Propagation phase-contrast micro-computed tomography allows laboratory-based three-dimensional imaging of articular cartilage down to the cellular level.

OBJECTIVE: High-resolution non-invasive three-dimensional (3D) imaging of chondrocytes in articular cartilage remains elusive. The aim of this study was to explore whether laboratory micro-computed tomography (micro-CT) permits imaging cells within articular cartilage. DESIGN: Bovine osteochondral plugs were prepared four ways: in phosphate-buffered saline (PBS) or 70% ethanol (EtOH), both with or without phosphotungstic acid (PTA) staining. Specimens were imaged with micro-CT following two protocols: 1) absorption contrast (AC) imaging 2) propagation phase-contrast (PPC) imaging. All samples were scanned in liquid. The contrast to noise ratio (C/N) of cellular features quantified scan quality and were statistically analysed. Cellular features resolved by micro-CT were validated by standard histology. RESULTS: The highest quality images were obtained using propagation phase-contrast imaging and PTA-staining in 70% EtOH. Cellular features were also visualised when stained in PBS and unstained in EtOH. Under all conditions PPC resulted in greater contrast than AC (p < 0.0001 to p = 0.038). Simultaneous imaging of cartilage and subchondral bone did not impede image quality. Corresponding features were located in both histology and micro-CT and followed the same distribution with similar density and roundness values. CONCLUSIONS: Three-dimensional visualisation and quantification of the chondrocyte population within articular cartilage can be achieved across a field of view of several millimetres using laboratory-based micro-CT. The ability to map chondrocytes in 3D opens possibilities for research in fields from skeletal development through to medical device design and treatment of cartilage degeneration.

Sexually dimorphic tibia shape is linked to natural osteoarthritis in STR/Ort mice.

OBJECTIVES: Human osteoarthritis (OA) is detected only at late stages. Male STR/Ort mice develop knee OA spontaneously with known longitudinal trajectory, offering scope to identify OA predisposing factors. We exploit the lack of overt OA in female STR/Ort and in both sexes of parental, control CBA mice to explore whether early divergence in tibial bone mass or shape are linked to emergent OA. METHOD: We undertook detailed micro-CT comparisons of trabecular and cortical bone, multiple structural/architectural parameters and finite element modelling (FEM) of the tibia from male and female STR/Ort and CBA mice at 8-10 (pre-OA), 18-20 (OA onset) and 40 + weeks (advanced OA) of age. RESULTS: We found higher trabecular bone mass in female STR/Ort than in either OA-prone male STR/Ort or non-prone CBA mice. Cortical bone, as expected, showed greater cross-sectional area in male than female CBA, which surprisingly was reversed in STR/Ort mice. STR/Ort also exhibited higher cortical bone mass than CBA mice. Our analyses revealed similar tibial ellipticity, yet greater predicted resistance to torsion in male than female CBA mice. In contrast, male STR/Ort exhibited greater ellipticity than both female STR/Ort and CBA mice at specific cortical sites. Longitudinal analysis revealed greater tibia curvature and shape deviations in male STR/Ort mice that coincided with onset and were more pronounced in late OA. CONCLUSION: Generalised higher bone mass in STR/Ort mice is more marked in non OA-prone females, but pre-OA divergence in bone shape is restricted to male STR/Ort mice in which OA develops spontaneously.

Regional diversity in the murine cortical vascular network is revealed by synchrotron X-ray tomography and is amplified with age.

Cortical bone is permeated by a system of pores, occupied by the blood supply and osteocytes. With ageing, bone mass reduction and disruption of the microstructure are associated with reduced vascular supply. Insight into the regulation of the blood supply to the bone could enhance the understanding of bone strength determinants and fracture healing. Using synchrotron radiation-based computed tomography, the distribution of vascular canals and osteocyte lacunae was assessed in murine cortical bone and the influence of age on these parameters was investigated. The tibiofibular junction from 15-week- and 10-month-old female C57BL/6J mice were imaged post-mortem. Vascular canals and three-dimensional spatial relationships between osteocyte lacunae and bone surfaces were computed for both age groups. At 15 weeks, the posterior region of the tibiofibular junction had a higher vascular canal volume density than the anterior, lateral and medial regions. Intracortical vascular networks in anterior and posterior regions were also different, with connectedness in the posterior higher than the anterior at 15 weeks. By 10 months, cortices were thinner, with cortical area fraction and vascular density reduced, but only in the posterior cortex. This provided the first evidence of age-related effects on murine bone porosity due to the location of the intracortical vasculature. Targeting the vasculature to modulate bone porosity could provide an effective way to treat degenerative bone diseases, such as osteoporosis.

The STR/ort mouse model of spontaneous osteoarthritis - an update.

Osteoarthritis is a degenerative joint disease and a world-wide healthcare burden. Characterized by cartilage degradation, subchondral bone thickening and osteophyte formation, osteoarthritis inflicts much pain and suffering, for which there are currently no disease-modifying treatments available. Mouse models of osteoarthritis are proving critical in advancing our understanding of the underpinning molecular mechanisms. The STR/ort mouse is a well-recognized model which develops a natural form of osteoarthritis very similar to the human disease. In this Review we discuss the use of the STR/ort mouse in understanding this multifactorial disease with an emphasis on recent advances in its genetics and its bone, endochondral and immune phenotypes.

The role of embryo movement in the development of the furcula.

The pectoral girdle is a complex structure which varies in its morphology between species. A major component in birds is the furcula, which can be considered equivalent to a fusion of the paired clavicles found in many mammals, and the single interclavicle found in many reptiles. These elements are a remnant of the dermal skeleton and the only intramembranous bones in the trunk. Postnatally, the furcula plays important mechanical roles by stabilising the shoulder joint and acting as a mechanical spring during flight. In line with its mechanical role, previous studies indicate that, unlike many other intramembranous bones, furcula growth during development can be influenced by mechanical stimuli. This study investigated the response of individual aspects of furcula growth to both embryo immobilisation and hypermotility in the embryonic chicken. The impact of altered incubation temperature, which influences embryo motility, on crocodilian interclavicle development was also explored. We employed whole-mount bone and cartilage staining and 3D imaging by microCT to quantify the impact of rigid paralysis, flaccid paralysis and hypermobility on furcula growth in the chicken, and 3D microCT imaging to quantify the impact of reduced temperature (32-28 °C) and motility on interclavicle growth in the crocodile. This revealed that the growth rates of the clavicular and interclavicular components of the furcula differ during normal development. Total furcula area was reduced by total unloading produced by flaccid paralysis, but not by rigid paralysis which maintains static loading of embryonic bones. This suggests that dynamic loading, which is required for postnatal bone adaptation, is not a requirement for prenatal furcula growth. Embryo hypermotility also had no impact on furcula area or arm length. Furcula 3D shape did, however, differ between groups; this was marked in the interclavicular component of the furcula, the hypocleideum. Hypocleideum length was reduced by both methods of immobilisation, and interclavicle area was reduced in crocodile embryos incubated at 28 °C, which are less motile than embryos incubated at 32 °C. These data suggest that the clavicular and interclavicle components of the avian furcula respond differently to alterations in embryo movement, with the interclavicle requiring both the static and dynamic components of movement-related loading for normal growth, while static loading preserved most aspects of clavicle growth. Our data suggest that embryo movement, and the mechanical loading this produces, is important in shaping these structures during development to suit their postnatal mechanical roles.

Prolonging disuse in aged mice amplifies cortical but not trabecular bones' response to mechanical loading.

OBJECTIVE: Short-term neurectomy-induced disuse (SN) has been shown to restore load responses in aged mice. We examined whether this restoration was further enhanced in both cortical and trabecular bone by simply extending the SN. METHODS: Following load:strain calibration, tibiae in female C57BL/J6 mice at 8, 14 and 20 weeks and 18 months (n=8/group) were loaded and bone changes measured. Effects of long-term SN examined in twenty-six 18 months-old mice, neurectomised for 5 or 100 days with/without subsequent loading. Cortical and trabecular responses were measured histomorphometrically or by micro-computed tomography. RESULTS: Loading increased new cortical bone formation, elevating cross-sectional area in 8, 14 and 20 week-old (p ⟨0.05), but not 18 month-old aged mice. Histomorphometry showed that short-term SN reinstated load-responses in aged mice, with significant 33% and 117% increases in bone accrual at 47% and 37%, but not 27% of tibia length. Cortical responses to loading was heightened and widespread, now evident at all locations, following prolonged SN (108, 167 and 98% at 47, 37 and 27% of tibial length, respectively). In contrast, loading failed to modify trabecular bone mass or architecture. CONCLUSIONS: Mechanoadaptation become deficient with ageing and prolonging disuse amplifies this response in cortical but not trabecular bone.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

The anabolic action of intermittent parathyroid hormone on cortical bone depends partly on its ability to induce nitric oxide-mediated vasorelaxation in BALB/c mice.

There is strong evidence that vasodilatory nitric oxide (NO) donors have anabolic effects on bone in humans. Parathyroid hormone (PTH), the only osteoanabolic drug currently approved, is also a vasodilator. We investigated whether the NO synthase inhibitor L-NAME might alter the effect of PTH on bone by blocking its vasodilatory effect. BALB/c mice received 28 daily injections of PTH[1-34] (80 µg/kg/day) or L-NAME (30 mg/kg/day), alone or in combination. Hindlimb blood perfusion was measured by laser Doppler imaging. Bone architecture, turnover and mechanical properties in the femur were analysed respectively by micro-CT, histomorphometry and three-point bending. PTH increased hindlimb blood flow by >30% within 10 min of injection (P < 0.001). Co-treatment with L-NAME blocked the action of PTH on blood flow, whereas L-NAME alone had no effect. PTH treatment increased femoral cortical bone volume and formation rate by 20% and 110%, respectively (P < 0.001). PTH had no effect on trabecular bone volume in the femoral metaphysis although trabecular thickness and number were increased and decreased by 25%, respectively. Co-treatment with L-NAME restricted the PTH-stimulated increase in cortical bone formation but had no clear-cut effects in trabecular bone. Co-treatment with L-NAME did not affect the mechanical strength in femurs induced by iPTH. These results suggest that NO-mediated vasorelaxation plays partly a role in the anabolic action of PTH on cortical bone.

Intermittent applied mechanical loading induces subchondral bone thickening that may be intensified locally by contiguous articular cartilage lesions.

OBJECTIVES: Changes in subchondral bone (SCB) and cross-talk with articular cartilage (AC) have been linked to osteoarthritis (OA). Using micro-computed tomography (micro-CT) this study: (1) examines changes in SCB architecture in a non-invasive loading mouse model in which focal AC lesions are induced selectively in the lateral femur, and (2) determines any modifications in the contralateral knee, linked to changes in gait, which might complicate use of this limb as an internal control. METHODS: Right knee joints of CBA mice were loaded: once with 2 weeks of habitual use (n = 7), for 2 weeks (n = 8) or for 5 weeks (n = 5). Both left (contralateral) and right (loaded) knees were micro-CT scanned and the SCB and trabecular bone analysed. Gait analysis was also performed. RESULTS: These analyses showed a significant increase in SCB thickness in the lateral compartments in joints loaded for 5 weeks, which was most marked in the lateral femur; the contralateral non-loaded knee also showed transient SCB thickening (loaded once and repetitively). Epiphyseal trabecular bone BV/TV and trabecular thickness were also increased in the lateral compartments after 5 weeks of loading, and in all joint compartments in the contralateral knee. Gait analysis showed that applied loading only affected gait in the contralateral himd-limb in all groups of mice from the second week after the first loading episode. CONCLUSIONS: These data indicate a spatial link between SCB thickening and AC lesions following mechanical trauma, and the clear limitations associated with the use of contralateral joints as controls in such OA models, and perhaps in OA diagnosis.

Non-invasive mouse models of post-traumatic osteoarthritis.

Animal models of osteoarthritis (OA) are essential tools for investigating the development of the disease on a more rapid timeline than human OA. Mice are particularly useful due to the plethora of genetically modified or inbred mouse strains available. The majority of available mouse models of OA use a joint injury or other acute insult to initiate joint degeneration, representing post-traumatic osteoarthritis (PTOA). However, no consensus exists on which injury methods are most translatable to human OA. Currently, surgical injury methods are most commonly used for studies of OA in mice; however, these methods may have confounding effects due to the surgical/invasive injury procedure itself, rather than the targeted joint injury. Non-invasive injury methods avoid this complication by mechanically inducing a joint injury externally, without breaking the skin or disrupting the joint. In this regard, non-invasive injury models may be crucial for investigating early adaptive processes initiated at the time of injury, and may be more representative of human OA in which injury is induced mechanically. A small number of non-invasive mouse models of PTOA have been described within the last few years, including intra-articular fracture of tibial subchondral bone, cyclic tibial compression loading of articular cartilage, and anterior cruciate ligament (ACL) rupture via tibial compression overload. This review describes the methods used to induce joint injury in each of these non-invasive models, and presents the findings of studies utilizing these models. Altogether, these non-invasive mouse models represent a unique and important spectrum of animal models for studying different aspects of PTOA.

Local origins impart conserved bone type-related differences in human osteoblast behaviour.

Osteogenic behaviour of osteoblasts from trabecular, cortical and subchondral bone were examined to determine any bone type-selective differences in samples from both osteoarthritic (OA) and osteoporotic (OP) patients. Cell growth, differentiation; alkaline phosphatase (TNAP) mRNA and activity, Runt-related transcription factor-2 (RUNX2), SP7-transcription factor (SP7), bone sialoprotein-II (BSP-II), osteocalcin/bone gamma-carboxyglutamate (BGLAP), osteoprotegerin (OPG, TNFRSF11B), receptor activator of nuclear factor-κß ligand (RANKL, TNFSF11) mRNA levels and proangiogenic vascular endothelial growth factor-A (VEGF-A) mRNA and protein release were assessed in osteoblasts from paired humeral head samples from age-matched, human OA/OP (n = 5/4) patients. Initial outgrowth and increase in cell number were significantly faster (p < 0.01) in subchondral and cortical than trabecular osteoblasts, in OA and OP, and this bone type-related differences were conserved despite consistently faster growth in OA. RUNX2/SP7 levels and TNAP mRNA and protein activity were, however, greater in trabecular than subchondral and cortical osteoblasts in OA and OP. BSP-II levels were significantly greater in trabecular and lowest in cortical osteoblasts in both OA and OP. In contrast, BGLAP levels showed divergent bone type-selective behaviour; highest in osteoblasts from subchondral origins in OA and trabecular origins in OP. We found virtually identical bone type-related differences, however, in TNFRSF11B:TNFSF11 in OA and OP, consistent with greater potential for paracrine effects on osteoclasts in trabecular osteoblasts. Subchondral osteoblasts (OA) exhibited highest VEGF-A mRNA levels and release. Our data indicate that human osteoblasts in trabecular, subchondral and cortical bone have inherent, programmed diversity, with specific bone type-related differences in growth, differentiation and pro-angiogenic potential in vitro.

Mechanoadaptation of developing limbs: shaking a leg.

The proportion of total limb length taken up by the individual skeletal elements (limb proportionality), varies widely between species. These diverse skeletal forms have evolved to allow for a range of limb uses and they first emerge as the embryo develops, to achieve the characteristic skeletal architecture of each species. During this time, the developing skeleton experiences mechanical loading as a result of embryonic muscle contraction. The possibility that adaptation to such mechanical input may allow embryos to coordinate the appearance of skeletal design with their expanding range of movements has so far received little attention. This is surprising, given the critical role exerted by embryo movement in normal skeletal development; stage-specific in ovo immobilisation of embryonic chicks results in joint contractures and a reduction in longitudinal bone growth in the limbs. Epigenetic mechanisms allow for selective activation of genes in response to environmental signals, resulting in the production of phenotypic complexity in morphogenesis; mechanical loading of bone during movement appears to be one such signal. It may be that 'mechanosensitive' genes under regulation of mechanical input adjust proportionality along the bone's proximo-distal axis, introducing a level of phenotypic plasticity. If this hypothesis is upheld, species with more elongated distal limb elements will have a greater dependence on mechanical input for the differences in their growth, and mechanosensitive bone growth in the embryo may have evolved as an additional source of phenotypic diversity during skeletal development.

Spontaneous osteoarthritis in Str/ort mice is unlikely due to greater vulnerability to mechanical trauma.

OBJECTIVE: Relative contributions of genetic and mechanical factors to osteoarthritis (OA) remain ill-defined. We have used a joint loading model found to produce focal articular cartilage (AC) lesions, to address whether genetic susceptibility to OA in Str/ort mice is related to AC vulnerability to mechanical trauma and whether joint loading influences spontaneous OA development. We also develop finite element (FE) models to examine whether AC thickness may explain any differential vulnerability to load-induced lesions. METHODS: Right knees of 8-week-old Str/ort mice were loaded, AC integrity scored and thickness compared to CBA mice. Mechanical forces engendered in this model and the impact of AC thickness were simulated in C57Bl/6 mice using quasi-static FE modelling. RESULTS: Unlike joints in non-OA prone CBA mice, Str/ort knees did not exhibit lateral femur (LF) lesions in response to applied loading; but exhibited thicker AC. FE modeling showed increased contact pressure and shear on the lateral femoral surface in loaded joints, and these diminished in joints containing thicker AC. Histological analysis of natural lesions in the tibia of Str/ort joints revealed that applied loading increased OA severity, proteoglycan loss and collagen type II degradation. CONCLUSION: Genetic OA susceptibility in Str/ort mice is not apparently related to greater AC vulnerability to trauma, but joint loading modifies severity of natural OA lesions in the medial tibia. FE modelling suggests that thicker AC in Str/ort mice diminishes tissue stresses and protects against load-induced AC lesions in the LF but that this is unrelated to their genetic susceptibility to OA.

Resolving trabecular metaphyseal bone profiles downstream of the growth plate adds value to bone histomorphometry in mouse models.

Introduction: Histomorphometry of rodent metaphyseal trabecular bone, by histology or microCT, is generally restricted to the mature secondary spongiosa, excluding the primary spongiosa nearest the growth plate by imposing an 'offset'. This analyses the bulk static properties of a defined segment of secondary spongiosa, usually regardless of proximity to the growth plate. Here we assess the value of trabecular morphometry that is spatially resolved according to the distance 'downstream' of-and thus time since formation at-the growth plate. Pursuant to this, we also investigate the validity of including mixed primary-secondary spongiosal trabecular bone, extending the analysed volume 'upstream' by reducing the offset. Both the addition of spatiotemporal resolution and the extension of the analysed volume have potential to enhance the sensitivity of detection of trabecular changes and to resolve changes occurring at different times and locations. Method: Two experimental mouse studies of trabecular bone are used as examples of different factors influencing metaphyseal trabecular bone: (1) ovariectomy (OVX) and pharmacological prevention of osteopenia and (2) limb disuse induced by sciatic neurectomy (SN). In a third study into offset rescaling, we also examine the relationship between age, tibia length, and primary spongiosal thickness. Results: Bone changes induced by either OVX or SN that were early or weak and marginal were more pronounced in the mixed primary-secondary upstream spongiosal region than in the downstream secondary spongiosa. A spatially resolved evaluation of the entire trabecular region found that significant differences between experimental and control bones remained undiminished either right up to or to within 100 µm from the growth plate. Intriguingly, our data revealed a remarkably linear downstream profile for fractal dimension in trabecular bone, arguing for an underlying homogeneity of the (re)modelling process throughout the entire metaphysis and against strict anatomical categorization into primary and secondary spongiosal regions. Finally, we find that a correlation between tibia length and primary spongiosal depth is well conserved except in very early and late life. Conclusions: These data indicate that the spatially resolved analysis of metaphyseal trabecular bone at different distances from the growth plate and/or times since formation adds a valuable dimension to histomorphometric analysis. They also question any rationale for rejecting primary spongiosal bone, in principle, from metaphyseal trabecular morphometry.

Expression of endothelial nitric oxide synthase protein is not necessary for mechanical strain-induced nitric oxide production by cultured osteoblasts.

UNLABELLED: Regulation of nitric oxide (NO) production is considered essential in mechanical load-related osteogenesis. We examined whether osteoblast endothelial NO synthase (eNOS)-derived NO production was regulated by HSP90. We found that HSP90 is essential for strain-related NO release but appears to be independent of eNOS in cultured osteoblasts. INTRODUCTION: NO is a key regulator of bone mass, and its production by bone cells is regarded as essential in mechanical strain-related osteogenesis. We sought to identify whether bone cell NO production relied upon eNOS, considered to be the predominant NOS isoform in bone, and whether this was regulated by an HSP90-dependent mechanism. METHODS: Using primary rat long bone-derived osteoblasts, the ROS 17/2.8 cell line and primary mouse osteoblasts, derived from wild-type and eNOS-deficient (eNOS(-/-)) mice, we examined by immunoblotting the expression of eNOS using a range of well-characterised antibodies and extraction methods, measured NOS activity by monitoring the conversion of radiolabelled L-arginine to citrulline and examined the production of NO by bone cells subjected to mechanical strain application under various conditions. RESULTS: Our studies have revealed that eNOS protein and activity were both undetectable in osteoblast-like cells, that mechanical strain-induced NO production was retained in bone cells from eNOS-deficient mice, but that this strain-related induction of NO production was, however, dependent upon HSP90. CONCLUSIONS: Together, our studies indicate that HSP90 activity is essential for strain-related NO release by cultured osteoblasts and that this is highly likely to be achieved by an eNOS-independent mechanism.

Regional variations in discrete collagen fibre mechanics within intact intervertebral disc resolved using synchrotron computed tomography and digital volume correlation.

Many soft tissues, such as the intervertebral disc (IVD), have a hierarchical fibrous composite structure which suffers from regional damage. We hypothesise that these tissue regions have distinct, inherent fibre structure and structural response upon loading. Here we used synchrotron computed tomography (sCT) to resolve collagen fibre bundles (∼5µm width) in 3D throughout an intact native rat lumbar IVD under increasing compressive load. Using intact samples meant that tissue boundaries (such as endplate-disc or nucleus-annulus) and residual strain were preserved; this is vital for characterising both the inherent structure and structural changes upon loading in tissue regions functioning in a near-native environment. Nano-scale displacement measurements along >10,000 individual fibres were tracked, and fibre orientation, curvature and strain changes were compared between the posterior-lateral region and the anterior region. These methods can be widely applied to other soft tissues, to identify fibre structures which cause tissue regions to be more susceptible to injury and degeneration. Our results demonstrate for the first time that highly-localised changes in fibre orientation, curvature and strain indicate differences in regional strain transfer and mechanical function (e.g. tissue compliance). This included decreased fibre reorientation at higher loads, specific tissue morphology which reduced capacity for flexibility and high strain at the disc-endplate boundary. STATEMENT OF SIGNIFICANCE: The analyses presented here are applicable to many collagenous soft tissues which suffer from regional damage. We aimed to investigate regional intervertebral disc (IVD) structural and functional differences by characterising collagen fibre architecture and linking specific fibre- and tissue-level deformation behaviours. Synchrotron CT provided the first demonstration of tracking discrete fibres in 3D within an intact IVD. Detailed analysis of regions was performed using over 200k points, spaced every 8 µm along 10k individual fibres. Such comprehensive structural characterisation is significant in informing future computational models. Morphological indicators of tissue compliance (change in fibre curvature and orientation) and fibre strain measurements revealed localised and regional differences in tissue behaviour.

Increased bone mass, altered trabecular architecture and modified growth plate organization in the growing skeleton of SOCS2 deficient mice.

Suppressor of cytokine signalling-2 (SOCS2) negatively regulates the signal transduction of several cytokines. Socs2(-/-) mice show increased longitudinal skeletal growth associated with deregulated GH/IGF-1 signalling. The present study examined the role of SOCS2 in endochondral ossification and trabecular and cortical bone formation, and investigated whether pro-inflammatory cytokines associated with pediatric chronic inflammatory disorders mediate their effects through SOCS2. Seven-week-old Socs2(-/-) mice were heavier (27%; P < 0.001) and longer (6%; P < 0.001) than wild-type mice. Socs2(-/-) tibiae were longer (8%; P < 0.001) and broader (18%; P < 0.001) than that of wild-type mice, and the Socs2(-/-) mice had wider growth plates (24%; P < 0.001) with wider proliferative and hypertrophic zones (10% (P < 0.05) and 14% (P < 0.001) respectively). Socs2(-/-) mice showed increased total cross-sectional bone area (16%: P < 0.001), coupled to increased total tissue area (17%; P < 0.05) compared to tibia from wild-type mice. Socs2(-/-) mice showed increased percent bone volume (101%; P < 0.001), trabecular number (82%; P < 0.001) and trabecular thickness (11%; P < 0.001), with associated decreases in trabecular separation (19%; P < 0.001). TNFalpha exposure to growth plate chondrocytes for 48 h increased SOCS2 protein expression. Growth of metatarsals from 1-day-old Socs2(-/-) and Socs2(+/+) mice, as well as expression of Aggrecan, Collagen Type II and Collagen Type X, were inhibited by TNFalpha, with no effect of genotype. Our data indicate that physiological levels of SOCS2 negatively regulate bone formation and endochondral growth. Our results further suggest that pro-inflammatory cytokines mediate their inhibitory effects on longitudinal bone growth through a mechanism that is independent of SOCS2.

Spatial relationship between bone formation and mechanical stimulus within cortical bone: Combining 3D fluorochrome mapping and poroelastic finite element modelling.

Bone is a dynamic tissue and adapts its architecture in response to biological and mechanical factors. Here we investigate how cortical bone formation is spatially controlled by the local mechanical environment in the murine tibia axial loading model (C57BL/6). We obtained 3D locations of new bone formation by performing 'slice and view' 3D fluorochrome mapping of the entire bone and compared these sites with the regions of high fluid velocity or strain energy density estimated using a finite element model, validated with ex-vivo bone surface strain map acquired ex-vivo using digital image correlation. For the comparison, 2D maps of the average bone formation and peak mechanical stimulus on the tibial endosteal and periosteal surface across the entire cortical surface were created. Results showed that bone formed on the periosteal and endosteal surface in regions of high fluid flow. Peak strain energy density predicted only the formation of bone periosteally. Understanding how the mechanical stimuli spatially relates with regions of cortical bone formation in response to loading will eventually guide loading regime therapies to maintain or restore bone mass in specific sites in skeletal pathologies.

Limb proportions show developmental plasticity in response to embryo movement.

Animals have evolved limb proportions adapted to different environments, but it is not yet clear to what extent these proportions are directly influenced by the environment during prenatal development. The developing skeleton experiences mechanical loading resulting from embryo movement. We tested the hypothesis that environmentally-induced changes in prenatal movement influence embryonic limb growth to alter proportions. We show that incubation temperature influences motility and limb bone growth in West African Dwarf crocodiles, producing altered limb proportions which may, influence post-hatching performance. Pharmacological immobilisation of embryonic chickens revealed that altered motility, independent of temperature, may underpin this growth regulation. Use of the chick also allowed us to merge histological, immunochemical and cell proliferation labelling studies to evaluate changes in growth plate organisation, and unbiased array profiling to identify specific cellular and transcriptional targets of embryo movement. This disclosed that movement alters limb proportions and regulates chondrocyte proliferation in only specific growth plates. This selective targeting is related to intrinsic mTOR (mechanistic target of rapamycin) pathway activity in individual growth plates. Our findings provide new insights into how environmental factors can be integrated to influence cellular activity in growing bones and ultimately gross limb morphology, to generate phenotypic variation during prenatal development.

Cartilage oligomeric matrix protein and hyaluronan levels in synovial fluid from horses with osteoarthritis of the tarsometatarsal joint compared to a control population.

REASONS FOR PERFORMING STUDY: Quantification of cartilage oligomeric matrix protein (COMP) levels within synovial fluid from the tarsometatarsal joint has not previously been reported and an effective synovial fluid marker would allow monitoring of disease progression and treatment. OBJECTIVES: To quantify levels of COMP and hyaluronan (HA) in synovial fluid from the tarsometatarsal joint, identify differences in levels from horses with osteoarthritis (OA) of the tarsometatarsal joint compared to a control population and to correlate levels with radiographic changes in horses with OA. METHODS: Synovial fluid was collected from the tarsometatarsal joint of 25 horses without hindlimb lameness (controls) and 25 lame horses, subjected to analgesia of the joint. COMP concentrations were measured using a homologous inhibition ELISA. Immunoblots of synovial fluid from 3 lame horses and 3 controls were performed to identify fragmentation of COMP. Hyaluronan (HA) concentration in synovial fluid was determined using a competition ELISA. Radiographs of the lame horses with OA were scored and correlated with levels of COMP and HA. RESULTS: Concentrations of COMP in OA of the tarsometatarsal joint were significantly lower than in the control samples. An additional fragment band of COMP (approximately 30 kDa) was identified on the immunoblots of the horses with OA and this fragment was not identified in controls. No significant difference was identified in the HA or HA:COMP ratio between lame and control horses. There was no correlation between levels of synovial fluid COMP and HA, and radiographic changes. CONCLUSIONS AND POTENTIAL RELEVANCE: Lowered levels of COMP in synovial fluid of tarsometatarsal joints correlates with the presence of osteoarthritis. However, a single value cannot be used to stage the disease process. Levels of HA may not be a useful marker for this disease. Decreased, rather than increased COMP levels, may reflect significant loss of cartilage in established osteoarthritis. A specific assay for the COMP fragment generated with osteoarthritis may allow the earlier detection of clinical cases.