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

Local fabric density in L2 vertebral body bone by high contrast resolution x-ray microtomography

Presentation from BRS Online 2020 MeetingLocal fabric density in L2 vertebral body bone by high contrast resolution x-ray microtomography. David Mills & Alan Boyde. Dental Physical Sciences, Queen Mary University of London. Many x-ray microtomography.(XMT) studies of human vertebral bone samples have been conducted, but none with high quality, high contrast resolution methodology, which necessarily requires long integration times and, therefore, stable samples. We accessed a collection of 69 ~2mm thick, parasagittal sections of L2 vertebral bodies which were preserved - by embedding in PMMA - under the aegis of the BioMed 1 European [long before Brexit] project [Bone quality in osteoporosis]. Prior studies using quantitative backscattered electron [qBSE-SEM] imaging had shown a wide spread of local mineralisation density values when sampled at one cubic micrometer [1 fl] resolution, but only in 2D section planes. To acquire 3D data, we used the QMUL MuCAT2 TDI microtomography system at 30 micron voxel size, 90kV, typically 72h scan time, each scan corrected for beam-hardening and calibrated with a multi-metal calibration carousel: Linear Attenuation Coefficient (LAC) accuracy better than 2%. Results shown are expressed in LAC (cm-1), equivalence to assumed HA concentration. Analysis using ImageJ Fiji. To avoid partial volume artefacts, we stripped, in 3D, one voxel from all free bone surfaces. We compared distributions for the whole bone slab and regions selected to contain only internal trabecular bone, avoiding cortices and end plates. Both showed extreme ranges, but whole slab values contained a higher proportion of highest density voxels. These results are in complete agreement with the earlier qBSE-SEM data which showed the highest values in calcified cartilage in end plates and calcified ligamentous inclusions and Sharpey fibre bone rich regions in cortices, NB the anterior cortex. 'Bone' in the vertebral body is not one tissue but has a variety of fabrics and fabric densities. Poster presentation in Muscle and Bone session Video poster available on-line until 8/8/2020 https://boneresearchsociety.org/resources/video/26/ or see file BRS 2020 L2 XMT David Mills & Alan Boyde 11.mp4 PDF copy of PowerPoint BRS 2020 L2 XMT David Mills Alan Boyde.pd

New method for quantitative polarised light microscopy of laser-ablation machined sections of bones and joints

Bone Research Society meeting, Bristol, 25-27 June 2017, ‘Late breaking’ abstract Abstract ID: 106 LBP11 New method for quantitative polarised light microscopy of laser-ablation machined sections of bones and joints Alan Boyde Dental Physical Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 4NS, UK Objectives: We have recently prepared very thin sections from the front face of bone blocks embedded in PMMA - which had previously been studied by backscattered electron scanning electron microscopy (BSE-SEM) and x-ray microtomography - by the new technique of laser-ablation machining. These sections justify the development and use of new 3D high resolution light microscopic methods. In conventional polarised light microscopy (PLM), positively birefringent crystals such as hydroxyapatite and/or negatively birefringent arrays of oriented molecules such as collagen appear brightest if they lie both in the plane of the section and at 45º to the axes of the crossed polarising filter elements. Birefringent elements appear black if they lie parallel to either polariser or analyser (or perpendicular to the plane of section). This situation prevents us from seeing the whole scene at once, because nothing can be seen in the dark sectors of the ‘Maltese cross’. Methods: We have overcome this problem by combining three grey-level PLM images. Digital images are recorded using green light with the polariser and analyser rotated 30º between each and used as red, green and blue components in a composite image. The colour maps the in-plane direction of the oriented molecular arrays irrespective of whether they are too small to be resolved. The intensity of the colour indicates the ‘strike’ of the molecules, i.e., the angle that they make to the plane of the section, brightest being parallel. An interpretive diagram has been developed which shows the colours for different orientations. This method has been applied to an array of large and small bone and joint samples. Results & Conclusion: Laser ablation microtomy produces high quality, thin sections of both hard, mineralised and dense fibrous connective tissues of any sort – even of single thin trabeculae – which can be studied with any light microscopic method as well as BSE-SEM. The novel 3x30º pseudocolour PLM images refine understanding of bone, cartilage, calcified cartilage, Sharpey fibre bone, ligament, calcified ligament, tendon, calcified tendon and fibrous periosteum structure in bones

New quantitative method for increasing information content in polarised light imaging of bone tissue

New quantitative method for increasing information content in polarised light imaging of bone tissue presentation from Bone Research Society BRS2020 Online, 6-8 July 2020. Bone Research Society Online meeting. 1Alan Boyde, 1David Mills, 2Alessandro Felder. 1. Dental Physical Sciences, Queen Mary University of London. 2. University College London. In linearly polarised light (LPL), birefringent structures appear brightest if they lie both in the plane of the section and at 45°/135° to the axes of the crossed polarising filter elements, but dark if perpendicular to the section plane or parallel to either polarizer or analyser, preventing measurement of the whole scene at once because nothing can be resolved in the dark sectors of the ‘Maltese cross’. This may be solved using circularly polarised light (CPL), when dip with respect to the section-plane may be quantified for plane parallel sections and we can use pseudocolour to produce dip maps. CPL, however, does not differentiate between in-plane orientations. We provide a new solution by combining numbers of PLM images to map orientations in 3D. We have automated the coupled rotations of polarising and analysing filters at, for example, 3°, 5°, 7.5°, 10° or 15° intervals through a range of 90° - with digital LPL images recorded at each orientation - and exploit digital processing. For in-plane orientation mapping display we use the colour circle sequence Red, Yellow, Green, Cyan, Blue, Magenta, where colour shows the orientation with 4 repeat cycles in 360º. Brightness is proportional to the cosine of the dip/strike angle with respect to section plane, being brightest in plane, and black when perpendicular to that plane, i.e., parallel to the optic axis. The dip value can be displayed in a pseudocoloured version of the sum of the separate monochrome LPL images using a Look-Up-Table with six 15° vertical orientation classes. The new method is powerful, label free and best used with unstained sections, but most stains do not interfere too much. Thus it may be used for much archival material. It proves to be excellent for undecalcified, uncalcified and decalcified tissue sections

Fragmenting densely mineralised acellular protrusions from articular calcified cartilage: a role in osteoarthritis?

Fragmenting densely mineralised acellular protrusions from articular calcified cartilage: a role in osteoarthritis? A. Boyde a, G.R. Davis a, D. Mills a, T. Zikmund a, V.L. Adams b, L.R. Ranganath b, N. Jeffery b, J.A. Gallagher b a Dental Physical Sciences, Oral Growth and Development, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK b Department of Musculoskeletal Biology, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK Objectives High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first discovered in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. If these fragment, they could make a significant contribution to joint destruction in osteoarthritis. We looked for them in human material. Methods Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were studied by MRI DESS at 0.23mm resolution and 26 micron resolution high contrast x-ray microtomography (XMT), then sectioned and embedded in PMMA, and block faces polished and the blocks re-imaged with 6 micron resolution XMT. Tissue mineralisation density was imaged qualitatively by backscattered electron SEM (BSE SEM) at 20kV using uncoated samples at 50Pa chamber pressure to achieve charge neutralisation. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. Block surfaces were sequentially repolished and restained. Results Figure: 3D rendering of 6 micron voxel resolution XMT data set showing HDMP complex projecting above subchondral bone plate. Human femoral head removed at arthroplasty. We found examples of HDMP in HAC in human hips. Their 3D shapes are complex and may show cutting blade forms. Their mineral content (a) exceeds that of articular calcified cartilage (ACC), otherwise the densest tissue in the joint and (b) is not uniform. The mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. Cracks within them are frequent. Dense fragments may be found within damaged HAC. Conclusions HDMP arise via the extrusion of an uncharacterised matrix into clefts in HAC. Little evidence of their existence remains after tissue has been decalcified with usual histological protocols. Their formation may be an extension of a normal but poorly recognised crack self-healing mechanism found in bone and ACC. They are surrounded by HAC, are dense and brittle and show innumerable fault lines within them. We provide evidence that they break in vivo by being able to find matching fragments in HAC. We conclude that these hard and sharp particles contribute to the shredding destruction of HAC. The osteoarthritis research community should be aware of their existence so that the frequency and possible clinical significance can be assessed in the future. Larger HDMP can be detected with the best MRI imaging

A new paradigm for bone formation in remarkable endosteal bone appositional rates in rat distal femur

A new paradigm for bone formation in remarkable endosteal bone appositional rates in rat distal femu. A Boyde, T Zikmund. Dental Physical Sciences, QMUL, London, UK. Poster LB14 presented at Bone Research Society/British Society for Matrix Biology Joint Meeting, 01-03 Sept 2015, Edinburgh. Objectives: to study details of bone growth rates and mechanisms in the distal femur of young rat using multiple combined 3D imaging methods. Methods: fluorescent mineralisation front labels - calcein was given at 12 days and tetracycline 3 days before termination. X-ray microtomography (microCT 8μm voxels) of right femurs was conducted before cutting the femurs longitudinally in the midline. The lateral halves were PMMA embedded for block face microscopy, using confocal LM for histology and to read labels. 20kV BSE SEM was used to image mineral content: more BSE imaging after iodine staining was also used for histology. Finally, all bone was dissolved to produce 3D casts of marrow and capillary blood vessel canal space. The medial halves were macerated for 3D BSE SEM. After analysis of the right-side data, we studied left femurs with 6 μm microCT, then cut them transversely to create thick 'rings' at defined distances from the distal condyles. The end faces were polished and used for imaging labels with confocal LM and the samples were then macerated for 3D SEM. All these types of image were cross correlated to produce composites which can be best understood by dynamic, sequential, repetitive display techniques. Results and Conclusions: remarkably high values for endosteal apposition were measured, with nearly matching high periosteal resorption rates to be assumed. In places, almost the entire 400μm thickness of the shaft was translocated in 15 days. This rapid endosteal bone growth is associated with the inclusion of capillary blood vessels which penetrate the osteoblastic layer at near normal incidence to the formative surface at ~50 micron spacing. This is a previously undescribed mode of compact cortical lamellar bone formation.Trabeculae drift centrally in parallel with cortical surfaces. Thus their double label intervals must be understood in a full 3D context. MicroCT imaging ‘loses’ small trabeculae which are physically retained even in macerated SEM samples. Real loss of trabeculae occurs by their burial in compact bone at endosteal growth surfaces as well as through resorption. The ‘gold standard’ must be held to be the combination several imaging methods

Supporting patients to get the best from their osteoporosis treatment: a rapid realist review of what works, for whom, and in what circumstance

Systematic reviews that examine effectiveness of interventions to improve medicines optimisation do not explain how or why they work. This realist review identified that interventions which effectively optimise medicines use in osteoporosis include opportunities to address patients' perceptions of illness and treatment and/or support primary care clinician decision making. INTRODUCTION: In people with osteoporosis, adherence to medicines is poorer than other diseases and patients report follow-up is lacking, and multiple unmet information needs. We conducted a rapid realist review to understand what contextual conditions and mechanisms enable interventions to support osteoporosis medication optimisation. METHODS: A primary search identified observational or interventional studies which aimed to improve medicines adherence or optimisation; a supplementary second search identified research of any design to gain additional insights on emerging findings. Extracted data was interrogated for patterns of context-mechanism-outcome configurations, further discussed in team meetings, informed by background literature and the Practicalities and Perception Approach as an underpinning conceptual framework. RESULTS: We identified 5 contextual timepoints for the person with osteoporosis (identifying a problem; starting medicine; continuing medicine) and the practitioner and healthcare system (making a diagnosis and giving a treatment recommendation; reviewing medicine). Interventions which support patient-informed decision making appear to influence long-term commitment to treatment. Supporting patients' practical ability to adhere (e.g. by lowering treatment burden and issuing reminders) only appears to be helpful, when combined with other approaches to address patient beliefs and concerns. However, few studies explicitly addressed patients' perceptions of illness and treatment. Supporting primary care clinician decision making and integration of primary and secondary care services also appears to be important, in improving rates of treatment initiation and adherence. CONCLUSIONS: We identified a need for further research to identify a sustainable, integrated, patient-centred, and cost- and clinically effective model of long-term care for people with osteoporosis

Fracture in the Elderly Multidisciplinary Rehabilitation (FEMuR): study protocol for a phase II randomised feasibility study of a multidisciplinary rehabilitation package following hip fracture

Objective: To conduct a rigorous feasibility study for a future definitive parallel-group randomised controlled trial (RCT) and economic evaluation of an enhanced rehabilitation package for hip fracture.Setting: Recruitment from 3 acute hospitals in North Wales. Intervention delivery in the community.Participants: Older adults (aged ≥65) who received surgical treatment for hip fracture, lived independently prior to fracture, had mental capacity (assessed by clinical team) and received rehabilitation in the North Wales area.Intervention: Remote randomisation to usual care (control) or usual care+enhanced rehabilitation package (intervention), including six additional home-based physiotherapy sessions delivered by a physiotherapist or technical instructor, novel information workbook and goal-setting diary.Primary and secondary outcome measures: Primary: Barthel Activities of Daily Living (BADL). Secondary measures included Nottingham Extended Activities of Daily Living scale (NEADL), EQ-5D, ICECAP capability, a suite of self-efficacy, psychosocial and service-use measures and costs. Outcome measures were assessed at baseline and 3-month follow-up by blinded researchers.Results: 62 participants were recruited, 61 randomised (control 32; intervention 29) and 49 (79%) completed 3-month follow-up. Minimal differences occurred between the 2 groups for most outcomes, including BADL (adjusted mean difference 0.5). The intervention group showed a medium-sized improvement in the NEADL relative to the control group, with an adjusted mean difference between groups of 3.0 (Cohen's d 0.63), and a trend for greater improvement in self-efficacy and mental health, but with small effect sizes. The mean cost of delivering the intervention was £231 per patient. There was a small relative improvement in quality-adjusted life year in the intervention group. No serious adverse events relating to the intervention were reported.Conclusions: The trial methods were feasible in terms of eligibility, recruitment and retention. The effectiveness and cost-effectiveness of the rehabilitation package should be tested in a phase III RCT

‘The MSK Grand National: What we have learnt about the musculoskeletal system from studying racehorses’.

Bone Research Society Annual Meeting Liverpool 13-15 April 2023 Programme Page 6 [Joint meeting with 50th ECTS Congress] www.boneresearchsociety.org Invited Lecture IL1 Friday 14th April 2023 ‘The MSK Grand National: What we have learnt about the musculoskeletal system from studying racehorses’. Alan Boyde, London U.K. I reviewed studies I have conducted over many years and jointly with many colleagues concerning calcified tissues in horses from: Scanning electron microscopy of primary membrane bone. Boyde A, Hobdell MH. Z Zellforsch Mikrosk Anat. 1969;99(1):98-108. doi: 10.1007/BF00338800. Through: Coronal cementogenesis in the horse. Jones SJ, Boyde A. Archs Oral Biol. 1974 Aug;19(8):605-14. doi: 10.1016/0003-9969(74)90128-9. To: The Bone Cartilage Interface and Osteoarthritis. Boyde A. Calcif Tissue Int. 2021 Sep;109(3):303-328. doi: 10.1007/s00223-021-00866-9. The PowerPoint slides are condensed into a PDF available on the QMRO site which I have also made available in the format of a 2024 and 2025 Calendar

Fleas and bites in bones.

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 increases in size by up to ten times to form a ‘neosome’. Within the penetrans group of Tunga, a new species, T. perforans, discovered by Ezquiaga et al (Medical and Veterinary Entomology 2015 29 196–204) lesions perforate the bones within the integument. We hypothesised – and show here - that the cavities eaten into the bone might be generated by recruitment of the host's osteoclasts and that they would resemble Howship's lacunae, being formed of multiples of small resorption pits. We studied one species without ‘flea-bites’, 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 ~3 mm diameter 'flea bite' holes in the external surfaces of the osteoderms. For BSE SEM [Zeiss EVO-MA10] samples were studied before and after treatment with sodium hypochlorite bleach to remove residual adherent soft tissue and contaminant soil or dust particles, washed, dried and imaged uncoated at 20kV, 50Pa chamber pressure. For x-ray microtomography (XMT: QMUL MuCat2 system, 90kV), larger samples consisting of many adherent osteoderms were supported with Araldite, cut to isolate regions containing the bony lesions and imaged at 10 micron voxel resolution. 3D rendering was performed using Drishti software. 3D BSE-SEM showed resorption pits characteristic of those made by osteoclasts. Lesions involved both the syndesmoses (sutures) between adjacent bones and the centres of bones. Many lesions showed extensive repair by infilling with new bone We conclude that the Tunga neosome creates a local host response which causes bone resorption, creating the space in which it can grow. Owing to the superficial location of the lesions, we speculate whether this might constitute a useful experimental/observational model in the future