The plate-to-rod transition in trabecular bone loss is elusive.

Changes in trabecular micro-architecture are key to our understanding of osteoporosis. Previous work focusing on structure model index (SMI) measurements have concluded that disease progression entails a shift from plates to rods in trabecular bone, but SMI is heavily biased by bone volume fraction. As an alternative to SMI, we proposed the ellipsoid factor (EF) as a continuous measure of local trabecular shape between plate-like and rod-like extremes. We investigated the relationship between EF distributions, SMI and bone volume fraction of the trabecular geometry in a murine model of disuse osteoporosis as well as from human vertebrae of differing bone volume fraction. We observed a moderate shift in EF median (at later disease stages in mouse tibia) and EF mode (in the vertebral samples with low bone volume fraction) towards a more rod-like geometry, but not in EF maximum and minimum. These results support the notion that the plate to rod transition does not coincide with the onset of bone loss and is considerably more moderate, when it does occur, than SMI suggests. A variety of local shapes not straightforward to categorize as rod or plate exist in all our trabecular bone samples.

Secondary osteons scale allometrically in mammalian humerus and femur.

Intra-cortical bone remodelling is a cell-driven process that replaces existing bone tissue with new bone tissue in the bone cortex, leaving behind histological features called secondary osteons. While the scaling of bone dimensions on a macroscopic scale is well known, less is known about how the spatial dimensions of secondary osteons vary in relation to the adult body size of the species. We measured the cross-sectional area of individual intact secondary osteons and their central Haversian canals in transverse sections from 40 stylopodal bones of 39 mammalian species (body mass 0.3-21 000 kg). Scaling analysis of our data shows that mean osteonal resorption area (negative allometry, exponent 0.23,R2 0.54,p<0.005) and Haversian canal area (negative allometry, exponent 0.31,R2 0.45,p<0.005) are significantly related to body mass, independent of phylogeny. This study is the most comprehensive of its kind to date, and allows us to describe overall trends in the scaling behaviour of secondary osteon dimensions, supporting the inference that the osteonal resorption area may be limited by the need to avoid fracture in smaller mammalian species, but the need to maintain osteocyte viability in larger mammalian species.

Altered lacunar and vascular porosity in osteogenesis imperfecta mouse bone as revealed by synchrotron tomography contributes to bone fragility.

Osteogenesis imperfecta (brittle bone disease) is caused by mutations in the collagen genes and results in skeletal fragility. Changes in bone porosity at the tissue level indicate changes in bone metabolism and alter bone mechanical integrity. We investigated the cortical bone tissue porosity of a mouse model of the disease, oim, in comparison to a wild type (WT-C57BL/6), and examined the influence of canal architecture on bone mechanical performance. High-resolution 3D representations of the posterior tibial and the lateral humeral mid-diaphysis of the bones were acquired for both mouse groups using synchrotron radiation-based computed tomography at a nominal resolution of 700nm. Volumetric morphometric indices were determined for cortical bone, canal network and osteocyte lacunae. The influence of canal porosity architecture on bone mechanics was investigated using microarchitectural finite element (µFE) models of the cortical bone. Bright-field microscopy of stained sections was used to determine if canals were vascular. Although total cortical porosity was comparable between oim and WT bone, oim bone had more numerous and more branched canals (p<0.001), and more osteocyte lacunae per unit volume compared to WT (p<0.001). Lacunae in oim were more spherical in shape compared to the ellipsoidal WT lacunae (p<0.001). Histology revealed blood vessels in all WT and oim canals. µFE models of cortical bone revealed that small and branched canals, typical of oim bone, increase the risk of bone failure. These results portray a state of compromised bone quality in oim bone at the tissue level, which contributes to its deficient mechanical properties.

Combined nanoindentation testing and scanning electron microscopy of bone and articular calcified cartilage in an equine fracture predilection site.

Condylar fracture of the third metacarpal bone (Mc3) is the commonest cause of racetrack fatality in Thoroughbred horses. Linear defects involving hyaline articular cartilage, articular calcified cartilage (ACC) and subchondral bone (SCB) have been associated with the fracture initiation site, which lies in the sagittal grooves of the Mc3 condyle. We discovered areas of thickened and abnormally-mineralised ACC in the sagittal grooves of several normal 18-month-old horses, at the same site that linear defects and condylar fracture occur in older Thoroughbreds and questioned whether this tissue had altered mechanical properties. We embedded bone slices in PMMA, prepared flat surfaces normal to the articular surface and studied ACC and SCB using combined quantitative backscattered electron scanning electron microscopy (qBSE) and nanoindentation testing: this allowed correlation of mineralisation density and tissue stiffness (E) at the micron scale. We studied both normal and affected grooves, and also normal condylar regions. Large arrays of indentations could be visualised as 2-dimensional maps of E with a limit to resolution of indentation spacing, which is much larger than qBSE pixel spacing. ACC was more highly mineralised but less stiff in early linear defects than in control regions, while subchondral bone was more highly mineralised and stiffer in specimens with early linear defects than those without. Thus both ACC and SCB mineralisation may be abnormal in a class of early linear defect in 18-month-old Thoroughbred horses, and this may possibly contribute to later fracture of the Mc3 condyle.

Changes in mineralised tissue at the site of origin of condylar fracture are present before athletic training in Thoroughbred horses.

AIM: To show that changes are present at the site of origin of metacarpal condylar fracture in young Thoroughbred horses before they enter race training. METHOD: Bone slices, 2 mm thick, in three mediolateral planes through the centre of rotation of the metacarpo-phalangeal joint (MCPJ) of both distal third metacarpal bones (Mc3) of 12 Thoroughbred horses aged 17 months, were imaged using point-projection digital X-ray imaging (muXR). RESULTS: In some horses, linear or ovoid radiolucency was found in articular calcified cartilage (ACC) and subchondral bone of the palmaro-distal aspect of the sagittal groove, exactly at the site of more advanced stages of condylar fatigue fracture. An incidental finding was ovoid radiolucency in the apex of the dorso-distal aspect of the sagittal ridge, with or without fragmentation or disturbance of the subchondral mineralised tissue line, resembling equine osteochondrosis. CONCLUSIONS AND CLINICAL RELEVANCE: The findings imply that the aetiology of condylar fatigue fracture in young Thoroughbred horses includes abnormality in development of the bone and joint that is present before athletic activity occurs.

Variations in articular calcified cartilage by site and exercise in the 18-month-old equine distal metacarpal condyle.

OBJECTIVES: To interrelate articular calcified cartilage thickness, mineralisation density, tidemark count and tidemark linear accretion rate by site in the equine third metacarpal distal condyle. To determine the effects of exercise during early life on articular calcified cartilage. METHOD: Six of 12 pasture-raised Thoroughbred horses were exercised from 10 days old. Calcein labels were given 19 and 8 days prior to euthanasia at 18 months old. Osteochondral specimens were cut from the distal third metacarpal condyle and imaged using confocal scanning light microscopy (CSLM) and quantitative backscattered electron scanning electron microscopy (qBSE). Articular calcified cartilage thickness and total thickness mineralisation density were measured on montaged qBSE image sets, and inter-label mineralisation density, tidemark count and linear accretion rate measured on registered CSLM-qBSE image pairs. RESULTS: Calcified cartilage thickness, mineralisation density, tidemark count and linear accretion rate varied significantly between sites. Regions with thinner calcified cartilage had greater linear accretion rates, hence rapid chondroclastic resorption. Mineralisation density was positively correlated with linear accretion rate. Fewer multiple tidemarks were counted in regions with greater linear accretion rates. Lag time between the tidemark and cement line was estimated (180 days; in the range of 0-648 days). Exercise had little effect on measured parameters. CONCLUSION: The major determinant of articular calcified cartilage thickness is the rate of chondroclastic resorption, not tidemark linear accretion rate. Our evidence supports coupled, mechanosensitive regulation of chondroclastic resorption and linear accretion rate in articular calcified cartilage. Exercising pasture-reared foals causes little additional adaptation in distal third metacarpal articular calcified cartilage.

Registration of confocal scanning laser microscopy and quantitative backscattered electron images for the temporospatial quantification of mineralization density in 18-month old thoroughbred racehorse articular calcified cartilage.

Combined backscattered electron scanning electron microscopy (BSE SEM) and confocal scanning laser microscopy (CSLM) have been used to put tissue mineralization data into the context of soft tissue histology and fluorescent label information. Mineralization density (Dm) and linear accretion rate (LAR) are quantifiable parameters associated with mineralizing fronts within calcified tissues. Quantitative BSE (qBSE) may be used to determine Dm, while CSLM may be used to detect label fluorescence from which LAR is calculated. Eighteen-month old Thoroughbred horses received single calcein injections 19 and 8 days prior to euthanasia, labeling sites of active mineralization with fluorescent bands. Confocal scanning laser microscopy images of articular calcified cartilage (ACC) from distal third metacarpal condyles were registered to qBSE images of the same sites using an in-house program. ImageJ and Sync Windows enabled the simultaneous collection of LAR and Dm data. The repeatability of the registration and measurement protocols was determined. Dm profiles between calcein labels were explored for an association with time. Dm was 119.7 +/- 24.5 (mean +/- standard deviation) gray levels (where 0 = backscattering from monobrominated and 255 from monoiodinated dimethacrylate standards, respectively), while modal and maximum LAR were 0.45 and 3.45 microm/day, respectively. Coefficients of variation (CV) for Dm were 0.70 and 0.77% with and without repeat registration, respectively; CVs for LAR were 1.90 and 2.26% with and without repeat registration, respectively. No relationship was identified between Dm and time in the 11-day interlabel interval. Registration of CSLM to qBSE images is sufficiently repeatable for quantitative studies of equine ACC.

Musculoskeletal responses of 2-year-old Thoroughbred horses to early training. 6. Bone parameters in the third metacarpal and third metatarsal bones.

AIM: To determine the effect of a known training regimen on the size and mineral content of the third metacarpal (Mc3) and third metatarsal (Mt3) bones of 2-year-old Thoroughbred horses trained on racetracks. METHODS: Mc3 and Mt3 of seven horses trained on grass and sand tracks were scanned at several sites using conventional quantitative and peripheral computed tomography (CT). Bone dimensions and density in the diaphysis and epiphysis were compared with those from seven untrained horses. Calcein label was injected in two clusters, during Weeks 9 and 12. The extent and rate of diaphyseal modelling was determined by confocal fluorescent microscopic examination of thin plane parallel sections of the mid-metacarpal region. RESULTS: Volumetric bone mineral density (BMDv) of the epiphysis was markedly higher and of the diaphysis was slightly higher in trained compared with untrained horses, but greater bone size in the trained horses had the greatest effect on an index of bone strength. Active osteons, defined as Haversian systems containing calcein label, were fewer, of smaller diameter at the time of calcein injection, and had a greater bone apposition rate in trained than in untrained horses. CONCLUSIONS: Conventional training of 2-year-old Thoroughbred racehorses over a 13-week period had a significant effect on bone size, density and strength index when compared to untrained horses. CLINICAL RELEVANCE: Bone responded rapidly to early training. The data provide reference values and sites for use in longitudinal studies of commercial training regimens.