Combining confocal and BSE SEM imaging for bone block surfaces

The present report presents a method for the correlation of qualitative and quantitative BSE SEM imaging with confocal scanning light microscopy (CSLM) imaging modes applied to bone samples embedded in PMMA. The SEM has a proper digital scan generator: we leave the BSE image unchanged, and match the CSLM image to it, because the CSLM scan mechanism is not digital, though the signal is digitised. Our overlapping program uses a linear transformation matrix which projects one system to the other, calculated by finding three corresponding points in BSE and CSLM pictures. BSE images are empty where cells and osteoid are present. Fluorescence mode CSLM fills in these gaps. The combination images enhance our understanding of what is going on - and re-establish the need for good cellular preservation

Nanoindentation of bone: Comparison of specimens tested in liquid and embedded in polymethylmethacrylate

Elastic modulus of bone was investigated by nanoindentation using common methods of sample preparation, data collection, and analysis, and compared to dynamic mechanical analysis (DMA: three-point bending) for the same samples. Nanoindentation (Berkovich, 5 μm and 21 μm radii spherical indenters) and DMA were performed on eight wet and dehydrated (100% ethanol), machined equine cortical bone beams. Samples were embedded in polymethylmethacrylate (PMMA) and mechanical tests repeated. Indentation direction was transverse to the bone long axis while DMA tested longitudinally, giving approximately 12% greater modulus in DMA. For wet samples, nanoindentation with spherical indenters revealed a low modulus surface layer. Estimates of the volume of material contributing to elastic modulus measurement showed that the surface layer influences the measured modulus at low loads. Consistent results were obtained for embedded tissue regardless of indenter geometry, provided appropriate methods and analysis were used. Modulus increased for nanoindentation (21 μm radius indenter) from 11.7 GPa ± 1.7 to 15.0 GPa ± 2.2 to 19.4 GPa ± 2.1, for wet, dehydrated in ethanol, and embedded conditions, respectively. The large increases in elastic modulus caused by replacing water with ethanol and ethanol with PMMA demonstrate that the role of water in fine pore space and its interaction with collagen strongly influence the mechanical behavior of the tissue

CORRELATIVE LIGHT MICROSCOPY AND X-RAY MICROTOMOGRAPHY OF GROUND SECTIONS OF MINERALISED TISSUES

Starting from scratch, if one wanted to correlate light microscopical (LM) and X-ray microtomographic (XMT, micro-CT) findings from the mineralized tissues - bone and calcified cartilage in the skeleton and dentine, enamel, and cementum in teeth - one could simply examine the same, resin embedded sample with at least one flat surface by confocal scanning reflection and/or fluorescence light microscopy and XMT. However, we are frequently presented with ready-made 'ground' sections mounted in Canada balsam or DPX on 25mm wide ~1mm thick glass slides with 0.17mm cover slips. Many such preparations are historical or are valuable by representing archival material from rare diseases or endangered species: all are inconvenient in form for XMT. We endeavored to economize on X-ray beam time by scanning a 25mm thick stack of slides, separating the relevant data from each sample and making exact matches with transmitted ordinary and polarized light microscopy images. Samples were selected to represent a wide range of sizes and skeletal and dental tissue types, including human femoral bone, human permanent teeth, dog carnassial tooth, narwhal mandible, black rhinoceros molars, sperm whale cementum and dentine, African elephant ivory, and prairie marmot molars. XMT was conducted using the QMUL Mucat-2 system [1], nominal voxel size 20μm, 90kV, 24 hours. Analysis used in-house analysis software TomView, ImageJ [2] and Drishti [3] software. In each case we were able to match XMT and light microscopy. We can now report mineralization densities for all the calcified tissues in the context of classical light microscopy imagery

New approach to increase information content in polarised light microscopy of skeletal and dental tissues.

Presentation and poster at Microscience Microscopy Congress, Manchester, July 2nd - 4th 201

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

OSTEOARTHRITIC SUBCHONDRAL BONE MARROW HISTOLOGY. LESSONS FROM CHANGES IN ALKAPTONURIA

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All that fractures is not bone: microscopic anatomy of vertebral bodies.

Abstract for poster P25 presented at Bone Research Society, Winchester 28/6/2018 All that fractures is not bone: microscopic anatomy of vertebral bodies. Alan Boyde and David Mills, Dental Physical Sciences, QMUL, London E1 4NS Objectives: To understand interface between cortical shell and cancellous bone in human vertebral bodies, age changes, and probable mechanical significance. Archival material, 3-4 mm mid-body vertical slices, 80 L2 embedded PMMA: blocks polished, carbon coated, 20 kV qBSE SEM; high contrast resolution x-ray microtomography (XMT: 44 hour scans); iodine vapour staining and further BSE SEM, uncoated. Some 10µm laser ablation machined sections from block surfaces for polarised light microscopy (PLM). 50 L4 macerated for 3D BSE SEM. Mineral concentration: cortex contains lamellar bone and more highly mineralised tissues: ligament, dense fibrous periosteum, or Sharpey fibre bone. 2D SEM with iodine staining, PLM: uncalcified osteoid, ligament, fibrous periosteum, and Sharpey fibres can be distinguished. 3D SEM: inimitable branching bundle morphology of bone collagen lamellae was displayed on all (re)modelled surfaces of trabeculae and endocortex, modified where penetrated by any Sharpey fibres at ectocortex. However, the greatest part of the most exterior cortex is composed of strictly parallel ─ non-branching ─ collagen, either mostly longitudinal ─ ligament ─ or decussating layers of dense fibrous periosteum. Ligament tissue becomes incorporated in the bone organ by calcification extending into it from the mineralising front of bone tissue proper. Owing to endocortical resorbtion, sections of the entire shell thickness can be composed of non-bone. Calcified tissues in vertebral cortical shells include matrices which are, emphatically, not bone: they have a different structure, their collagen is not made by osteoblasts, and generally reach a higher level of mineralisation than bone: they will be assessed as ‘thicker bone’ with CT. Further, these phases cannot be recognised from bone by clinical imaging, and it is highly improbable that they will be distinguished using decalcification and staining LM histology. If lamellar structure is ideal, then the anterior cortex in particular is not. The proportion of calcified ligament tissue masquerading as bone dramatically increases in anterior collapse, often in hand with thickening of this cortex. It remains to be elucidated whether failure is favoured by this less auspicious and perhaps more fragile structure. A vicious circle

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