Assessment of subchondral bone microdamage quantification using contrast-enhanced imaging techniques.

Bone microdamage is common at subchondral bone (SCB) sites subjected to repeated high rate and magnitude of loading in the limbs of athletic animals and humans. Microdamage can affect the biomechanical behaviour of bone under physiological loading conditions. To understand the effects of microdamage on the mechanical properties of SCB, it is important to be able to quantify it. The extent of SCB microdamage had been previously estimated qualitatively using plain microcomputed tomography (µCT) and a radiocontrast quantification method has been used for trabecular bone but this method may not be directly applicable to SCB due to differences in bone structure. In the current study, SCB microdamage detection using lead uranyl acetate (LUA) and quantification by contrast-enhanced µCT and backscattered scanning electron microscopy (SEM) imaging techniques were assessed to determine the specificity of the labels to microdamage and the accuracy of damaged bone volume metrices. SCB specimens from the metacarpus of racehorses, with the hyaline articular cartilage (HAC) removed, were grouped into two with one group subjected to ex vivo uniaxial compression loading to create experimental bone damage. The other group was not loaded to preserve the pre-existing in vivo propagated bone microdamage. A subset of each group was stained with LUA using an established or a modified protocol to determine label penetration into SCB. The µCT and SEM images of stained specimens showed that penetration of LUA into the SCB was better using the modified protocol, and this protocol was repeated in SCB specimens with intact hyaline articular cartilage. The percentage of total label localised to bone microdamage was determined on SEM images, and the estimated labelled bone volume determined by µCT in SCB groups was compared. Label was present around diffuse and linear microdamage as well as oblique linear microcracks present at the articular surface, except in microcracks with high-density mineral infills. Bone surfaces lining pores with recent mineralisation were also labelled. Labelled bone volume fraction (LV/BV) estimated by µCT was higher in the absence of HAC. At least 50% of total labels were localised to bone microdamage when the bone area fraction (B.Ar/T.Ar) of the SCB was greater than 0.85 but less than 30% when B.Ar/T.Ar of the SCB was less than 0.85. To adjust for LUA labels on bone surfaces, a measure of the LV/BV corrected for bone surface area (LV/BV BS-1) was used to quantify damaged SCB. In conclusion, removal of HAC and using a modified labelling protocol effectively stained damaged SCB of the metacarpus of racehorses and represents a technique useful for quantifying microdamage in SCB. This method can facilitate future investigations of the effects of microdamage on joint physiology.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Chondrocytic ephrin B2 promotes cartilage destruction by osteoclasts in endochondral ossification.

The majority of the skeleton arises by endochondral ossification, whereby cartilaginous templates expand and are resorbed by osteoclasts then replaced by osteoblastic bone formation. Ephrin B2 is a receptor tyrosine kinase expressed by osteoblasts and growth plate chondrocytes that promotes osteoblast differentiation and inhibits osteoclast formation. We investigated the role of ephrin B2 in endochondral ossification using Osx1Cre-targeted gene deletion. Neonatal Osx1Cre.Efnb2(Δ/Δ) mice exhibited a transient osteopetrosis demonstrated by increased trabecular bone volume with a high content of growth plate cartilage remnants and increased cortical thickness, but normal osteoclast numbers within the primary spongiosa. Osteoclasts at the growth plate had an abnormal morphology and expressed low levels of tartrate-resistant acid phosphatase; this was not observed in more mature bone. Electron microscopy revealed a lack of sealing zones and poor attachment of Osx1Cre.Efnb2(Δ/Δ) osteoclasts to growth plate cartilage. Osteoblasts at the growth plate were also poorly attached and impaired in their ability to deposit osteoid. By 6 months of age, trabecular bone mass, osteoclast morphology and osteoid deposition by Osx1Cre.Efnb2(Δ/Δ) osteoblasts were normal. Cultured chondrocytes from Osx1Cre.Efnb2(Δ/Δ) neonates showed impaired support of osteoclastogenesis but no significant change in Rankl (Tnfsf11) levels, whereas Adamts4 levels were significantly reduced. A population of ADAMTS4(+) early hypertrophic chondrocytes seen in controls was absent from Osx1Cre.Efnb2(Δ/Δ) neonates. This suggests that Osx1Cre-expressing cells, including hypertrophic chondrocytes, are dependent on ephrin B2 for their production of cartilage-degrading enzymes, including ADAMTS4, and this might be required for attachment of osteoclasts and osteoblasts to the cartilage surface during endochondral ossification.

Keratinocyte-specific ablation of protease-activated receptor 2 prevents gingival inflammation and bone loss in a mouse model of periodontal disease.

Chronic periodontitis is characterised by gingival inflammation and alveolar bone loss. A major aetiological agent is Porphyromonas gingivalis, which secretes proteases that activate protease-activated receptor 2 (PAR2 ). PAR2 expressed on oral keratinocytes is activated by proteases released by P. gingivalis, inducing secretion of interleukin 6 (IL-6), and global knockout of PAR2 prevents bone loss and inflammation in a periodontal disease model in mice. To test the hypothesis that PAR2 expressed on gingival keratinocytes is required for periodontal disease pathology, keratinocyte-specific PAR2 -null mice were generated using K14-Cre targeted deletion of the PAR2 gene (F2rl1). These mice were subjected to a model of periodontitis involving placement of a ligature around a tooth, combined with P. gingivalis infection ("Lig + Inf"). The intervention caused a significant 44% decrease in alveolar bone volume (assessed by microcomputed tomography) in wildtype (K14-Cre:F2rl1wt/wt ), but not littermate keratinocyte-specific PAR2 -null (K14-Cre:F2rl1fl/fl ) mice. Keratinocyte-specific ablation of PAR2 prevented the significant Lig + Inf-induced increase (2.8-fold) in the number of osteoclasts in alveolar bone and the significant up-regulation (2.4-4-fold) of the inflammatory markers IL-6, IL-1ß, interferon-γ, myeloperoxidase, and CD11b in gingival tissue. These data suggest that PAR2 expressed on oral epithelial cells is a critical regulator of periodontitis-induced bone loss and will help in designing novel therapies with which to treat the disease.

The antiepileptic medications carbamazepine and phenytoin inhibit native sodium currents in murine osteoblasts.

OBJECTIVE: Fracture risk is a serious comorbidity in epilepsy and may relate to the use of antiepileptic drugs (AEDs). Many AEDs inhibit ion channel function, and the expression of these channels in osteoblasts raises the question of whether altered bone signaling increases bone fragility. We aimed to confirm the expression of voltage-gated sodium (NaV ) channels in mouse osteoblasts, and to investigate the action of carbamazepine and phenytoin on NaV channels. METHODS: Immunocytochemistry was performed on primary calvarial osteoblasts extracted from neonatal C57BL/6J mice and additional RNA sequencing (RNASeq) was included to confirm expression of NaV . Whole-cell patch-clamp recordings were made to identify the native currents expressed and to assess the actions of carbamazepine (50 µm) or phenytoin (50 µm). RESULTS: NaV expression was demonstrated with immunocytochemistry, RNA sequencing, and functionally, with demonstration of robust tetrodotoxin-sensitive and voltage-activated inward currents. Application of carbamazepine or phenytoin resulted in significant inhibition of current amplitude for carbamazepine (31.6 ± 5.9%, n = 9; p < 0.001), and for phenytoin (35.5 ± 6.9%, n = 7; p < 0.001). SIGNIFICANCE: Mouse osteoblasts express NaV , and native NaV currents are blocked by carbamazepine and phenytoin, supporting our hypothesis that AEDs can directly influence osteoblast function and potentially affect bone strength.

EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis.

Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the ephrinB2/EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast-specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2-fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex- and age-matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2-fold increase in annexin V staining and 7-fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.

Hyaluronan synthesis and myogenesis: a requirement for hyaluronan synthesis during myogenic differentiation independent of pericellular matrix formation.

Exogenous hyaluronan is known to alter muscle precursor cell proliferation, migration, and differentiation, ultimately inhibiting myogenesis in vitro. The aim of the current study was to investigate the role of endogenous hyaluronan synthesis during myogenesis. In quantitative PCR studies, the genes responsible for synthesizing hyaluronan were found to be differentially regulated during muscle growth, repair, and pathology. Although all Has genes (Has1, Has2, and Has3) were differentially regulated in these models, only Has2 gene expression consistently associated with myogenic differentiation. During myogenic differentiation in vitro, Has2 was the most highly expressed of the synthases and increased after induction of differentiation. To test whether this association between Has2 expression and myogenesis relates to a role for Has2 in myoblast differentiation and fusion, C2C12 myoblasts were depleted of Has2 by siRNA and induced to differentiate. Depletion of Has2 inhibited differentiation and caused a loss of cell-associated hyaluronan and the hyaluronan-dependent pericellular matrix. The inhibition of differentiation caused by loss of hyaluronan was confirmed with the hyaluronan synthesis inhibitor 4-methylumbelliferone. In hyaluronan synthesis-blocked cultures, restoration of the pericellular matrix could be achieved through the addition of exogenous hyaluronan and the proteoglycan versican, but this was not sufficient to restore differentiation to control levels. These data indicate that intrinsic hyaluronan synthesis is necessary for myoblasts to differentiate and form syncytial muscle cells, but the hyaluronan-dependent pericellular matrix is not sufficient to support differentiation alone; additional hyaluronan-dependent cell functions that are yet unknown may be required for myogenic differentiation.

Contractile properties of slow and fast skeletal muscles from protease activated receptor-1 null mice.

INTRODUCTION: Protease-activated receptors (PARs) may play a role in skeletal muscle development. We compared the contractile properties of slow-twitch soleus muscles and fast-twitch extensor digitorum longus (EDL) muscles from PAR-1 null and littermate control mice. METHODS: Contractile function was measured using a force transducer system. Fiber type proportions were determined using immunohistochemistry. RESULTS: Soleus muscles from PAR-1 null mice exhibited longer contraction times, a leftward shift in the force-stimulation frequency relationship, and decreased fatiguability compared with controls. PAR-1 null soleus muscles also had increased type 1 and decreased type IIb/x fiber numbers compared with controls. In PAR-1 null EDL muscles, no differences were found, except for a slower rate of fatigue compared with controls. CONCLUSIONS: The absence of PAR-1 results in a slower skeletal muscle contractile phenotype, likely due to an increase in type I and a decrease in type IIb/x fiber numbers. Muscle Nerve 50: 991-998, 2014.

Differences in bone turnover markers and injury risks between local and international horses: A Victorian Spring Racing Carnival study.

BACKGROUND: Musculoskeletal injuries (MSI) are common in racehorses and have been of increasing concern in horses travelling internationally to compete. Understanding the differences in bone turnover between local horses and international horses following long-distance air transportation may inform MSI prevention strategies. OBJECTIVES: To understand the differences in bone turnover markers and risk of MSI between local horses and international horses following long-distance air transportation. STUDY DESIGN: Prospective cohort. METHODS: The concentrations of bone turnover markers (OCN and CTXI), markers of stress (cortisol), inflammation (serum amyloid A) and circadian rhythm (melatonin), and bisphosphonates were determined in blood samples collected twice (14-17 days apart), from horses following international travel (n = 69), and from local horses (n = 79). The associations between markers, long-distance travel and MSI were determined using multivariable generalised linear regression models. RESULTS: Within 3-5 days post-transport, concentrations of cortisol in international horses were higher than those of local horses (main effect, Coef. 0.39; 95% CI 0.24, 0.54; p < 0.001) but they decreased and were not different to those of local horses at the second timepoint (interaction effect, Coef. -0.27; 95% CI -0.46, -0.07; p = 0.007). After adjusting for age and sex, OCN and CTXI were not significantly different between international and local horses; however, OCN was lower in international horses at timepoint 2 (interaction effect, Coef. -0.16; 95% CI -0.31, -0.01; p = 0.043). The prevalence of MSI was higher in the international (26%; 95% CI 16, 38%) compared with local horses (8%; 95% CI 3, 16%; p < 0.001), with all severe MSI sustained by the international horses. At the second timepoint compared with the first timepoint post-transport, cortisol remained high or increased (interaction effect, Coef. 0.43; 95% CI 0.24, 0.61; p < 0.001) and OCN increased (interaction effect, Coef. 0.26; 95% CI 0.08, 0.44; p = 0.006) in the horses that sustained severe MSI. MAIN LIMITATIONS: Horse population and racing career parameters differed between groups. Bone turnover markers have low sensitivity to detect local bone changes. CONCLUSIONS: Most horses showed minimal effects of long-distance air transport within 2 weeks relative to local horses as assessed by stress and bone turnover markers. Screening for persistent high cortisol and evidence of net bone formation after long-distance air transportation may help to identify racehorses at high risk of catastrophic MSI.

CONTEXTE: Les blessures musculosquelettiques (MSI) sont communes chez les chevaux de course et demeurent une source d'inquiétude pour les chevaux voyageant à l'international. Comprendre les différences de remodelage osseux entre les chevaux locaux et ceux voyageant suivant un trajet aérien longue distance pourrait aider au développement de stratégies de prévention des dommages musculosquelettiques. OBJECTIFS: Comprendre les différences de marqueurs de remodelage osseux et de risques de MSI entre les chevaux locaux et ceux voyageant à l'international suivant un transport aérien de longue distance. TYPE D'ÉTUDE: Étude de cohorte prospective. MÉTHODES: Les concentrations des marqueurs de remodelage osseux (OCN et CTXI), de stress (cortisol), d'inflammation (serum amyloid A), de rythme circadien (melatonin) et les bisphosphonates ont été mesurés dans des échantillons sanguins à deux reprises (14­17 jours à part) chez des chevaux ayant été à l'international (n = 69) et étant restés localement (n = 79). L'association entre les marqueurs, le transport longue distance et les MSI a été déterminée par modèles de régression linéaire multivarié généralisé. RÉSULTATS: Entre 3 à 5 jours suivant le transport, les concentrations de cortisol chez les chevaux internationaux étaient supérieures aux chevaux locaux (effet primaire, Coef. 0.39; 95% CI 0.24, 0.54; P < 0.001), mais ont diminué par la suite jusqu'à ne plus être différent de ceux des chevaux locaux à la deuxième mesure (effet interaction, Coef. −0.27; 95% CI −0.46, −0.07; P = 0.007). Après ajustement pour l'âge et le sexe, OCN et CTXI n'étaient pas significativement différents entre les chevaux internationaux et locaux. Cependant, OCN était inférieur chez les chevaux internationaux à la deuxième mesure (effet interaction, Coef. −0.16; 95% CI −0.31, −0.01; P = 0.043). La prévalence de MSI était plus élevée chez les chevaux internationaux (26%; 95% CI 16, 38%) comparativement aux chevaux locaux (8%; 95% CI 3, 16%; p < 0.001), avec toutes les MSI sévères subi par les chevaux internationaux. Au moment de la deuxième mesure comparée à la première mesure après le transport, le cortisol est demeuré élevé ou a augmenté (effet interaction, Coef. 0.43; 95% CI 0.24, 0.61; P < 0.001) et l'OCN a augmenté (effet interaction, Coef. 0.26; 95% CI 0.08, 0.44; P = 0.006) chez les chevaux ayant subi une MSI sévère. LIMITES PRINCIPALES: La population équine et leurs paramètres de course diffèrent entre les groupes. Les marqueurs de remodelage osseux ont une faible sensibilité pour la détection de changements osseux localisés. CONCLUSION: En deux semaines, les effets de transport aérien longue distance ont été minimaux pour la majorité des chevaux comparativement aux chevaux locaux, tel que démontré par les marqueurs de stress et de remodelage osseux. La détection de niveau élevé de cortisol de façon persistante et d'évidence d'os néoformé suivant un transport aérien de longue distance pourrait aider à détecter les chevaux de course à haut risque de MSI.

Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics.

Articular cartilage is indispensable for joint function but has limited capacity for self-repair. Engineering of neocartilage in vitro is therefore a major target for autologous cartilage repair in arthritis. Previous analysis of neocartilage has targeted cellular organization and specific molecular components. However, the complexity of extracellular matrix (ECM) development in neocartilage has not been investigated by proteomics. To redress this, we developed a mouse neocartilage culture system that produces a cartilaginous ECM. Differential analysis of the tissue proteome of 3-week neocartilage and 3-day postnatal mouse cartilage using solubility-based protein fractionation targeted components involved in neocartilage development, including ECM maturation. Initially, SDS-PAGE analysis of sequential extracts revealed the transition in protein solubility from a high proportion of readily soluble (NaCl-extracted) proteins in juvenile cartilage to a high proportion of poorly soluble (guanidine hydrochloride-extracted) proteins in neocartilage. Label-free quantitative mass spectrometry (LTQ-Orbitrap) and statistical analysis were then used to filter three significant protein groups: proteins enriched according to extraction condition, proteins differentially abundant between juvenile cartilage and neocartilage, and proteins with differential solubility properties between the two tissue types. Classification of proteins differentially abundant between NaCl and guanidine hydrochloride extracts (n = 403) using bioinformatics revealed effective partitioning of readily soluble components from subunits of larger protein complexes. Proteins significantly enriched in neocartilage (n = 78) included proteins previously not reported or with unknown function in cartilage (integrin-binding protein DEL1; coiled-coil domain-containing protein 80; emilin-1 and pigment epithelium derived factor). Proteins with differential extractability between juvenile cartilage and neocartilage included ECM components (nidogen-2, perlecan, collagen VI, matrilin-3, tenascin and thrombospondin-1), and the relationship between protein extractability and ECM ultrastructural organization was supported by electron microscopy. Additionally, one guanidine extract-specific neocartilage protein, protease nexin-1, was confirmed by immunohistochemistry as a novel component of developing articular cartilage in vivo. The extraction profile and matrix-associated immunostaining implicates protease nexin-1 in cartilage development in vitro and in vivo.

Intracortical remodelling and porosity in the distal radius and post-mortem femurs of women: a cross-sectional study.

BACKGROUND: Osteoporosis research has focused on vertebral fractures and trabecular bone loss. However, non-vertebral fractures at predominantly cortical sites account for 80% of all fractures and most fracture-related morbidity and mortality in old age. We aimed to re-examine cortical bone as a source of bone loss in the appendicular skeleton. METHODS: In this cross-sectional study, we used high-resolution peripheral CT to quantify and compare cortical and trabecular bone loss from the distal radius of adult women, and measured porosity using scanning electron microscopy. Exclusion criteria were diseases or prescribed drugs affecting bone metabolism. We also measured bone mineral density of post-mortem hip specimens from female cadavers using densitometry. Age-related differences in total, cortical, and trabecular bone mass, trabecular bone of cortical origin, and cortical and trabecular densities were calculated. FINDINGS: We investigated 122 white women with a mean age of 62.8 (range 27-98) years. Between ages 50 and 80 years (n=89), 72.1 mg (95% CI 67.7-76.4) hydroxyapatite (68%) of 106.5 mg hydroxyapatite of bone lost at the distal radius was cortical and 34.3 mg (30.5-37.8) hydroxyapatite (32%) was trabecular; 17.1 mg (11.7-22.5) hydroxyapatite (16%) of total bone loss occurred between ages 50 and 64 years (n=34) and 89.4 mg (83.7-101.1) hydroxyapatite (84%) after age 65 years (n=55). Remodelling within cortex adjacent to the marrow accounted for 49.9 mg (45.4-53.7) hydroxyapatite (47%) of bone loss. Between ages 50-64 years (n=34) and 80 years and older (n=33), cortical density decreased by 127.8 mg (93.1-162.1) hydroxyapatite per cm(3) (15%, p<0.0001) before porosity trabecularising the cortex was included, but 374.3 mg (318.2-429.5) hydroxyapatite per cm(3) (43%, p<0.0001) after; trabecular density decreased by 18.2 mg (-1.4 to 38.2) hydroxyapatite per cm(3) (14%, p=0.06) before cortical remnants were excluded, but 68.7 mg (37.7-90.4) hydroxyapatite per cm(3) (52%, p<0.0001) after. INTERPRETATION: Accurate assessment of bone structure, especially porosity producing cortical remnants, could improve identification of individuals at high and low risk of fracture and therefore assist targeting of treatment. FUNDING: Australia National Health and Medical Research Council.

Protease-activated receptor-1 down-regulates the murine inflammatory and humoral response to Helicobacter pylori.

BACKGROUND & AIMS: Helicobacter pylori infection results in a diversity of pathologies, from asymptomatic gastritis to adenocarcinoma. The reason for these diverse outcomes is multifactorial and includes host factors that regulate severity of Helicobacter-induced gastritis. Protease-activated receptors (PAR) are environmental sensors that can detect tissue damage and pathogens. Whereas PAR-2 has proinflammatory activity and PAR-1 can protect the gastric mucosa against chemical damage, neither has previously been examined for their potential roles in regulating Helicobacter pathogenesis. METHODS: PAR-1(-/-), PAR-2(-/-), and wild-type mice were infected with H pylori for up to 2 months then colonization levels determined by colony-forming assay, gastritis by histology, and serum antibody levels by enzyme-linked immunosorbent assay. Responsiveness of primary epithelial cells to PAR-1 activation was assessed by calcium mobilization assay. Primary epithelial cells, macrophages, and dendritic cells were cocultured with H pylori and nuclear factor (NF)-kappaB, and cytokine secretion was determined by enzyme-linked immunosorbent assay. RESULTS: Two months postinfection, H pylori levels were significantly reduced in PAR-1(-/-) and increased in PAR-2(-/-) mice. This effect on colonization was inversely correlated with inflammation severity. Infection of PAR-1(-/-) mice induced an increased serum antibody response. Primary epithelial cells were activated by a PAR-1-activating peptide. H pylori stimulation of primary epithelial cells, but not macrophages or dendritic cells, from PAR-1(-/-) mice induced increased levels of NF-kappaB and the proinflammatory cytokine macrophage-inflammatory protein (MIP)-2. PAR-1 also down-regulated MIP-2 secretion in response to cag pathogenicity island activity. CONCLUSIONS: PAR-1 protects the host against severe Helicobacter-induced gastritis. This may be mediated by suppressing the production of proinflammatory cytokines such as MIP-2.

Microstructural properties of the proximal sesamoid bones of Thoroughbred racehorses in training.

BACKGROUND: Proximal sesamoid bone fractures are common catastrophic injuries in racehorses. Understanding the response of proximal sesamoid bones to race training can inform fracture prevention strategies. OBJECTIVES: To describe proximal sesamoid bone microstructure of racehorses and to investigate the associations between microstructure and racing histories. STUDY DESIGN: Cross-sectional. METHODS: Proximal sesamoid bones from 63 Thoroughbred racehorses were imaged using micro-computed tomography. Bone volume fraction (BVTV) and bone material density (BMD) of the whole bone and four regions (apical, midbody dorsal, midbody palmar and basilar) were determined. Generalised linear regression models were used to identify the associations between bone parameters and race histories of the horses. RESULTS: The mean sesamoid BVTV was 0.79 ± 0.08 and BMD was 806.02 ± 24.66 mg HA/ccm. BVTV was greater in medial sesamoids compared with lateral sesamoids (0.80 ± 0.07 vs 0.79 ± 0.08; P < .001) predominantly due to differences in the apical region (medial-0.76 ± 0.08 vs lateral-0.72 ± 0.07; P < .001). BVTV in the midbody dorsal region (0.86 ± 0.06) was greater than other regions (midbody palmar-0.79 ± 0.07, basilar-0.78 ± 0.06 and apical-0.74 ± 0.08; P < .001). BVTV was greater in sesamoids with more microcracks on their articular surface (Coef. 0.005; 95% CI 0.001, 0.009; P = .01), greater extent of bone resorption on their abaxial surface (Grade 2-0.82 ± 0.05 vs Grade 1-0.80 ± 0.05 or Grade 0-0.79 ± 0.06; P = .006), in horses with a low (0.82 ± 0.07) or mid handicap rating (0.78 ± 0.08) compared with high rating (0.76 ± 0.07; P < .001), in 2- to 5-year-old horses (0.81 ± 0.07) compared with younger (0.68 ± 0.08) or older horses (0.77 ± 0.08; P < .001) and in horses that commenced their racing career at less than 4 years of age (0.79 ± 0.08 vs 0.77 ± 0.77; P < .001). BMD was greater in the midbody dorsal (828.6 ± 19.6 mg HA/ccm) compared with other regions (apical-805.8 ± 21.8, midbody palmar-804.7 ± 18.4 and basilar-785.0 ± 17.1; P < .001), in horses with a handicap rating (low-812.1 ± 20.0, mid-821.8 ± 21.3 and high-814.6 ± 19.4) compared with those with no rating (791.08 ± 24.4, P < .001), in females (806.7 ± 22.0) and geldings (812.2 ± 22.4) compared with entires (792.7 ± 26.2; P = .02) and in older horses (<2-year-old-763.7 ± 24.8 vs 2- to 5-year-old-802.7 ± 23.4, and 6- to 12-year-old-817.8 ± 20.0; P = .002). MAIN LIMITATIONS: Data were cross-sectional. CONCLUSIONS: Densification of the proximal sesamoid bones is associated with the commencement of racing in younger horses and the presence of bone fatigue-related pathology. Lower sesamoid BVTV was associated with longevity and better performance.

Protease-activated receptor 2 has pivotal roles in cellular mechanisms involved in experimental periodontitis.

The tissue destruction seen in chronic periodontitis is commonly accepted to involve extensive upregulation of the host inflammatory response. Protease-activated receptor 2 (PAR-2)-null mice infected with Porphyromonas gingivalis did not display periodontal bone resorption in contrast to wild-type-infected and PAR-1-null-infected mice. Histological examination of tissues confirmed the lowered bone resorption in PAR-2-null mice and identified a substantial decrease in mast cells infiltrating the periodontal tissues of these mice. T cells from P. gingivalis-infected or immunized PAR-2-null mice proliferated less in response to antigen than those from wild-type animals. CD90 (Thy1.2) expression on CD4(+) and CD8(+) T-cell-receptor beta (TCRbeta) T cells was significantly (P < 0.001) decreased in antigen-immunized PAR-2-null mice compared to sham-immunized PAR-2-null mice; this was not observed in wild-type controls. T cells from infected or antigen-immunized PAR-2-null mice had a significantly different Th1/inflammatory cytokine profile from wild-type cells: in particular, gamma interferon, interleukins (interleukin-2, -3, and -17), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha demonstrated lower expression than wild-type controls. The absence of PAR-2 therefore appears to substantially decrease T-cell activation and the Th1/inflammatory response. Regulation of such proinflammatory mechanisms in T cells and mast cells by PAR-2 suggests a pivotal role in the pathogenesis of the disease.

Proteinase-activated receptor-2 (PAR2) and mouse osteoblasts: regulation of cell function and lack of specificity of PAR2-activating peptides.

1. Using synthetic proteinase-activated receptor-2 (PAR(2))-activating peptides (PAR(2)APs) corresponding to the tethered ligand domain of the extracellular N-terminus of PAR(2) to mimic the actions of activating proteinases and using primary cultures of calvarial osteoblasts derived from both wild-type (WT) and PAR(2)-null (KO) mice, we investigated the potential role of PAR(2) in regulating osteoblast function. 2. Primary calvarial osteoblasts from WT and KO mice were evaluated for their growth kinetics and mineralization in the absence of PAR(2) agonists and for their responses in a variety of functional assays to the PAR(2)APs Ser-Leu-Ile-Gly-Arg-Leu-amide (SLIGRL-NH(2)) and 2-furoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (2-fLIGRLO-NH(2)), as well as to trypsin. 3. In contrast with WT cells, PAR(2)-KO osteoblasts did not exhibit increased collagen Type I mRNA expression in response to SLIGRL-NH(2). When grown in serum-containing medium, KO cells increased in number more rapidly than WT cells, an effect that could be attributed to decreased apoptosis rather than increased proliferation. Surprisingly, in both WT and KO osteoblasts, the two PAR(2)APs induced mobilization of intracellular calcium stores. Similarly, the PAR(2)APs inhibited serum deprivation-induced apoptosis and parathyroid hormone-, 1,25-dihydroxyvitamin D(3)- or interleukin-11-induced mineralization in WT and KO cells. 4. We conclude that PAR(2) plays a role in osteoblast survival and collagen Type I mRNA induction and that osteoblasts can respond to the PAR(2)APs via both PAR(2)-dependent and -independent mechanisms.

Associations between the radiographic appearance of vascular channels in proximal sesamoid bones, their microstructural characteristics and past racing performance in Thoroughbreds.

BACKGROUND: Abnormalities in vascular channel appearance within the proximal sesamoid bone (PSB) are the most common findings in Thoroughbred yearling presale radiographs and are often evaluated on radiographs of adult racehorses. Despite this, their pathogenesis and clinical significance are poorly understood, and associations with racing performance are inconsistent. OBJECTIVES: To determine microstructural characteristics of the PSBs associated with the radiographic appearance of vascular channels using microcomputed tomography (µCT) and to determine associations with past racing performance in mature horses. STUDY DESIGN: Cross-sectional. METHODS: One pair of PSBs were isolated from a forelimb of 59 Thoroughbred racehorses undergoing post-mortem examination. Each PSB (n = 118) was radiographed, assigned a vascular channel grade using previously published and novel grading systems, then imaged using µCT. Associations between radiographic, µCT and performance variables were investigated with uni- and multivariable generalised linear models. RESULTS: All PSBs had at least one vascular channel (mean 3.6 ± 0.89) observed on µCT originating from the abaxial border, yet in only 63.6% (75/118) were channels observed radiographically. Proximal sesamoid bones with a higher bone volume fraction (odds ratio [OR] 1.08; 95% confidence interval [CI] 1.01-1.15; P = .03) and wider channel diameter (mm) on µCT (OR 20.67; 95% CI 3.29-130.00; P = .001) were more likely to have vascular channels identified on radiographs. Greater radiographic channel number (OR 0.96; 95% CI 0.92-1.00; P = .04) and channel diameter (mm; OR 0.96; 95% CI 0.92-1.00; P = .04) were associated with fewer career placings. MAIN LIMITATIONS: Radiographs of isolated bones avoided the normal superimposition of tissue encountered in the live horse. CONCLUSIONS: The ability to identify vascular channels radiographically indicates widening of channels and densification of the PSB. More radiographic channels and greater channel diameter were associated with similar or poorer measures of past performance, suggesting that these changes are not desirable.

Normal inflammation and regeneration of muscle following injury require osteopontin from both muscle and non-muscle cells.

BACKGROUND: Osteopontin is secreted by skeletal muscle myoblasts and macrophages, and its expression is upregulated in muscle following injury. Osteopontin is present in many different structural forms, which vary in their expression patterns and effects on cells. Using a whole muscle autograft model of muscle injury in mice, we have previously shown that inflammation and regeneration of muscle following injury are delayed by the absence of osteopontin. The current study was undertaken to determine whether muscle or non-muscle cells provide the source of osteopontin required for its role in muscle regeneration. METHODS: The extensor digitorum longus muscle of wild-type and osteopontin-null mice was removed and returned to its bed in the same animal (autograft) or placed in the corresponding location in an animal of the opposite genotype (allograft). Grafts were harvested at various times after surgery and analysed by histology, flow cytometry and quantitative polymerase chain reaction. Data were analysed using one- or two-way ANOVA or Kruskal-Wallis test. RESULTS: Immunohistochemistry confirmed that osteopontin was expressed by macrophages in osteopontin-null muscle allografts in wild-type hosts and by myoblasts in wild-type allografts in osteopontin-null hosts. The decrease in muscle fibre number observed in wild-type autografts following grafting and the subsequent appearance of regenerating fibres were delayed in both types of allografts to a similar extent as in osteopontin-null autografts. Infiltration of neutrophils, macrophages and M1 and M2 macrophage subtypes were also delayed by lack of osteopontin in the muscle and/or host. While the proportion of macrophages showing the M1 phenotype was not affected, the proportion showing the M2 phenotype was decreased by osteopontin deficiency. Expression of tumour necrosis factor-α and interleukin-4 was lower in osteopontin-null than in wild-type autografts, and there was no difference between the osteopontin-null graft types. CONCLUSIONS: Osteopontins from muscle and non-muscle sources are equally important in the acute response of muscle to injury, and cannot substitute for each other, suggesting that they play distinct roles in regulation of cell behaviour. Future studies of mechanisms of osteopontin's roles in acute muscle inflammation and regeneration will need to investigate responses to osteopontins derived from both myoblasts and macrophages.

Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

Mineralized bone forms when collagen-containing osteoid accrues mineral crystals. This is initiated rapidly (primary mineralization), and continues slowly (secondary mineralization) until bone is remodeled. The interconnected osteocyte network within the bone matrix differentiates from bone-forming osteoblasts; although osteoblast differentiation requires EphrinB2, osteocytes retain its expression. Here we report brittle bones in mice with osteocyte-targeted EphrinB2 deletion. This is not caused by low bone mass, but by defective bone material. While osteoid mineralization is initiated at normal rate, mineral accrual is accelerated, indicating that EphrinB2 in osteocytes limits mineral accumulation. No known regulators of mineralization are modified in the brittle cortical bone but a cluster of autophagy-associated genes are dysregulated. EphrinB2-deficient osteocytes displayed more autophagosomes in vivo and in vitro, and EphrinB2-Fc treatment suppresses autophagy in a RhoA-ROCK dependent manner. We conclude that secondary mineralization involves EphrinB2-RhoA-limited autophagy in osteocytes, and disruption leads to a bone fragility independent of bone mass.

Author Correction: Increased autophagy in EphrinB2-deficient osteocytes is associated with elevated secondary mineralization and brittle bone.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.