlated directly with that produced by both in vivo and in vitro short-term tetracycline labelling in the same teeth: it also resembled the patterns previously demonstrated in a) horseradish peroxidase penetration into lateral intercellular spaces between maturation ameloblasts, b) the distribution of enamel labelling in vivo with 45Ca, c) of etching of enamel surfaces demineralized with EDTA with the glutaraldehyde fixed enamel organ in situ and d) in the distribution of smooth-ended and ruffled border types of cell specializations. We conclude that these bands demonstrate further and previously unrecognised aspects of the cyclical phenomena in enamel maturation-mineralization and show cyclical differences in the physico-chemical status of the organic matrix in the maturation process. A second, incremental growth type of banding pattern occurred at 200-225 micron intervals in the lower incisors and had the same distribution at the beginning and end of the maturation zone.

Animals↗
PubMed1981

Morphological correlations with dimensional change during SEM specimen preparation.

We have previously reported details of the dimensional changes taking place during the processing of soft tissue specimens for scanning electron microscopy. Mouse embryo limbs were used for many of these measurements and the present paper deals with the associated morphological findings. Effects of fixation, dehydration and drying are considered. Freeze drying and critical point drying of glutaraldehyde and glutaraldehyde and osmium fixed samples give perfectly acceptable results for scanning electron microscopy. The best volume retention with freeze dried material is matched by the best morphological appearance of the specimen surface, except when ice crystal damage occurs due to a failure to freeze the tissue rapidly enough. For CPD tissues, perforation of the plasmalemma may occur in glutaraldehyde-only fixed tissue, this being prevented by post-osmication if the glutaraldehyde fixation is not unduly prolonged. This perforation may be due to the extraction of some plasmalemma component during dehydration or further solvent substitution on critical point drying. Solvent evaporation drying usually causes recognizable distortion due to shrinkage: this is minimal in the case of solvent evaporation drying in a nearly saturated atmosphere of the same solvent if this is a very volatile solvent. The examples of Freon 113 and diethyl ether are given here. Swelling during early stages of ethanol dehydration can be prevented by using 70% or 100% ethanol as the first step, with marginal reduction in the post CPD volume and no apparent differences in the SEM. The severe swelling causing sample disruption which can occur with GA + OsO4 fixed tissue can also be prevented by treating the sample with divalent cations, such as Ca++ or Cu++, at any stage.

Animals↗
PubMed1981

Display of maturation cycles in rat incisor enamel with tetracycline labelling.

Tetracycline was incorporated within seconds of intracardiac injection to form bright yellow fluorescent bands under UV irradiation in maturation zone enamel. The bands were narrow, widening with time. At several hours after subcutaneous injection, the fluorescent bands were wide and of low intensity. It is concluded that tetracycline enters maturing enamel opposite the narrow bands of non-striated border ameloblasts which are probably a main exit route for organic matrix remnants. Tetracycline distribution patterns at hours after injection reflect the diffusion of this substance within enamel and the pattern of its removal, which also occurs in relation to the non-striated border, smooth-ended maturation ameloblasts.

Aging↗
PubMed1981

Autoradiographic evidence of cyclical entry of calcium into maturing enamel of the rat incisor tooth.

Adult rats were injected intraperitoneally with radioactive calcium and allowed to survive for periods up to 60 min. Animals were then killed and the upper incisors were removed. Soft tissue was removed from the teeth by dissection and with Clorox. Teeth were then coated with photographic emulsion and, after 17 days, the emulsion on the specimens was developed. Black bands of reduced silver indicated areas of radioactivity where calcium had entered the enamel. These bands were separated by narrower bands of non-radioactive enamel. It was judged that calcium did not gain entry into the enamel in these areas of enamel at this particular time. The patterns made by the bands of calcium entry and restriction were similar to patterns which show where striated border and non-striated border maturation ameloblasts cover the maturing enamel. Maturation ameloblasts with a striated border cover a larger area of the maturing enamel than do maturation ameloblasts without a striated border. It is considered that the maturation ameloblasts with a striated border are engaged in calcium entry into the maturing enamel.

Ameloblasts↗
PubMed1981

Freeze-drying shrinkage of glutaraldehyde fixed liver.

Dimensional changes were recorded during the freeze drying (FD) of 1 mm cubes of glutaraldehyde (GA) fixed adult mouse liver. The areas of the front faces of these blocks was measured using a Quantimet 720 image analysing computer system. Interpolating the first straight line portion of the graphs of size versus time backwards to the origin allowed the determination of the original size even if the specimen was covered with some surface-water ice at the beginning of the experiment. GA fixed mouse liver shrinks 7.3% linearly during freeze drying. This gross shrinkage is not increased if the cold stage temperature is raised to approximately 263 K from approximately 223 K, but the rate of drying as monitored by the rate of shrinkage is greatly increased. If morphological specimens are to be prepared for scanning electron microscopy (SEM), freeze drying can be completed rapidly after an initial period at approximately 223 K, since ice recrystallization leading to increased ice crystal artefact will occur in the deep layers which will not be visible in the SEM. Shrinkage of single cells followed during freeze drying in the SEM showed similar gross dimensional changes of about 7.5% linear shrinkage to occur at and below 198 K.

Animals↗
PubMed1981

The use of stereophotogrammetry to measure acetabular and femoral anteversion.

Stereoradiography with a base shift of the source of illumination was used to produce pairs of radiographs to be measured by stereophotogrammetric techniques. The direction of shift was parallel with the longitudinal axis of the body, so that each radiograph in the stereopair could be used for other clinical purposes. A base shift of 10 centimetres with a distance of 100 centimetres between the focus and the film gave acceptable value of stereoscopic parallax. The radiographs were measured using a Hilger and Watts medical stereometer. This method was checked with test specimens, namely an osteotomised pelvis in which one acetabulum could be rotated and an osteotomised femur in which the whole upper portion could be rotated against the shaft. Measurements made on the acetabulum and its radiographs showed a correlation coefficient of 0.9838 over the range 0 to 30 degrees of anteversion, with a mean error +2.54 degrees and a standard deviation of +/- 1.52 degrees (n = 21). For the femoral neck, over the range from 10 degrees of retroversion to 80 degrees of anteversion, the correlation coefficient was 0.9979, the mean error +2.46 and the standard deviation +/- 1.48 degrees (n = 30).

Acetabulum↗