Alan Boyde

By the commonly accepted rules of nomenclature, cells forming hard tissue which become incorporated within the inter- and extra-cellular tissue mass which they secrete are called ‘-cytes’: the spaces in which these cells sit are called lacunae. Hence, osteoblasts become osteocytes in osteocytic lacunae, chondroblasts become chondrocytes, cementoblasts cementocytes, even odontoblasts become odontocytes in some mammalian species. We provide evidence that enamel spindles are cell bodies of ameloblasts which have become entrapped and buried in the enamel matrix produced by the surrounding cells, to produce amelocytic lacunae: it is no life for a cell in a rigid, dense and dead tissue, and we do not suppose that amelo cytes live once they are set on that route. Spindles, normally only found at the enamel-dentine junction, are absent when the enamel at the junction is free of prisms, i.e., the ameloblasts had no Tomes’ processes. Spindles have an organic content, the amelocyte residue which can be stained - and removed by bleach, after which the spindle space no longer stains. Spindles are extensions of enamel tubules which result from the extension of a fine ameloblastic process [from the major Tomes’ process] which unites with an odontoblast process. In marsupial mammals, the spindle-like ends of the enamel tubules are commonly located well within the enamel. Spindles/amelocytes may form at the neonatal line in human deciduous teeth in cases of severe neonatal stress, and these features simply represent encapsulation of moribund ameloblasts. The frequency of spindles at the enamel dentine junction is greater in cases where the tooth germ is severely deformed by growth pressure from the forming root of a neighbouring tooth.

Although healthy teeth are never really white, bleaching agents are used cosmetically to whiten teeth. We studied the in vitro effects of some bleaching agents and acids on the Vickers Hardness (VH) of human dental enamel using a 100 gram [981 mN] load applied for 15 s and examined treated surfaces with backscattered electron mode scanning electron microscopy (BSE SEM; 20 kV, uncoated, chamber pressure 50 Pa). Rows of indents were made in the untreated condition and intervening rows placed after treatment. Thus, the samples served as their own controls. Gel bleaching agents − 35% hydrogen peroxide or carbamide peroxide - and 30% hydrogen peroxide solutions caused substantial reductions in microhardness. Weaker peroxide gels and solutions caused lesser changes. A 5% available chlorine sodium hypochlorite bleach treatment resulted in no significant change. Prism boundary discontinuities in flat polished surfaces are normally thin and dark in BSE-SEM imaging because of the lower mineral content in the region where crystallite orientation changes abruptly – they are also not joined up. Strong peroxide bleached surfaces, however, showed wider, whiter and conjoined boundaries as in acetic acid treated samples. This appearance is the hallmark of subsurface demineralisation and remineralisation, wherein the boundary region is widened and extended by dissolution of mineral, with reprecipitation of dissolved mineral in surrounding partially demineralised tissue and infilling of the original boundary defect. Identical sequences are identified in natural and artificial carious lesions and in acid etched enamel. This appearance was not generated by 6% peroxide gel. The softening of enamel by peroxide bleaching agents is explained both by the removal and redistribution of the hard mineral phase, accounted for by their acidic (i.e. low pH) nature; and the removal of the enamel protein matrix ‘glue’ holding the crystallites together.

This chapter discusses methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Shipping wet bone samples between labs is best done in glycerol. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE-SEM imaging of bone samples. PMMA is recommended for embedding because it is easily micromilled or polished. Iodine vapor staining of PMMA blocks gives excellent histology for cells and unmineralized matrix with BSE-SEM. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including confocal scanning light microscopy, point projection X-ray microscopy, microradiography and microtomography, is important. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Laser ablation microtomy allows preparation of samples as slides for any method of light microscopy combined with SEM. Control of the vacuum pressure in the SEM sample chamber can be used to eliminate “charging” problems without the need to apply a surface conductive coating, making SEM quick and easy to use.

Counting growth layers in dentine and/or secondary cementum is widely used for age determination in wild mammals but the underlying seasonal changes in the structure and degree of mineralisation of dental tissue have not been well characterised. We embedded first (m1) and second (m2) mandibular permanent molar teeth from a 12‐year‐old female Svalbard reindeer (Rangifer tarandus platyrhynchus) in PolyMethylMethAcrylate (PMMA), prepared cut and polished surfaces coated with evaporated carbon and used 20 kV back‐scattered electron imaging in a scanning electron microscope (BSE‐SEM) to study aspects of dental tissue structure which depend on the degree of mineralisation at the micron and sub‐micron scale. BSE‐SEM revealed differences between the mineral content of growth layers (annulations) in the secondary cementum and the primary and secondary dentine, the latter, incidentally, still forming at death in m1. Wide bands of less well mineralised tissue formed in the cementum during active appositional phases. Thin, denser bands formed by maturation‐mineralisation of existing tissue when growth slowed in winter. This maturation mimics the processes seen in lamellar bone and articular cartilage. Counter to previous suggestions, there was evidence of substantial resorption and repair of the secondary cementum and of formation of dentine throughout life. Secondary dentine is layered by mineral content like cementum. In the crown, this was mainly tubular dentine with well‐marked interglobular dentine layers. In the lower pulp chamber and root, it was largely without tubules. Substantial non‐mineralised spaces found at the cement‐dentine junction in the root apical regions in m2 represent inclusions of the Hertwig's Epithelial Root Sheath (HERS) or the Epithelial Rests of Malassez (ERM) between the two tissues, a phenomenon which has previously only been identified in Muridae. The anatomical changes which result in the formation of the incremental lines (annulations) in dental tissues of reindeer, identified here for the first time at the micrometre level, are likely to be common across most if not all long‐lived species of mammals living in seasonal environments.

Resorption within cortices of long bones removes excess mass and damaged tissue and increases during periods of reduced mechanical loading. Returning to high‐intensity exercise may place bones at risk of failure due to increased porosity caused by bone resorption. We used point‐projection X‐ray microscopy images of bone slices from highly loaded (metacarpal, tibia) and minimally loaded (rib) bones from 12 racehorses, 6 that died during a period of high‐intensity exercise and 6 that had a period of intense exercise followed by at least 35 days of rest prior to death, and measured intracortical canal cross‐sectional area (Ca.Ar) and number (N.Ca) to infer remodelling activity across sites and exercise groups. Large canals that are the consequence of bone resorption (Ca.Ar >0.04 mm²) were 1.4× to 18.7× greater in number and area in the third metacarpal bone from rested than exercised animals (p = 0.005–0.008), but were similar in number and area in ribs from rested and exercised animals (p = 0.575–0.688). An intermediate relationship was present in the tibia, and when large canals and smaller canals that result from partial bony infilling (Ca.Ar >0.002 mm²) were considered together. The mechanostat may override targeted remodelling during periods of high mechanical load by enhancing bone formation, reducing resorption and suppressing turnover. Both systems may work synergistically in rest periods to remove excess and damaged tissue.

Armadillos are bitten by several species of flea. Females of the genus Tunga penetrate the epidermis and when in place are fertilised by males, after which the abdomen swells enormously to form a ‘neosome’. Within the penetrans group, T. perforans, makes lesions that perforate the osteoderms within the integument to form ~3 mm diameter cavities occupied by a discoid neosome. We examined these lesions in carapace material from animals which had died in the wild to see whether we could recruit evidence as to how they may be generated, either by the insect or by the host. We studied one species without such lesions, the nine‐banded armadillo Dasypus novemcinctus, and two species with, the greater hairy armadillo Chaetophractus villosus and the southern three‐banded armadillo Tolypeutes matacus, both showing the characteristic ‘flea bite’ holes in the external surfaces of the osteoderms. Samples were studied by three‐dimensional backscattered electron mode scanning electron microscopy and X‐ray microtomography. Both methods showed resorption pit complexes in the external surfaces of the osteoderms characteristic of those made by osteoclasts in active bone resorption. Lesions involved both the syndesmoses (sutures) between adjacent bones and the central regions of the osteoderms. Many lesions showed extensive repair by infilling with new bone. We conclude that the T. perforans neosome creates a local host response which causes bone resorption, creating the space in which it can grow.

Resorption within cortices of long bones removes excess mass and damaged tissue, and increases during periods of reduced mechanical loading. Returning to high-intensity exercise may place bones at risk of failure due to increased porosity caused by bone resorption. We used microradiographs of bone slices from highly-loaded (metacarpal, tibia) and minimally-loaded (rib) bones from 12 racehorses, 6 that died during a period of high-intensity exercise and 6 that had a period of intense exercise followed by at least 35 days of rest prior to death, and measured intracortical canal cross-sectional area (Ca.Ar) and number (N.Ca) to infer remodelling activity across sites and exercise groups. Large canals that are the consequence of bone resorption (Ca.Ar > 0.04 mm ² ) were 1.4× to 18.7× greater in number and area in the third metacarpal bone from rested than exercised animals (p = 0.005– 0.008), but were similar in number and area in ribs from rested and exercised animals (p = 0.575–0.688). An intermediate relationship was present in the tibia, and when large canals and smaller canals that result from partial bony infilling (Ca.Ar > 0.002 mm ² ) were considered together. The mechanostat may override targeted remodelling during periods of high mechanical load by enhancing bone formation, reducing resorption and suppressing turnover. Both systems may work synergistically in rest periods to remove excess and damaged tissue.