Early Scanning Electron Microscopic Studies of Hard Tissue Resorption: Their Relation to Current Concepts Reviewed

This paper highlights some observations made by the authors in SEM studies of hard tissue resorption and considers their significance in relation to current concepts. All mammalian mineralised tissues may undergo physiological resorption, the resulting surface reflecting the density of mineralisation and the organic matrix chemistry, organisation and orientation. Resorption-repair coupling may follow the resorption of any tissue, but SEM studies first noted this process in the case of the dental tissues. The difference between fetal and adult bone formation and resorption provided evidence against the concept of osteocytic osteolysis. SEM stereophotogrammetric methods for the quantitation of individual resorption lacunae are now much quicker and have been extended to the study of in vitro resorption by mammalian and avian osteoclasts isolated from bone and seeded into new substrates. Experimental studies using SEM were first conducted on the osteotropic hormonal effects on bones forming in vivo and extended to the in vitro situation. The effects observed underlined the several actions of PTH on osteoblasts and indicated their important role in the control of bone resorption. Immunological marking techniques monitored by SEM first established that osteoclasts had no Fc or C3 receptors, although other cells in the vicinity did. The study of osteoclasts resorbing substrates other than bone in vitro has increased our understanding of the essential components of a resorbable substrate. Experiments growing separated bone cells and marrow cells on calcified substrates have shown that such cells will continue to resorb for at least six weeks

The Interface of Cells and Their Matrices in Mineralized Tissues: A Review

The interface between cells and matrices in mineralized tissues formed in vivo has been studied mainly by looking at the matrix surface, which is easily prepared, and not at the cell surface, which presents problems. Vertebrate calcified tissues range from being acellular to highly cellular, but for all the tissues the formative cells lay down and organise a cell-specific matrix, although this may be deposited initially on a different tissue-type. The formation of hard tissues is a group activity of many cells; resorption is the province of one cell, though it may be controlled by others in the vicinity. Cell-matrix interfaces that develop in vitro have also mainly been studied at the matrix side. The main difficulty with in vitro studies of hard tissue interfaces is that the eel ls do not have the same activity or even cellular functions as they had in vivo under the complex control of physiological regulation. The question of osteoblastic osteoclasis falls into this category. It is possible to provide new substrata for both formative and resorptive hard tissue cells to test for the interaction between the cells and the \u27matrix\u27 on to which they are seeded. The changing cell-matrix interface may also be modelled using computer simulation of osteoclastic movement across a substrate based on known patterns exhibited by other cell types in vitro. Comparison with the shapes of complex resorption pits shows a surprising match, This suggests that the track of the osteoclast due to cell motility and the bone resorptive mechanism resulting in pits along that track are likely to be separately controlled phenomena

Experiment K-6-01. Distribution and biochemistry of mineral and matrix in the femurs of rats

Previous analyses of the composition of mineral and matrix in the bone of young rats following space flight has revealed deficits in calcium, phosphorus, and osteocalcin, a non-collagenous protein, without an associated decrease in collagen. To characterize the location and nature of this mineralization defect in a weight bearing long bone, the femur, researchers attempted to relate the spatial distribution of mineral in situ in the proximal, central and distal thirds of the femoral diaphysis to the biochemical composition of bone from the same area. Biochemical analyses revealed lower concentrations of calcium, phosphorus and osteocalcin but not collagen only in the central third of the diaphysis of the flight animals (F) compared to synchronous controls (S). Collagen concentration was reduced only in the proximal third of the diaphysis, where all 3 crosslinks, expressed as nM/mol collagen were higher in F than S. A new technique, x ray microtomography, with a resolution of 26 microns, was used to obtain semi-quantitative data on mineral distribution in reconstructed sections of wet whole bone. To improve the resolution of the mineral density distribution, images of the surfaces of cut sections were analyzed by backscattered electrons in a scanning electron microscope (BSE). There was good agreement between the results of the two stereochemical techniques which revealed distinct patterns of mineralization in transverse and longitudinal directions of the diaphysis. The novel methodology developed for this flight experiment shows considerable promise in elucidating the biochemical nature of what appear to be regional alterations in the mineralization of long bones of animals exposed to spaceflight

Marsupial and Monotreme Enamel Structure

We present some recent developments in our understanding of two basic questions: the origin, extent, nature and course of marsupial enamel tubules; and the characterisation of monotreme enamel, more particularly, the prismatic nature of platypus enamel. Methods used included SEM of methacrylate casts of marsupial enamel tubules, worn and cut surfaces of whole marsupial teeth, developing and erupted platypus teeth, and a well-developed molar of the newly discovered Miocene ornithorhynchid Obdurodon sp., and tandem scanning reflected light microscopy of intact marsupial teeth. We conclude that there are significant species differences with respect to prism shape, row formation and tubule disposition in marsupials and, moreover, that these features change in a consistent way through the thickness of the enamel. Consideration of enamel prism course in incisor and molar enamel of Macropus eugenii, together with the tubule casts, enables us to conclude that there is a fundamental relationship of tubule to prism in the body of marsupial enamel. This and previously reported data put beyond dispute the essential relationship of the marsupial tubule to the formative ameloblast. The enamel of Ornithorhynchus anatinus is shown to be prismatic only in part, with well-formed regular prisms not being a primary structural feature. The enamel of the fossil monotreme is prismatic and tubular and displays large areas of Pattern 2 prism packing. Monotreme enamel has been interpreted as representing a structural stage intermediate between that of known multituberculates and extant tribosphenid mammals

In-hospital cardiac arrests: Effect of amended Australian Resuscitation Council 2006 guidelines

Objective: To evaluate cardiac arrest outcomes following the introduction of the Australian Resuscitation Council (ARC) 2006 amended guidelines for basic and advanced life support. Methods: A retrospective study of all consecutive cardiac arrests during a 3-year phase pre-implementation (2004–06) and a 3-year phase post-implementation (2007–09) of the ARC 2006 guidelines was conducted at a tertiary referral hospital in Brisbane, Australia. Results: Over the 6-year study phase 690 cardiac arrests were reported. Resuscitation was attempted in 248 patients pre-implementation and 271 patients post-implementation of the ARC 2006 guidelines. After adjusting for significant prognostic factors we found no significant change in return of spontaneous circulation (ROSC) (odds ratio 1.21, 95% confidence interval 0.80–1.85, P = 0.37) or survival to discharge (odds ratio 1.49, 95% confidence interval 0.94–2.37, P = 0.09) after the implementation of the ARC 2006 guidelines. Factors that remained significant in the final model for both outcomes included having an initial shockable rhythm, a shorter length of time from collapse to arrival of cardiac arrest team, location of the patient in a critical-care area, shorter length of resuscitation and a day-time arrest (0700–2259 hours). In addition the arrest being witnessed was significant for ROSC and younger age was significant for survival to discharge. Conclusions: There are multiple factors that influence clinical outcomes following an in-hospital cardiac arrest and further research to refine these significant variables will assist in the future management of cardiac arrests. What is known about this topic?: The evaluation of outcomes from in-hospital cardiac arrests focuses on immediate survival expressed as ROSC and survival to hospital discharge. These clinical outcomes have not improved substantially over the last two decades. What does this paper add?: This paper identifies the factors that are related to ROSC and survival to discharge following the implementation of the ARC 2006 guidelines, which included a refocus on providing quality cardiopulmonary resuscitation with minimal interruptions. What are the implications for practitioners?: Given that multiple factors can influence clinical outcomes following an in-hospital cardiac arrest, focusing on maximising a range of factors surrounding cardiopulmonary resuscitation is essential to improve outcomes

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

Variation in the Sizes of Resorption Lacunae made in Vitro

The assessment of in vitro osteoclastic activity has, until recently, been dependent on the analysis of organ culture experiments. We have developed a single cell resorption assay so that the resorptive function of individual osteoclasts could be studied. This paper examines the biological variation in the sizes of resorption lacunae produced by bone cell cultures derived from neonate rats and rabbits, and prehatch or hatchling chicks. Cultures were run for 24h for all species; and in addition for 48h for rat, 9 or 12 hours for rabbit and 3-7 hours for chick. The numbers of the nuclei of osteoclasts seeded on to plastic were counted for all three species. SEM stereophotogrammetry was used to measure areas, volumes, and maximum and average depths of the lacunae using specially designed instruments and software. Rat osteoclasts were smallest, and more chick osteoclasts were very large. There was a species difference in the onset of resorption and the sizes of pits produced, the chick osteoclasts being more vigorous resorbers than the rabbit ones, and the rat least so. For a given plan area, chick lacunae were deeper. There was a high correlation between area and volume. The range of maximum depths for a given area was high, however. Thus the mean of a few measurements of depths should not be used to calculate volume from area. At 24 hours, 77% of the rat, 47% of the rabbit and 28% of the chick lacunae were less than 1,000 μm3 in volume; and 11% of the rat, 17% of the rabbit and 22% of the chick lacunae were between 1,000 and 2,000μm in volume. The mean values at 24 hours were 981, 2796, and 4582 μm3 for rat, rabbit and chick lacunae respectively

Scanning Electron Microscopy in Bone Pathology: Review of Methods, Potential and Applications

This article reviews the applications of SEM methods to human bone pathologies referring to studies made at UCL. We consider the methods which may be most suitable; these prove to be not routine in the context of most bio-medical applications of SEM. Valuable information can be obtained from a bone sample if its edges are ground flat, before making either (a) a matrix surface preparation by washing away all the cells or (b) a mineralising front preparation, by also dissolving the osteoid -for which hydrogen peroxide is recommended to produce a robust specimen. BSE contrast from a cut block surface can be used to measure bone phase volume. SE contrasts from natural surfaces (trabeculae, canals and lacunae) can be used to study forming, resting and resorbing* surfaces both qualitatively and quantitatively (*except in the case of histological osteomalacia, where the existence of osteoid will go undetected and reversal lines will be difficult to distinguish from recently resorbed surfaces). We also recommend the use of PMMA embedded bone blocks, which can be used as obtained from the pathologist, but are better embedded by a more rigorous procedure. BSE image analysis can be used to quantitate bone density fractions opening up a completely new investigative method for the future. Osteoid can be measured automatically using CL if the bone sample is block stained with brilliant sulphaflavine before embedding or if a scintillant is added to the embeddant. We give examples of observations made from a number of bone diseases: vitamin D resistant rickets, ostegenesis imperfecta; osteomalacia; osteoporosis; hyperparathyroidism; fluorosis; Paget \u27s disease; tumour metastasis to bone

Imaging hard – inside the skeleton

PDF of article embargoed by the publisher until march 2019PDF of article embargoed by the publisher until march 2019PDF of article embargoed by the publisher until march 2019Vertebrate hard tissue biologists study the surface and below-surface microanatomical features and compositional characteristics of bones and teeth of the skeleton (fish scales also deposit calcium phosphate and calcium carbonate into their structure, and we include them among the tissues we study). The variability in bone and tooth histology rivals that of all other organ systems, making it an ideal tissue for understanding the development, function, and physiology of organisms. What is more, in deference to soft tissues, bones and teeth survive as fossils, permitting all that we can know from the skeleton about an organism living today to be extrapolated to animals living millions of years ago