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.

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.

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.

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.

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.

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.

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.

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 vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation.

Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase) V0 subunit d2 (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.

A T cell-specific knockout reveals an important role for protease-activated receptor 2 in lymphocyte development.

Activation of protease-activated receptor-2 (PAR2) expressed by T cells has been linked to the bone loss associated with periodontitis. We generated PAR2 conditional-null mice and crossed these with mice expressing Cre recombinase under control of the Lck proximal promoter, to produce T cell-specific PAR2-null mice in order to further study the cellular mechanism involved in periodontitis. Here we report that efficient deletion of PAR2 in thymocytes isolated from T cell-specific PAR2-null mice resulted in thymic and splenic hypoplasia and a reduction in the cells of the cortex and a loss of distinction between the cortex and the medulla of the thymus. FACS analysis confirmed significant reductions in CD4 and CD8 double negative (DN3 and DN4) sub-populations, as well as double positive and single positive T cells, in T cell-specific PAR2-null mice compared to Cre expressing PAR2 wild-type mice. The proportion of annexin V positive and propidium iodide negative cells was increased in CD4 and CD8 double negative, double positive and single positive T cells from T cell-specific PAR2-null mice. No change in the proportion of Ki67 positive cells was observed in sections of thymus from T cell-specific PAR2-null mice, suggesting that the depletion of T cell sub-populations in T cell-specific PAR2-null mice resulted from increased apoptosis rather than reduced proliferation. Together, these results demonstrate that PAR2 plays an important and previously unrecognised anti-apoptotic role in T cell development and suggest that the PAR2 conditional-null mouse will be an important resource for determining tissue and cell specific effects of PAR2.

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.

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.

Identification of novel osteochondrosis--Associated genes.

During the early stages of articular osteochondrosis, cartilage is retained in subchondral bone, but the pathophysiology of this condition of growing humans and domestic animals is poorly understood. A subtractive hybridization study was undertaken to compare gene expression between the cartilage of early experimentally induced equine osteochondrosis lesions and control cartilage. Of the many putative differentially expressed genes identified, eight were confirmed by quantitative PCR analysis as differentially expressed, in addition to those already known to be associated with early lesions. Genes encoding vacuolar H(+)-ATPase V0 subunit d2 (ATP6V0D2), cathepsin K, integrin-binding sialoprotein, integrin αV, low density lipoprotein receptor-related protein 4, lumican, osteopontin, and thymosin ß4 (TMSB4) were expressed at higher levels in lesions than in control cartilage. These genes included 34 genes not previously identified in cartilage. Some genes identified as associated with early lesions are known chondrocyte hypertrophy-associated genes, and in transmission electron microscopy studies normal hypertrophic chondrocytes were observed in lesions. Differential expression of ATP6V0D2 and TMSB4 in the cartilage of early naturally occurring osteochondrosis lesions was confirmed by immunohistochemistry. These results identify novel osteochondrosis-associated genes and provide evidence that articular osteochondrosis does not necessarily result from failure of chondrocytes to undergo hypertrophy.

Tumour progression and cancer-induced pain: a role for protease-activated receptor-2?

The role of proteases in modifying the microenvironment of tumour cells has long been recognised. With the discovery of the protease-activated receptor family of G protein-coupled receptors a mechanism for cells to sense and respond directly to proteases in their microenvironment was revealed. Many early studies described the roles of protease-activated receptors in the cellular events that occur during blood coagulation and inflammation. More recently, studies have begun to focus on the roles of protease-activated receptors in the establishment, progression and metastasis of a variety of tumours. This review will focus on the expression of protease-activated receptor-2 and its activators by normal and neoplastic tissues, and describe current evidence that activation of protease-activated receptor-2 is an important event at multiple stages of tumour progression and in pain associated with cancer.

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.

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.

Osteopontin, inflammation and myogenesis: influencing regeneration, fibrosis and size of skeletal muscle.

Osteopontin is a multifunctional matricellular protein that is expressed by many cell types. Through cell-matrix and cell-cell interactions the molecule elicits a number of responses from a broad range of target cells via its interaction with integrins and the hyaluronan receptor CD44. In many tissues osteopontin has been found to be involved in important physiological and pathological processes, including tissue repair, inflammation and fibrosis. Post-natal skeletal muscle is a highly differentiated and specialised tissue that retains a remarkable capacity for regeneration following injury. Regeneration of skeletal muscle requires the co-ordinated activity of inflammatory cells that infiltrate injured muscle and are responsible for initiating muscle fibre degeneration and phagocytosis of necrotic tissue, and muscle precursor cells that regenerate the injured muscle fibres. This review focuses on the current evidence that osteopontin plays multiple roles in skeletal muscle, with particular emphasis on its role in regeneration and fibrosis following injury, and in determining the severity of myopathic diseases such as Duchenne muscular dystrophy.