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Biomedical subjects

A Boyde

Publications and source records attributed to A Boyde.

At least 55 records · Page 3Linked to original sources
PubMed1998

Age changes in bone.

Changes in bone structure as a function of age have been studied by simple inspection, x-ray imaging, stereo-photography, deep field optical microscopy, circularly polarised light microscopy, and scanning electron microscopy (SEM), including both topographic and compositional backscattered electron (BSE) imaging modes. The study of bone as a three-dimensional object, rather than in thin sections, enables us to envisage modelling and remodelling processes in context. The study of ultra-flat block surfaces permits the acquisition of data from an effectively very thin layer in the block face, and to examine bone as a spectrum of tissue types varying in the degree of mineralisation. Particular attention has been paid in our earlier studies to the iliac crest, lumbar vertebral bodies, femoral mid-shaft, neck and head and parietal and frontal skull bones. Recently, we have compared findings from these sites with observations on the mandible. We conclude, from our new imaging data, that common generalisations about the changes in bone in ageing and osteoporosis are too simplified, and that the mandible differs sufficiently from post-cranial skeletal sites that it would be unwise to extrapolate from findings in the jaw to the circumstances elsewhere.

Age Factors↗
PubMed1998

Autofluorescence and mineral content of carious dentine: scanning optical and backscattered electron microscopic studies.

The aim of this study was to investigate the extent of natural intrinsic fluorescence in carious human dentine and any correspondence of such autofluorescence (AF) to the mineral distribution within the lesion. Two investigative techniques were used, both employing the same sample set and fields in diamond-polished block surfaces of polymethylmethacrylate-embedded carious teeth. AF at emission wavelengths >515 nm, excited by 488-nm laser light, was assessed using a confocal laser scanning optical microscope (CLSM), the recordings made under standard operating conditions. The relative mineral content was assessed using digital backscattered scanning electron microscopy (20 kV BSE-SEM). The AF intensity correlated with the level of demineralisation as seen by BSE, but the depth of the lesion as seen by CLSM was significantly greater implying that the AF is not directly related to the mineral component.

Bromides↗
PubMed1997

Microstructure of enamel.

Enamel is a composite material consisting of mineral and organic phases. The properties of the mineral phase are modulated dramatically by its division into microscopic crystals, cemented together by the organic matrix protein polymer. A good concept of the 3D orientations of the crystals derives from visualizing their growth perpendicular to the surface in which they develop, which is pitted by the secretory poles of the ameloblasts. The arrangement of the crystals is the cause of the discontinuities, known as the prism boundaries or junctions, in the otherwise continuous structure. These locations acquire a more concentrated organic matrix during maturation, and they are both crack stoppers and crack propagation tracks in the adult tissue. Any tendency of prisms to cleave may be reduced by their varicosities, which reflect daily variations in the rate of production; their cross-sectional shape; the non-parallelism of adjacent groups, which develops through translocation of groups of cells across the surface during development; and the support of any one microscopic tissue element by other tissue, including dentine, placed to resist an applied load. Incremental growth lines are preferential cleavage planes within the enamel. Failure patterns of enamel in normal and abnormal use can be explained by these parameters, with additional consideration of functional variation and fatigue.

Ameloblasts↗
PubMed1997

Cancellous bone structure in the growing and aging lumbar spine in a historic Nubian population.

There is abundant data on cancellous bone in the aging human spine, but little relating to the growing vertebral cancellous bone in childhood an adolescence. The purpose of this study was to map vertebral cancellous bone in a growth and age series of historic skeletal samples and to make comparisons with data published on recent material. Lumbar vertebral bodies were collected from 65 skeletons (0-60 years) from a medieval Nubian population. Ethnohistoric information was collected to interpret conditions that might have influenced bone structure and metabolism. The cancellous bone was studied three dimensionally, using stereophotography and scanning electron microscopy and morphometrically by performing a semiautomatic structural analysis on digitized backscattered electron images of polymethacrylate-embedded material. The cancellous bone structure in the children consisted mainly of a densely packed, uniform network of small rodlike trabeculae. The greatest bone volume fraction with small, more platelike trabeculae was observed during adolescence. In young adults, larger platelike trabeculae were present in the central zone and smaller trabeculae in the superior and inferior zones, as described for modern skeletal material. Structural changes associated with aging were observed much sooner than in modern man. by the estimated age of approximately 50-60 years, the predominant architectural elements were slender rarified rods in both sexes. The ethnohistorical data suggest that this was essentially a black African population of physically active peasants, not likely to suffer Vitamin D insufficiency or deficient calcium intake. Thus an earlier onset of the biological age changes in cancellous bone found in modern populations was probably relevant.

Adult↗
PubMed1997

A quantitative backscattered electron imaging study of hypomineralization and hypoplasia in fluorosed dental enamel of deer.

Mineral content and distribution of fluorosed and unfluorosed (control) dental enamel of roe deer and red deer cheek teeth were analyzed using digital backscattered electron (BSE) imaging of PMMA-embedded specimens. Compared to the controls, the fluorosed enamel exhibited various aberrations resulting from a fluoride-induced disturbance of the processes involved in enamel formation. Thus, the presence of surface hypoplasias and an enhancement of the incremental pattern in the fluorosed enamel are evidence of a fluoride impact on the secretory ameloblasts, whereas a (subsurface) hypomineralization of different depth and extent is indicative of a fluoride effect on the maturation stage of amelogenesis. The marked variation in the severity of enamel hypomineralization seen along the coronocervical axis of a specimen pointed to a fluoride impact of varying intensity during this period of tooth development. Our observations further indicated that, in some locations, ameloblasts severely affected by fluoride during enamel matrix formation were able to recover from this insult and to function quite normally during the maturation stage of amelogenesis. A major advantage of the BSE imaging technique used in the present study over other methods is that it allows for a combination of micromorphological information with quantitative data on the mineralization of the analyzed tissue, which proved to be very useful for the characterization of fluoride-induced changes in dental enamel.

Ameloblasts↗
PubMed1997

Purinergic transmitters inhibit bone formation by cultured osteoblasts.

Adenosine triphosphate (ATP) and other purinoceptor agonists cause a transient rise in [Ca2+]i in cultured osteoblast-like cells and have a mitogenic effect, as does parathyroid hormone (PTH), and there is evidence that ATP and PTH can act synergistically on osteoblasts. The likelihood that nucleotides, acting through purinoceptors, are important local factors in bone remodeling is therefore considerable. However, their effect on bone formation is unknown. We recently developed a culture system in which appositional bone formation occurs only in narrow grooves cut in a substratum. We have used this as an assay to measure the effects of ATP (50 and 500 mumol/L), ATP gamma S (20 mumol/L), 2-MeSATP (2 and 20 mumol/L), uridine triphosphate (UTP) (0.2, 2, and 20 mumol/L), adenosine (20 mumol/L), bovine PTH (0.25 and 0.5 IU/mL), rat PTH1-34 (10(-8) and 10(-7) mol/L), and rat PTHrP1-40 (10(-9) and 10(-8) mol/L) on bone formation by rat calvarial osteoblasts. The culture medium was renewed 3 times/week (every 2 or 3 days), and the number of bone loci and length and area of Alizarin red-stained mineralized bone formed in the grooves of each specimen in 16-29 days were measured. Compared with controls, ATP gamma S, 2-MeSATP, and ATP reduced the amount of bone formed in a 2-3 week culture period. Adenosine had no effect, and UTP either had no effect or at 2 mumol/L stimulated bone formation. PTH and PTHrP completely abolished bone formation in 4 week cultures. Our findings are consistent with evidence for more than one P2 purinoceptor subtype in bone, and show for the first time that the effect of ATP on appositional bone formation by osteoblasts in vitro is, like PTH and PTHrP, inhibitory.

Adenosine↗
PubMed1996

Scanning electron microscopy of bone: instrument, specimen, and issues.

There are many ways available now to maximise and analyse the information that can be obtained on the structure and constitution of bone using SEM. This paper considers a range of methods and the problems that arise relating to instrumentation and methodology as they apply to the use of SEM in the study of bone. In addition to the review content, some novel technical approaches to the SEM of bone are considered here for the first time; these include low kV imaging for the detection of new surface bone packets (and residual demineralized matrix after resorption), low kV BSE imaging of uncoated, embedded, and unembedded samples, environmental SEM for the study of wet tissue, low distortion, very low magnification imaging for the study of cancellous bone architecture, the use of multiple detectors for fast electrons in improving the imaging of porous samples, and high resolution, low voltage imaging for the study of collagen degradation during bone resorption.

Adolescent↗
PubMed1996

Characterization of bone mineral crystals in horse radius by small-angle X-ray scattering.

The size and the orientation of the bone salt (mineral) crystals in the cranial and caudal zones in the transverse midshaft section of the equine radius were investigated by small-angle X-ray scattering (SAXS). The results are interpreted as indicating that the crystals had an elongated shape with an average thickness of T = 3.17 +/- 0. 15 nm in the caudal region and T = 3.79 +/- 0.20 nm in the cranial region. Their orientation was predominantly in the longitudinal direction of the bone. There was no preferential orientation within the transverse plane. The distribution of tilt angles with respect to the longitudinal direction was determined directly from the SAXS data: the average angle was about 30 degrees for the cranial region and 45 degrees for the caudal region. Assuming that the needle-like crystals are parallel with the collagen fibrils, the angular distribution of the crystals is in good agreement with previous measurements of collagen orientation using circularly polarized light microscopy.

Animals↗
PubMed1996

Topographically induced bone formation in vitro: implications for bone implants and bone grafts.

We have investigated the influence of substrate topography on the timing and location of bone formation by rat osteoblasts. 250 mu m thick slabs of dental tissues were used intact or had a rectangular grid of grooves (350 mu m wide and of variable depth) cut with a diamond wheel. They were then seeded with rat calvarial osteoblasts and cultured in MEM with 10% FCS at 37 degrees C in 5% CO(2). Ascorbic acid 50 mu g/mL and beta-glycero-phosphate 2 mmol/L were added at confluence. Cultures were observed daily from 2 to 4 weeks, until fixation (and storage) in 70% ethanol. Most were stained with alizarin red S to visualize the newly formed bone. The presence of gap junctions in the bone nodules was determined using connexin-43 immunolabeling and confocal microscopy. Two specimens were embedded in polymethylmethacrylate (PMMA): micromilled blockfaces were coated with carbon and examined by digital backscattered electron (BSE) microscopy. Bone formation began in the second week, preferentially wherever cellular condensation was favored: these locations were (a) within the grooves; (b) at the junction between the slab and the bottom of the culture dish; (c) at the periphery of the dish; and (d) in cracks where dissimilar tissues had separated. In the grooves, a grid of aligned bone developed, the deeper trenches showing bone formation earlier than shallower ones, with bone formation tapering off as a groove became shallower. BSE images showed that the bone formed was well mineralized and contained a high volume proportion of osteocytes. Mean and median values for the mean BSE coefficients were: in vitro bone in grooves 0.138528, 0.141484; in vivo aged bone (2 year old rat mandible) 0.143431, 0.144206; and in vivo young bone (neonate rat cranium) 0.129011, 0.132696. Connexin-43 gap junctions were immunolocalized on osteocytes fully enclosed within bone and on osteoblasts overlying it. We conclude first that local topography is an important factor in the location and timing of bone formation in vitro, and that it is likely to be equally important in vivo in normal bone turnover, fracture repair and the incorporation of bone grafts. Second, the mineral density of the bone formed in vitro is consistent with its being true bone.

Animals↗
PubMed1996

Mice lacking tartrate-resistant acid phosphatase (Acp 5) have disrupted endochondral ossification and mild osteopetrosis.

Mature osteoclasts specifically express the purple, band 5 isozyme (Acp 5) of tartrate-resistant acid phosphatase, a binuclear metalloenzyme that can generate reactive oxygen species. The function of Acp 5 was investigated by targeted disruption of the gene in mice. Animals homozygous for the null Acp 5 allele had progressive foreshortening and deformity of the long bones and axial skeleton but apparently normal tooth eruption and skull plate development, indicating a rôle for Acp 5 in endochondral ossification. Histomorphometry and mineralization density analysis of backscattered electron imaging revealed widened and disorganized epiphyseal growth plates with delayed mineralization of cartilage in 6- to 8-week-old mutant mice. The membrane bones of the skull showed increased density at all ages examined, indicating defective osteoclastic bone turnover. Increased mineralization density was observed in the long bones of older animals which showed modelling deformities at their extremities: heterozygotes and homozygous Acp 5 mutant mice had tissue that was more mineralized and occupied a greater proportion of the bone in all regions. Thus the findings reflect a mild osteopetrosis due to an intrinsic defect of osteoclastic modelling activity that was confirmed in the resorption pit assay in vitro. We conclude that this bifunctional metalloprotein of the osteoclast is required for normal mineralization of cartilage in developing bones; it also maintains integrity and turnover of the adult skeleton by a critical contribution to bone matrix resorption.

Acid Phosphatase↗
PubMed1996

Video rate confocal laser scanning reflection microscopy in the investigation of normal and neoplastic living cell dynamics.

The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the liver or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 microns. A series of mesenchyme derived cell lines--from normal cells to sarcoma cells of different malignancy--was used to compare behaviour of the observed intracellular structures and results of fixation. We contrasted the dynamic behaviour of fine features in the cytoplasm of normal and neoplastic living cells and changes induced by various treatments. The tubulomembraneous 3D structure of cytoplasm in living cells is dynamic with motion observable at the new limits of resolution provided by VRCLSM. All organelles appear integrated into one functional compartment supporting the continuous 3D trafficking of small particles (vesicles). This integrated dynamic spatial network (IDSN) was found to be largest in neoplastic cells.

Animals↗
PubMed1995

Pattern of collagen fiber orientation in the ovine calcaneal shaft and its relation to locomotor-induced strain.

BACKGROUND: Gebhardt (1905. Arch. Entwickl. Org., 20:187-322) originated the hypothesis that the direction of collagen fibers in bone is a structural response to the type of mechanical load to which the bone is subjected. He proposed that collagen fibers aligned parallel to the loading axis are best suited to withstand tensile strain, whereas fibers oriented perpendicular to the loading axis are best able to resist compressive strain. Research comparing load patterns with fiber alignment in bone have tended to support Gebhardt's hypothesis. The aim of the present study is to further test this hypothesis by assessing the correspondence between the distribution of strain and the distribution of collagen fiber orientation in a bone that is subjected to compound loading (i.e., both tension and compression at different phases during the loading cycle). The ovine calcaneum was selected to meet this criterion. METHODS: Calcaneum surface strain distributions were obtained from experimental results reported by Lanyon (1973. J. Biomech. 6:41-49). Histological sections of the calcaneal shaft were prepared and observed using circularly polarized light (CPL) microscopy to determine the distribution of collagen fiber alignment. The observed alignment pattern was then compared with the predicted pattern based on Gebhardt's hypothesis. RESULTS: Contrary to previous studies, our findings show no clear correspondence between the strain type of greatest magnitude and the direction of collagen fibers. Areas of bone characterized by high compression and low tension showed predominantly longitudinal collagen alignment (contra to Gebhardt). CONCLUSIONS: It is argued that even small magnitudes of tension operating on local areas of bone may be sufficient to induce collagen alignment favorable to this type of strain, even when greater magnitudes of compressive strain are acting on the same bone volume.

Animals↗
PubMed1995

The rate of osteoclastic destruction of calcified tissues is inversely proportional to mineral density.

This study examined the relative ease with which three dissimilar mineralized tissues from one individual organ were resorbed by osteoclasts in vitro. Cells released from the long bones of prehatch chicks by agitating fragments of the chopped bones in medium were cultured for 24 hours on slices cut from an Elephas maximus molar so that enamel, dentine, and coronal cementum were present in bands on the surface of the slice. The resultant pits were measured using a video-rate, line-confocal reflection light microscope system. Variations in tissue mineralization were characterized by analysis of digital backscattered electron images. The enamel pits were smaller than the dentine and the cementum pits, but the dentine and cementum pits were not significantly different from each other. The sizes of the pits correlated with the relative mineral densities of the three tissues, showing that the rate of osteoclastic destruction of calcified tissues is inversely proportional to mineral density. This indicates that the initial step in osteoclasis, the removal of the mineral phase, determines the volume removed and is the rate-limiting step.

Animals↗
PubMed1995

Volumes of chick and rat osteoclasts cultured on glass.

We have examined the relationship between the number of nuclei of an osteoclast and its volume. Chick and rat cells were released from long bones by chopping the shafts and flushing the fragments in Eagle's Minimum Essential Medium with added 10% fetal calf serum. The bone cell suspension was seeded onto glass coverslips. In Experiment 1, rat and chick cells were allowed to settle for 15 minutes, more medium was then added, and the cells were cultured in 5% CO2 at 37 degrees C for 4 hours. In Experiment 2, only rat cells were used, and the cells were cultured in the presence or absence of 10(-6) M 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (APD) in the medium for 4 or 6 hours. The coverslips were washed in 37 degrees C phosphate-buffered saline and fixed for 24 hours in 2.5% glutaraldehyde in isotonic cacodylate buffer (initially 37 degrees C). The chick cells were critical point dried (CPD) or freeze dried (FD); all rat cells were FD. After drying, cells were coated with gold by vacuum evaporation. The volumes and areas of osteoclasts were measured using a video-rate, line-confocal reflection laser scanning microscope and the number of nuclei in each cell was counted. The volumes and volumes per nucleus of the FD cells were larger than those of the CPD cells but there was no significant difference in plan-areas. Rat osteoclasts were larger than chick cells in all the measured parameters except the mean number of nuclei/cell. The correlation coefficients for the areas, volumes, and the numbers of nuclei for rat and chick cells were all high (r > 0.725). The volumes and volumes per nucleus, but not the areas or areas per nucleus, of the osteoclasts cultured with APD were significantly smaller than control cells. We conclude that FD causes less shrinkage than CPD; chick osteoclasts are about two-thirds the size of rat osteoclasts; and 10(-6) M APD caused a reduction of rat osteoclast volume and volume per nucleus of 21%.

Animals↗
PubMed1995

Stereoscopic and biplanar microphotography of Golgi-impregnated neurons: a correlative study using conventional and real-time, direct-image confocal microscopies.

A correlative study of neuronal reconstruction methods was made using both conventional (non-confocal) and real-time confocal microscopies. Simple and sophisticated (totally automated) methods are described for making both biplanar microphotographs using conventional transmitted light, and stereoscopic microphotographs using real-time confocal microscopy of Golgi-impregnated neurons. Confocal microscopy discriminates against out-of-focus layers to produce optical sections which can be summed on photographic film to obtain neuronal reconstructions. Biplanar images are obtained by fusion, using a stereo-viewer, of two adjacent optical sections obtained with a conventional transmitted light microscope. Stereoscopic and biplanar microphotographs of 3-day-old chick neurons are presented.

Animals↗