Topographically guided hierarchical mineralization.

Material platforms based on interaction between organic and inorganic phases offer enormous potential to develop materials that can recreate the structural and functional properties of biological systems. However, the capability of organic-mediated mineralizing strategies to guide mineralization with spatial control remains a major limitation. Here, we report on the integration of a protein-based mineralizing matrix with surface topographies to grow spatially guided mineralized structures. We reveal how well-defined geometrical spaces defined within the organic matrix by the surface topographies can trigger subtle changes in single nanocrystal co-alignment, which are then translated to drastic changes in mineralization at the microscale and macroscale. Furthermore, through systematic modifications of the surface topographies, we demonstrate the possibility of selectively guiding the growth of hierarchically mineralized structures. We foresee that the capacity to direct the anisotropic growth of such structures would have important implications in the design of biomineralizing synthetic materials to repair or regenerate hard tissues.

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.

Evaluation of laser ablation microtomy for correlative microscopy of hard tissues.

Laser ablation machining or microtomy (LAM) is a relatively new approach to producing slide mounted sections of translucent materials. We evaluated the method with a variety of problems from the bone, joint and dental tissues fields where we require thin undecalcified and undistorted sections for correlative light microscopy (LM) and backscattered electron scanning electron microscopy (BSE SEM). All samples were embedded in poly-methylmethacrlate (PMMA) and flat block surfaces had been previously studied by BSE-SEM and confocal scanning light microscopy (CSLM). Most were also studied by X-yay microtomography (XMT). The block surface is stuck to a glass slide with cyanoacrylate adhesive. Setting the section thickness and levelling uses inbuilt optical coherence tomographic imaging. Tight focusing of near-infrared laser radiation in the sectioning plane gives extreme intensities causing photodisruption of material at the focal point. The laser beam is moved by a fast scanner to write a cutting line, which is simultaneously moved by an XY positioning unit to create a sectioning plane. The block is thereby released from the slide, leaving the section stuck to the slide. Light, wet polishing on the finest grade (4000 grit) silicon carbide polishing paper is used to remove a 1-2 µm thick damaged layer at the surface of the section. Sections produced by laser cutting are fine in quality and superior to those produced by mechanical cutting and can be thinner than the 'voxel' in most laboratory X-ray microtomography systems. The present extensive pilot studies have shown that it works to produce samples which we can study by both light and electron microscopy.

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis.

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh(-120/+)) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh(-120/+) mice. We also find a much larger fibril strain/tissue strain ratio in Crh(-120/+) mice (~1.5) compared to the wild-type mice (~0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.

Detection of early osteoarthritis in the centrodistal joints of Icelandic horses: Evaluation of radiography and low-field magnetic resonance imaging.

REASONS FOR PERFORMING STUDY: Validated noninvasive detection methods for early osteoarthritis (OA) are required for OA prevention and early intervention treatment strategies. OBJECTIVES: To evaluate radiography and low-field magnetic resonance imaging (MRI) for the detection of early stage OA osteochondral lesions in equine centrodistal joints using microscopy as the reference standard. STUDY DESIGN: Prospective imaging of live horses and imaging and microscopy of cadaver tarsal joints. METHODS: Centrodistal (distal intertarsal) joints of 38 Icelandic research horses aged 27-29 months were radiographed. Horses were subjected to euthanasia approximately 2 months later and cadaver joints examined with low-field MRI. Osteochondral joint specimens were classified as negative or positive for OA using light microscopy histology or scanning electron microscopy. Radiographs and MRIs were evaluated for osteochondral lesions and results compared with microscopy. RESULTS: Forty-two joints were classified OA positive with microscopy. Associations were detected between microscopic OA and the radiography lesion categories; mineralisation front defect (P<0.0001), joint margin lesion (P<0.0001), central osteophyte (P = 0.03) and the low-field MRI lesion categories; mineralisation front defect (P = 0.01), joint margin lesion (P = 0.02) and articular cartilage lesion (P = 0.0003). The most frequent lesion category detected in microscopic OA positive joints was the mineralisation front defect in radiographs (28/42 OA positive joints, specificity 97%, sensitivity 67%). No significant differences were detected between the sensitivity and specificity of radiography and low-field MRI pooled lesion categories, but radiography was often superior when individual lesion categories were compared. CONCLUSIONS: Early stage centrodistal joint OA changes may be detected with radiography and low-field MRI. Detection of mineralisation front defects in radiographs may be a useful screening method for detection of early OA in centrodistal joints of young Icelandic horses.

Calcium silicate cement-induced remineralisation of totally demineralised dentine in comparison with glass ionomer cement: tetracycline labelling and two-photon fluorescence microscopy.

Two-photon fluorescence microscopy, in combination with tetracycline labelling, was used to observe the remineralising potentials of a calcium silicate-based restorative material (Biodentine(TM) ) and a glass ionomer cement (GIC:​Fuji​IX) on totally demineralised dentine. Forty demineralised dentine discs were stored with either cement in three different solutions: phosphate buffered saline (PBS) with tetracycline, phosphate-free tetracycline, and tetracycline-free PBS. Additional samples of demineralised dentine were stored alone in the first solution. After 8-week storage at 37 °C, dentine samples were imaged using two-photon fluorescence microscopy and Raman spectroscopy. Samples were later embedded in PMMA and polished block surfaces studied by 20 kV BSE imaging in an SEM to study variations in mineral concentration. The highest fluorescence intensity was exhibited by the dentine stored with Biodentine(TM) in the PBS/tetracycline solution. These samples also showed microscopic features of matrix remineralisation including a mineralisation front and intra- and intertubular mineralisation. In the other solutions, dentine exhibited much weaker fluorescence with none of these features detectable. Raman spectra confirmed the formation of calcium phosphate mineral with Raman peaks similar to apatite, while no mineral formation was detected in the dentine stored in cement-free or PBS-free media, or with GIC. It could therefore be concluded that Biodentine(TM) induced calcium phosphate mineral formation within the dentine matrix when stored in phosphate-rich media, which was selectively detectable using the tetracycline labelling.

On fragmenting, densely mineralised acellular protrusions into articular cartilage and their possible role in osteoarthritis.

High density mineralised protrusions (HDMP) from the tidemark mineralising front into hyaline articular cartilage (HAC) were first described in Thoroughbred racehorse fetlock joints and later in Icelandic horse hock joints. We now report them in human material. Whole femoral heads removed at operation for joint replacement or from dissection room cadavers were imaged using magnetic resonance imaging (MRI) dual echo steady state at 0.23 mm resolution, then 26-µm resolution high contrast X-ray microtomography, sectioned and embedded in polymethylmethacrylate, blocks cut and polished and re-imaged with 6-µm resolution X-ray microtomography. Tissue mineralisation density was imaged using backscattered electron SEM (BSE SEM) at 20 kV with uncoated samples. HAC histology was studied by BSE SEM after staining block faces with ammonium triiodide solution. HDMP arise via the extrusion of an unknown mineralisable matrix into clefts in HAC, a process of acellular dystrophic calcification. Their formation may be an extension of a crack self-healing mechanism found in bone and articular calcified cartilage. Mineral concentration exceeds that of articular calcified cartilage and is not uniform. It is probable that they have not been reported previously because they are removed by decalcification with standard protocols. Mineral phase morphology frequently shows the agglomeration of many fine particles into larger concretions. HDMP are surrounded by HAC, are brittle, and show fault lines within them. Dense fragments found within damaged HAC could make a significant contribution to joint destruction. At least larger HDMP can be detected with the best MRI imaging ex vivo.

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.

Horse-, training- and race-level risk factors for palmar/plantar osteochondral disease in the racing Thoroughbred.

REASONS FOR PERFORMING STUDY: Palmar/plantar osteochondral disease (POD) is a common, debilitating condition in Thoroughbred racehorses; however, training- and racing-related factors associated with this disease are unknown. OBJECTIVES: To determine horse-, racing- and training-related risk factors for POD. The general hypotheses were that early training and racing, and increased intensity of racing and training, lead to increased severity of POD. METHODS: The metacarpo/metatarsophalangeal joints of 164 Thoroughbred racehorses were examined at post mortem and graded for third metacarpal and metatarsal POD. The relationships between training- and racing-related factors and grade of POD in each condyle were determined using multilevel, multivariable, ordinal logistic regression models. RESULTS: A total of 1288 condyles were graded. Factors associated with higher grades of POD were the total lifetime number of races, an increase in gallop sessions in the previous season, racing before import to Hong Kong and an increase in the number of short (8-16 weeks) between-race intervals per season. Horses in their first racing season were more likely to have lower POD grades, while horses that had a long between-race interval (greater than 16 weeks) in the season prior to euthanasia were also more likely to have lower POD grades. Lower POD grades were significantly more likely as days since last race increased up to 400 days. Age at first race was not significantly associated with grade of POD. CONCLUSIONS AND POTENTIAL RELEVANCE: Cumulative racing exposure and training intensity in the previous season were associated with higher grades of POD, supporting the hypothesis that the disease is due to repetitive loading. Longer between-race intervals and increased time since racing were associated with lower POD grades, which may indicate that lesions heal. Further work is required to enable optimisation of racing and training programmes to reduce the frequency and severity of this disease.

Pathological and clinical features associated with palmar/plantar osteochondral disease of the metacarpo/metatarsophalangeal joint in Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: Palmar/plantar osteochondral disease (POD) of the metacarpo/metatarsophalangeal (Mc/MtPh) joints is common in Thoroughbred racehorses, but there is little information on predilection sites, associated pathologies and clinical signs. OBJECTIVES: To describe the distribution of POD at post mortem examination and to examine the association of POD with other pathologies and clinical histories. METHODS: The Mc/MtPh joints of 102 Thoroughbred racehorses were examined post mortem. Gross pathology of associated osteochondral elements, suspensory ligament and flexor tendons was recorded. Clinical data were extracted from computerised records. Relationships between all Mc/MtPh joint pathologies, clinical data and grade of POD in each condyle were determined using multilevel, ordinal logistic regression models. RESULTS: Palmar/plantar osteochondral disease was recorded (grade 1-3 in at least one condyle) in 80.4% (95% confidence interval 71-88) of horses. The grade of POD was significantly higher in the forelimb, where the medial condyle had higher grades, than in the hindlimb, where the POD grade was higher in the lateral condyle. There was a significant positive relationship between POD grade and multiple other joint pathologies, whereas grades of linear fissures had a negative relationship with grade of POD. There was no relationship between grade of POD and tendon and suspensory ligament gross pathology, although horses with a history of tendon injury had significantly lower POD grades. Horses that had a clinical history of a Mc/MtPh joint problem had higher POD grades. In addition, horses that had been retired from racing for more than 2 months at the date of death had significantly lower POD grades. CONCLUSIONS AND POTENTIAL RELEVANCE: Palmar/plantar osteochondral disease has specific predilection sites. Horses with a clinical history of Mc/MtPh-associated lameness had higher POD grades, whereas those with a history of superficial digital flexor tendon injury and retired horses had lower grades. The progression of POD and its ability to heal requires further evaluation.

Symmetrically reduced stiffness and increased extensibility in compression and tension at the mineralized fibrillar level in rachitic bone.

In metabolic bone diseases, the alterations in fibrillar level bone-material quality affecting macroscopic mechanical competence are not well-understood quantitatively. Here, we quantify the fibrillar level deformation in cantilever bending in a mouse model for hereditary rickets (Hpr). Microfocus in-situ synchrotron small-angle X-ray scattering (SAXS) combined with cantilever bending was used to resolve nanoscale fibril strain in tensile- and compressive tissue regions separately, with quantitative backscattered scanning electron microscopy used to measure microscale mineralization. Tissue-level flexural moduli for Hpr mice were significantly (p<0.01) smaller compared to wild-type (~5 to 10-fold reduction). At the fibrillar level, the fibril moduli within the tensile and compressive zones were significantly (p<0.05) lower by ~3- to 5-fold in Hpr mice compared to wild-type mice. Hpr mice have a lower mineral content (24.2±2.1Cawt.% versus 27.4±3.3Ca wt.%) and its distribution was more heterogeneous compared to wild-type animals. However, the average effective fibril modulus did not differ significantly (p>0.05) over ages (4, 7 and 10weeks) between tensile and compressive zones. Our results indicate that incompletely mineralized fibrils in Hpr mice have greater deformability and lower moduli in both compression and tension, and those compressive and tensile zones have similar moduli at the fibrillar level.

Staining plastic blocks with triiodide to image cells and soft tissues in backscattered electron SEM of skeletal and dental tissues.

Backscattered electron scanning electron microscopy (BSE SEM) is an invaluable method for studying the histology of the hard, mineralised components of poly-methyl methacrylate (PMMA) or other resin embedded skeletal and dental tissues. Intact tissues are studied in micro-milled or polished block faces with an electron-optical section thickness of the order of a half to one micron and with the area of the section as big as a whole--large or small--bone organ. However, BSE SEM does not give information concerning the distribution of uncalcified, 'soft', cellular and extracellular matrix components. This can be obtained by confocal microscopy of the same block and the two sorts of images merged but the blocks have to be studied in two microscope systems. The present work shows a new, simple and economic approach to visualising both components by using the triiodide ion in Lugol's iodine solution to stain the block surface prior to the application of any conductive coating--and the latter can be omitted if charging is suppressed by use of poor vacuum conditions in the SEM sample chamber. The method permits the use of archival tissue, and it will be valuable in studies of both normal growth and development and pathological changes in bones and joints, including osteoporosis and osteoarthritis, and tissue adaptation to implants.

Identification of trabecular excrescences, novel microanatomical structures, present in bone in osteoarthropathies.

It is widely held that bone architecture is finely regulated in accordance with homeostatic requirements. Aberrant remodelling (hyperdensification and/or cyst formation in the immediately subchondral region) has previously been described in bone underlying cartilage in arthropathies. The present study examined the trabecular architecture of samples of bone, initially in the severe osteoarthropathy of alkaptonuria, but subsequently in osteoarthritis using a combination of light microscopy, 3D scanning electron microscopy and quantitative backscattered electron scanning electron microscopy. We report an extraordinary and previously unrecognised bone phenotype in both disorders, including novel microanatomical structures. The underlying subchondral trabecular bone contained idiosyncratic architecture. Trabecular surfaces had numerous outgrowths that we have termed "trabecular excrescences", of which three distinct types were recognised. The first type arose from incomplete resorption of branching secondary trabeculae arising from the deposition of immature (woven) bone in prior marrow space. These were characterised by very deeply scalloped surfaces and rugged edges. The second type had arisen in a similar way but been smoothed over by new bone deposition. The third type, which resembled coarse stucco, probably arises from resting surfaces that had been focally reactivated. These were poorly integrated with the prior trabecular wall. We propose that these distinctive microanatomical structures are indicative of abnormal osteoclast/osteoblast modelling in osteoarthropathies, possibly secondary to altered mechanical loading or other aberrant signalling. Identification of the mechanisms underlying the formation of trabecular excrescences will contribute to a better understanding of the role of aberrant bone remodelling in arthropathies and development of new therapeutic strategies.

The role of calcified cartilage and subchondral bone in the initiation and progression of ochronotic arthropathy in alkaptonuria.

OBJECTIVE: Alkaptonuria is a genetic disorder of tyrosine metabolism, resulting in elevated circulating concentrations of homogentisic acid. Homogentisic acid is deposited as a polymer, termed ochronotic pigment, in collagenous tissues, especially cartilages of weight-bearing joints, leading to a severe osteoarthropathy. We undertook this study to investigate the initiation and progression of ochronosis from the earliest detection of pigment through complete joint failure. METHODS: Nine joint samples with varying severities of ochronosis were obtained from alkaptonuria patients undergoing surgery and compared to joint samples obtained from osteoarthritis (OA) patients. Samples were analyzed by light and fluorescence microscopy, 3-dimensional scanning electron microscopy (SEM), and the quantitative backscattered electron mode of SEM. Cartilage samples were mechanically tested by compression to determine Young's modulus of pigmented, nonpigmented, and OA cartilage samples. RESULTS: In alkaptonuria samples with the least advanced ochronosis, pigment was observed intracellularly and in the territorial matrix of individual chondrocytes at the boundary of the subchondral bone and calcified cartilage. In more advanced ochronosis, pigmentation was widespread throughout the hyaline cartilage in either granular composition or as blanket pigmentation in which there is complete and homogenous pigmentation of cartilage matrix. Once hyaline cartilage was extensively pigmented, there was aggressive osteoclastic resorption of the subchondral plate. Pigmented cartilage became impacted on less highly mineralized trabeculae and embedded in the marrow space. Pigmented cartilage samples were much stiffer than nonpigmented or OA cartilage as revealed by a significant difference in Young's modulus. CONCLUSION: Using alkaptonuria cartilage specimens with a wide spectrum of pigmentation, we have characterized the progression of ochronosis. Intact cartilage appears to be resistant to pigmentation but becomes susceptible following focal changes in calcified cartilage. Ochronosis spreads throughout the cartilage, altering the mechanical properties. In advanced ochronosis, there is aggressive resorption of the underlying calcified cartilage leading to an extraordinary phenotype in which there is complete loss of the subchondral plate. These findings should contribute to better understanding of cartilage-subchondral interactions in arthropathies.

Cartilage damage involving extrusion of mineralisable matrix from the articular calcified cartilage and subchondral bone.

Arthropathy of the distal articular surfaces of the third metacarpal (Mc3) and metatarsal (Mt3) bones in the Thoroughbred racehorse (Tb) is a natural model of repetitive overload arthrosis. We describe a novel pathology that affects the articular calcified cartilage (ACC) and subchondral bone (SCB) and which is associated with hyaline articular cartilage degeneration. Parasagittal slices cut from the palmar quadrant of the distal condyles of the left Mc3/Mt3 of 39 trained Tbs euthanized for welfare reasons were imaged by point projection microradiography, and backscattered electron (BSE) scanning electron microscopy (SEM), light microscopy, and confocal scanning light microscopy. Mechanical properties were studied by nanoindentation. Data on the horses' training and racing career were also collected. Highly mineralised projections were observed extending from cracks in the ACC mineralising front into the hyaline articular cartilage (HAC) up to two-thirds the thickness of the HAC, and were associated with focal HAC surface fibrillation directly overlying their site. Nanoindentation identified this extruded matrix to be stiffer than any other mineralised phase in the specimen by a factor of two. The presence of projections was associated with a higher cartilage Mankin histology score (P<0.02) and increased amounts of gross cartilage loss pathologically on the condyle (P<0.02). Presence of projections was not significantly associated with: total number of racing seasons, age of horse, amount of earnings, number of days in training, total distance galloped in career, or presence of wear lines.

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 bone mineral and matrix in response to a soft diet.

Alterations in the magnitude of habitual mechanical loads upon the skeleton may not only affect bone architecture, but also influence the nature of the bone matrix. We tested the hypothesis that changing the mechanical consistency of the diet affects both the mineral and non-mineralized moieties of bone matrix. Female rats were fed a soft diet (powdered chow as a paste), while control animals were fed the standard chow. After 8 or 20 wks, animals were killed. Cranial (mandible, maxilla, parietal, and frontal) bones and ulnae were analyzed for mineralization density by quantitative backscattered electron microscopy, and sulphated glycosaminoglycan levels with alcian blue staining were measured by microdensitometry. The soft diet group showed a significant increase in mineralization density distribution at almost all cranial sites and a reduction in alcian blue staining in alveolar bone. Altering the consistency of the diet significantly affects mineral concentration and glycosaminoglycan content of alveolar bone.

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.

Ovariectomy vs. hypofunction: their effects on rat mandibular bone.

Previous studies have suggested that the mandible may be more influenced by mechanical loading than by circulating hormone levels. We tested the hypothesis that hypofunction has a greater influence than ovariectomy on mandibular bone. Two-month-old rats were ovariectomized (OVX) or had maxillary molars removed from one side to induce unilateral mandibular hypofunction. Control animals remained untreated. After 5 months, animals were killed, and bones were assessed by micro-tomography (muCT), quantitative back-scattered electron analysis in an SEM (qBSE-SEM), and light microscopy. Mineralization density was reduced in calvarial, maxillary, and mandibular alveolar bone following OVX, yet was increased in lingual mandibular alveolar bone of the hypo-function animals compared with controls. OVX caused a reduction in osteocyte density in alveolar bone, while hypofunction showed an increase compared with controls. Hypofunction led to alveolar bone becoming more highly mineralized and more cellular, while ovariectomy caused a reduction in both mineralization density and osteocyte numbers.

Post mortem evaluation of palmar osteochondral disease (traumatic osteochondrosis) of the metacarpo/metatarsophalangeal joint in Thoroughbred racehorses.

REASONS FOR PERFORMING STUDY: Thoroughbred racehorses are commonly affected by subchondral bone injury, but the exact prevalence and the distribution of palmar/plantar osteochondral disease (POD) lesions are unknown. The relationship between pathologies has not been elucidated, although it is widely accepted that POD is a manifestation of traumatic overload arthrosis. HYPOTHESIS: There is an association between grade of POD and other pathologies affecting the third metacarpal and metatarsal (MC/MTIII) condyles (wear lines, cartilage loss, marginal remodelling, dorsal impact injuries and linear fissures). OBJECTIVES: To evaluate the pathology found affecting the distal MC/MTIII condyles of Thoroughbred racehorses at post mortem examination, to describe the prevalence and distribution of POD lesions within a population of racing Thoroughbreds and to determine relationships between pathologies of the distal condyles of the third metacarpal and metatarsal bones. METHODS: The metacarpo/metatarsophalangeal joints of 64 Thoroughbred racehorses were examined at routine post mortem examination and graded for third metacarpal and metatarsal condylar pathology. Associations between pathologies were determined. RESULTS: POD had a within horse prevalence of 67%. There was a significant linear relationship between grade of POD and grades of wear lines, cartilage ulceration and dorsal impact injuries. There was a significant relationship, but this was not linear, between grade of POD and grade of linear fissures. Using ordinal logistic regression, compared to condyles with grade 0 or grade 2 linear fissures, condyles with grade 1 linear fissures were found to be more likely to have a lower POD grade. POTENTIAL RELEVANCE: POD can be considered to be a manifestation of traumatic overload arthrosis, but the role of subchondral bone adaptation is complex and warrants further investigation.