The structure and development of mammalian enamel.

PhDEnamel development and structure have been studied in a number of placental and marsupial mammals, by light microscopy; electron-microscopy; and scanning electron microscopy. The relationship between the formative cells of the enamel and its structural organisation into "prisms" and interprismatic regions has been studied in particular. The crystallites in developing enamel tend to be oriented perpendicular to its mineralising front; but their orientation may be modified by either the translatory movement which may occur between certain surfaces of the TOMES' processes of the ameloblasts and the mineralising front, or the self directed growth of groups of groups of crystallites. The presence of a repetitive (prism) pattern of crystallite orientation in formed enamel is determined by changes of orientation of and within the mineralising front: these changes are 1) the result of the peculiar mode of secretion of the enamel precursor substances from and about projections from the ameloblasts; and 2) absent during the formation of the first and last layers of enamel (formed at the enamel-dentine junction and the true enamel surface respectively) by a given group of ameloblasts: hence there are no prisms in these regions. Abrupt changes in orientation of the mineralising front determine abrupt changes in crystallite orientation in the enamel (equivalent to the "prism-sheaths" of adult enamel). The secretory territories of individual ameloblasts are only equivalent to prisms in one particular pattern: one ameloblast may be related to more than one prism. Decussation of prisms is associated with the depressions in the mineralising front filling in from alternate sides in "zones". Zone formation begins as a spiral over cusp centres. Light scattering from enamel depends on 1) the size; and 2) the orientation of its ultrastructural elements and 3) the wavelength of the incident radiation; blue light being scattered preferentially; hence the visibility of: - 1) the incremental striae; and 2) the decussating zones of prisms; and 3) the brown colour of the incremental striae when viewed by transmitted light. The calcium content in developing enamel measured by the x-ray emission microanalytical method was found to increase steadily, from the surface of the developing enamel inwards

Tandem Scanning Reflected Light Microscopy of Primate Enamel

Studies of the cross sectional packing arrangements of primate enamel prisms have been used in a number of recent studies in attempts to determine their taxonomic utility. Credibility of the results has been greatly influenced by the methods employed to examine enamel prism packing patterns and also by the limited sampling. We report here the use of a technique for the non destructive examination, in depth, of enamel prism packing patterns in modern and fossil primate teeth which has considerable advantages over any others so far used, and the preliminary results of a survey of enamel structural diversity in the Order Primates. The phylogenetic implications of these findings are also discussed. A novel microscope, the Tandem Scanning Reflected Light Microscope (TSM) has been used. This instrument has allowed these data to be obtained non destructively which has permitted the inclusion of rare fossil primates in this survey. The technique has many advantages relating to the interpretation of the results as the specimens are not etched or otherwise prepared. Primates exhibit all three major prism packing arrangements known for recent mammals. The distribution of these permits the recognition of haplorhine from strepsirhine primates and also cercopithecoid monkeys from other catarrhines

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 live or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 μ.m. 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

Tandem Scanning Reflected Light Microscopy: Applications in Clinical Dental Research

The Tandem Scanning Reflected Light Microscope (TSRLM) enables the investigation of microscopic structures both at and deep to the surface of intact objects. The present paper reviews studies undertaken to determine whether the TSRLM would be usable and useful in the investigation of natural and restorative dental materials in vitro and in vivo. It was found that the TSRLM could be used to study normal and diseased dental tissues and the new materials which are used to replace lost substance. More importantly, it could be used to characterize the interface between tooth and optically translucent materials in bulk samples, giving high resolution information from not only a shallow depth of field, but at planes below cut surfaces. This makes it possible to study interfacial regions in three dimensions without the risk of delamination that must accompany the preparation of a microscopic section. The use of fluorescent markers enables more information to be derived from the tooth/adhesive interface. Studies to date indicate the need for the development of adhesion promoting agents which incorporate a fluorescent radical in their molecular structure. Preliminary work using the instrument for observation of cutting interactions between a high speed bur and a tooth indicates some useful potential in the study of cavity preparation techniques and tissue failure mechanisms. Recent developments of the TSRLM for three dimensional imaging in other dental applications are outlined. This microscope is an important advance in the microscopic assessment of adaptation of biomaterials to hard tissues

Scanning Microscopic Observations on Dental Caries

This paper presents findings made using special techniques of imaging and/or of specimen preparation to investigate the changes in tooth structure which occur in caries. We have studied both coronal and root caries in enamel, dentine and cementum using scanning electron and confocal scanning optical microscopy. In preparation for backscattered electron (BSE) imaging in the SEM, teeth were stored in 70% ethanol until further dehydration in ethanol and embedding in polymethylmethacrylate (PMMA). Longitudinally cut surfaces were diamond polished and coated with carbon or silver before BSE imaging. Important changes in the distribution of densities in both enamel and dentine occurred during caries, and could be correlated with prior published studies using polarised light and microradiography to study demineralization in these tissues. However, the resolution of the BSE imaging technique is much higher than that of these previous methods. A new method was used for demonstrating local variations in microhardness with special relevance to the changes occurring in dental caries. Sectioned surfaces were subjected to treatment with a jet of soft abrasive particles, resulting in the selective removal of carious enamel, and enhanced removal of carious dentine. The tandem scanning reflected light microscope (TSRLM) has also been shown to be useful in characterising the spread of caries in the dental tissues. Teeth only need to be cut once, because the image is formed on looking into a bulk specimen. Fluorescent dyes can be used to study the distribution of pore volume, making use of the high resolution in depth of this confocal microscope

Mineralization of Normal and Rachitic Chick Growth Cartilage: Vascular Canals, Cartilage Calcification and Osteogenesis

This paper reviews recent work in the authors\u27 laboratories that has led to new observations and thoughts concerning the mineralization of normal and rachitic chick growth cartilage. The proximal tibial growth cartilages of normal and rachitic chicks were rapidly frozen and prepared for SEM and biochemical studies. Using a scanning microfluorimetric technique we showed that at the mineralization front of normal and rachitic cartilage there is an abrupt change in chondrocyte metabolism. Thus cells in this region exhibited an increase in NADH and oxidative metabolism. In rickets, there was a decrease in the reduced pyridine nucleotide content of each of the zones. The reversal in chondrocyte metabolism was not due to low oxygen tension. SEM observations indicated that this region of cartilage was well supplied with vascular channels; moreover, mineral was first seen deposited in matrix in close proximity to the blood supply. Indeed these vascular channels appeared to be a basic architectural feature of normal cartilage, although disorganized in the rachitic state. The morphological studies also showed that gaps existed in the continuity of the mineral phase in normal cartilage. Although the rachitic cartilage does mineralize, discontinuities in the mineral distribution are much more severe, with the general failure of fusion of adjacent mineral clusters. These structures would serve as pathways for transport of nutritional factors and gases to chondrocytes that are distant from the vascular channels. Observation of hypertrophic eel ls reinforced the view that some osteoblasts represented a terminal stage in the maturation of chondrocytes

Optical and Scanning Electron Microscopy in the Single Osteoclast Resorption Assay

The present studies relate to the single or isolated osteoclastic resorption function assay which we introduced in 1983 to overcome objections to assays based upon measurements of calcium release from bones, in which it was never strictly controlled whether the mechanism involved the destruction of bone with the formation of classical Howship\u27s lacunae. The method may prove to be quite popular in the near future and has already been adopted by other research groups. In previous work, we had utilised stereo-photogrammetry of scanning electron micrographs to measure the depth, volume and other parameters of the individual lacunae. However, increasing experience with the method has suggested that we can await a wide range of biological variability in single cell function in any one experiment. We have therefore tested other methods from which data could be obtained more rapidly to permit a better statistical analysis, albeit with reduced accuracy, of each resorption complex. The main aim of the studies reported here was to evaluate various methods of optical and scanning electron microscopy that can be used for the visualization of osteoclasts and their associated resorption lacunae generated in vitro in slabs of dentine and bone. Optical microscopy was found to be complementary to SEM, enabling vital microscopy of unstained and stained cells. In particular, oblique illumination LM and tandem scanning reflected LM (TSRLM) proved to be of paramount value for this purpose. Fixed coated specimens could be most rapidly scanned for resorption lacunae using darkfield reflected LM or TSRLM

Tandem Scanning Reflected Light Microscopy

Most attempts at optical microscopy of bulk living objects have failed because of reflections from optical components and the object surface, and overlapping of unsharp out-of-focus images with the one (weak) sharp in-focus image. The optical signal in the image plane thus consists of a strong d.c. component, a weak noise and a still weaker signal. Most of the first two components can be suppressed by scanning in concordance with both the illuminating and the image-forming rays. In the first scan the focussed-on-object plane is illuminated only in several very fine discrete spots. In the second scan only that light is permitted to reach the image plane which has been reflected in the illuminated points of the object plane. Both scans are performed by a single rotating aperture disc and the whole field is covered within 0.05 sec. Because the total number of scanning lines is about 10,000 in the field of the microscope objective - giving a line period for the strongest objectives of 10nm - the resolution is not impaired by this scanning. On the contrary, it follows from theory that the resolution is better because the contrast is also improved. The following living biological objects have been successfully observed: Eye tissues - cornea (epithelium, stroma, endothelium), lens, all layers of the retina: nerves and nerve fibres: brain cells in whole brain: muscle fibres and nerve endings in striated muscle, heart muscle eel ls (in juvenile mice, through epicardium): stratum corneum of human skin, frog skin eel ls: spermatozoa: blood cells: cartilage, bone and dental tissues: plant eel ls. Fossil teeth and bones, protozoa and bacteria in flint, insects in amber, and fossil plant tissues have also been successfully studied

Osteochondral lesions in distal tarsal joints of Icelandic horses reveal strong associations between hyaline and calcified cartilage abnormalities

Osteochondral lesions in the joints of the distal tarsal region of young Icelandic horses provide a natural model for the early stages of osteoarthritis (OA) in low-motion joints. We describe and characterise mineralised and non-mineralised osteochondral lesions in left distal tarsal region joint specimens from twenty-two 30 ±1 month-old Icelandic horses. Combinations of confocal scanning light microscopy, backscattered electron scanning electron microscopy (including, importantly, iodine staining) and three-dimensional microcomputed tomography were used on specimens obtained with guidance from clinical imaging. Lesion-types were described and classified into groups according to morphological features. Their locations in the hyaline articular cartilage (HAC), articular calcified cartilage (ACC), subchondral bone (SCB) and the joint margin tissues were identified and their frequency in the joints recorded. Associations and correlations between lesion-types were investigated for centrodistal joints only. In centrodistal joints the lesion-types HAC chondrocyte loss, HAC fibrillation, HAC central chondrocyte clusters, ACC arrest and ACC advance had significant associations and strong correlations. These lesion-types had moderate to high frequency in centrodistal joints but low frequencies in tarsometatarsal and talocalcaneal-centroquartal joints. Joint margin lesion-types had no significant associations with other lesion-types in the centrodistal joints but high frequency in both the centrodistal and tarsometatarsal joints. The frequency of SCB lesion-types in all joints was low. Hypermineralised infill phase lesion-types were detected. Our results emphasise close associations between HAC and ACC lesions in equine centrodistal joints and the importance of ACC lesions in the development of OA in low-motion compression-loaded equine joints

What does the arthropathy of alkaptonuria teach us about disease mechanisms in osteoarthritis and ageing of joints? Lessons from a rare disease

AKU Society, the Rosetrees Foundation, the Childwick Trust, the Big Lottery and EUFP