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Objective:\ud \ud Joint injury involving destabilisation of the joint and damage to the articular cartilage (e.g., sports-related injury) can result in accelerated post-traumatic osteoarthritis (PTOA). Destabilised medial meniscotibial ligament (DMM) surgery is one of the most commonly used murine models and whilst it recapitulates Osteoarthritis (OA) pathology, it does not necessarily result in multi-tissue injury, as occurs in PTOA. We hypothesised that simultaneous cartilage damage and joint destabilisation would accelerate the onset of OA pathology.\ud Methods:\ud \ud OA was induced in C57BL/6 mice via (a) DMM, (b) microblade scratches of articular cartilage (CS) or (c) combined DMM and cartilage scratch (DCS). Mice were culled 7, 14 and 28 days post-surgery. Microcomputed tomography (μCT) and histology were used to monitor bone changes and inflammation. Dynamic weight bearing, an indirect measure of pain, was assessed on day 14.\ud Results:\ud \ud Osteophytogenesis analysis via μCT revealed that osteophytes were present in all groups at days 7 and 14 post-surgery. However, in DCS, osteophytes were visually larger and more numerous when compared with DMM and cartilage scratch (CS). Histological assessment of cartilage at day 14 and 28, revealed significantly greater damage in DCS compared with DMM and CS. Furthermore, a significant increase in synovitis was observed in DCS. Finally, at day 14 osteophyte numbers correlated with changes in dynamic weight bearing.\ud Conclusion:\ud \ud Joint destabilisation when combined with simultaneous cartilage injury accelerates joint deterioration, as seen in PTOA. Thus, DCS provides a novel and robust model for investigating multiple pathological hallmarks, including osteophytogenesis, cartilage damage, synovitis and OA-related pain.

The emergence of bone as an endocrine regulator has prompted a re-evaluation of the role of bone mineralization factors in the development of metabolic disease. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) controls bone mineralization through the generation of pyrophosphate, and levels of NPP1 are elevated both in dermal fibroblast cultures and muscle of individuals with insulin resistance. We investigated the metabolic phenotype associated with impaired bone metabolism in mice lacking the gene that encodes NPP1 (Enpp1(-/-) mice). Enpp1(-/-) mice exhibited mildly improved glucose homeostasis on a normal diet but showed a pronounced resistance to obesity and insulin resistance in response to chronic high-fat feeding. Enpp1(-/-) mice had increased levels of the insulin-sensitizing bone-derived hormone osteocalcin but unchanged insulin signalling within osteoblasts. A fuller understanding of the pathways of NPP1 could inform the development of novel therapeutic strategies for treating insulin resistance.

Objective Proteinase-activated receptor 2 (PAR2) deficiency protects against cartilage degradation in experimental osteoarthritis (OA). The wider impact of this pathway upon OA-associated pathologies such as osteophyte formation and pain is unknown. Herein, we investigated early temporal bone and cartilage changes in experimental OA in order to further elucidate the role of PAR2 in OA pathogenesis.\ud \ud Methods OA was induced in wild-type (WT) and PAR2-deficient (PAR2−/−) mice by destabilisation of the medial meniscus (DMM). Inflammation, cartilage degradation and bone changes were monitored using histology and microCT. In gene rescue experiments, PAR2−/− mice were intra-articularly injected with human PAR2 (hPAR2)-expressing adenovirus. Dynamic weight bearing was used as a surrogate of OA-related pain.\ud \ud Results Osteophytes formed within 7 days post-DMM in WT mice but osteosclerosis was only evident from 14 days post induction. Importantly, PAR2 was expressed in the proliferative/hypertrophic chondrocytes present within osteophytes. In PAR2−/− mice, osteophytes developed significantly less frequently but, when present, were smaller and of greater density; no osteosclerosis was observed in these mice up to day 28. The pattern of weight bearing was altered in PAR2−/− mice, suggesting reduced pain perception. The expression of hPAR2 in PAR2−/− mice recapitulated osteophyte formation and cartilage damage similar to that observed in WT mice. However, osteosclerosis was absent, consistent with lack of hPAR2 expression in subchondral bone.\ud \ud Conclusions This study clearly demonstrates PAR2 plays a critical role, via chondrocytes, in osteophyte development and subchondral bone changes, which occur prior to PAR2-mediated cartilage damage. The latter likely occurs independently of OA-related bone changes.

Osteoarthritis is a global musculoskeletal disease, with no disease-modifying drugs, leaving only analgesia and ultimately surgery to reduce pain and disability, affecting up to 80% of people older than 65 years old. There are strong functional interactions among the cartilage, synovium, and subchondral bone, leading to cartilage damage when mechanical loading is excessive chronic or repetitive, by inducing the expression of proinflammatory cytokines, and matrix metalloproteinases. PAR2 has recently been identified as a novel upstream mediator of catabolic events in the osteoarthritic chondrocytes, where its expression is enhanced, but the mechanism by which it promotes osteoarthritis has yet to be clarified. This protein is activated through the cleavage of the N-terminal domain, which can be mimicked by addition of a SLIGKV external peptide and also inhibited by using antibodies that avoid the cleavage. Fluid shear stress onto cell culture has been previously reported as good model of osteoarthritic in vitro studies, inducing the expression of COX2 which through PGE2 produces an increase of proinflammatory cytokines and matrix metalloproteinases, and also the inhibition of PAR2 signal transduction by decreasing PAR2 surface expression by a still unknown mechanism. To gain a deeper understanding of the role of PAR2 as one of the important factors in osteoarthritis, PAR2 protein levels and its location, as well as its gene expression and other important markers were studied in SW1353 chondrocyte-like cells in response to the different types and intensities of fluid shear stress. Results could suggest a direct relation between PAR2 and osteoarthritis, whose protein levels might be dependent on the type and intensity of flow. It was also found that PAR2 was internalized after FSS, suggesting that it could be in response to PGE2. Furthermore, the effect of PAR2 activation and inhibition on the gene expression of other important pathways was also investigated. RESUMEN La osteoartritis (o artrosis) es una enfermedad musculoesquelética, que afecta hasta el 80% de la población mayor de 65 años, para la cual no hay ningún tratamiento curativo basado en fármacos, utilizándose únicamente analgésicos para aliviar el dolor, y la cirugía como última opción. Existen fuertes interacciones entre el cartílago, el liquido sinovial, y el hueso subcondral, que hacen que cuando la carga mecánica es excesiva o crónica, se induzca la expresión de citoquinas proinflamatorias y metaloproteasas de la matriz, que producen daño en el cartílago. PAR2, gen cuya expresión aumenta en la osteoartritis, ha sido identificado recientemente como un nuevo mediador de los eventos catalíticos de la enfermedad, cuyo mecanismo de acción se desconoce. La proteína que codifica, es activada a través del corte de su dominio N-terminal, lo que puede ser imitado por la adición de un péptido externo SLIGKV, o puede ser inhibido usando anticuerpos que impidan la rotura. El uso de un esfuerzo cortante generado por fluido, sobre un cultivo celular, ha demostrado ser un buen modelo para el estudio in vitro de la osteoartritis, induciendo la expresión de COX2, que a través de PGE2 produce un incremento de citoquinas proinflamatorias y metaloproteinasas de la matriz, así como la inhibición de la señal de transducción de PAR2, reduciendo su expresión en la membrana por un mecanismo que se desconoce. Con el objetivo de conocer mejor el papel que juega PAR2, como uno de los factores importantes, en la osteoartritis, sus niveles de expresión proteica y localización, así como su expresión génica y la de otros marcadores importantes, fueron estudiados en células condrocíticas SW1353 en respuesta a diferentes tipos e intensidades de esfuerzos de corte generados por fluido. Los resultados sugieren que puede tratarse de una relación directa entre PAR2 y osteoartritis, siendo la cantidad de proteína PAR2 dependiente del tipo e intensidad del flujo. También se observó que PAR2 aparecía internalizado después del esfuerzo de corte, sugiriendo que podría ser en respuesta a PGE2. Además, también se investigó el efecto de la activación y la inhibición de PAR2 sobre la expresión génica en otras rutas importantes para la osteoartritis.

PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) have non-redundant functions during skeletal mineralization. While TNAP deficiency (Alpl(-/-) mice) leads to hypophosphatasia, caused by accumulation of the mineralization inhibitor inorganic pyrophosphate (PPi ), comparably elevated levels of PPi in Phospho1(-/-) mice do not explain their stunted growth, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis. We have previously shown that elevated PPi in Alpl(-/-) mice is accompanied by elevated osteopontin (OPN), another potent mineralization inhibitor, and that the amount of OPN correlates with the severity of hypophosphatasia in mice. Here we demonstrate that plasma OPN is elevated and OPN expression is upregulated in the skeleton, particularly in the vertebrae, of Phospho1(-/-) mice. Liquid chromatography/tandem mass spectrometry showed an increased proportion of phosphorylated OPN (p-OPN) peptides in Phospho1(-/-) mice, suggesting that accumulation of p-OPN causes the skeletal abnormalities in Phospho1(-/-) mice. We also show that ablation of the OPN gene, Spp1, leads to improvements in the skeletal phenotype in Phospho1(-/-) as they age. In particular, their scoliosis is ameliorated at 1 month of age and is completely rescued at 3 months of age. There is also improvement in the long bone defects characteristic of Phospho1(-/-) mice at 3 months of age. Mineralization assays comparing [Phospho1(-/-) ; Spp1(-/-) ], Phospho1(-/-) and Spp1(-/-) chondrocytes display corrected mineralization by the double knockout cells. Expression of chondrocyte differentiation markers was also normalized in the [Phospho1(-/-) ; Spp1(-/-) ] mice. Thus, while Alpl and Phospho1 deficiencies lead to similar skeletal phenotypes and comparable changes in the expression levels of PPi and OPN, there is a clear dissociation in the hierarchical roles of these potent inhibitors of mineralization, with elevated PPi and elevated p-OPN levels causing the respective skeletal phenotypes in Alpl(-/-) and Phospho1(-/-) mice. © 2014 American Society for Bone and Mineral Research.

The emergence of bone as an endocrine regulator has prompted a re-evaluation of the role of bone mineralization factors in the development of metabolic disease. Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) controls bone mineralization through the generation of pyrophosphate and is elevated in dermal fibroblast cultures and muscle of patients with insulin resistance. We investigated the metabolic phenotype associated with impaired bone metabolism in mice lacking the NPP1 gene (Enpp1(-/-) mice). Enpp1(-/-) mice exhibited mildly improved glucose homeostasis on a normal diet but showed a pronounced resistance to obesity and insulin resistance in response to chronic high fat feeding. Enpp1(-/-) mice had increased levels of the insulin sensitising bone-derived hormone osteocalcin but unchanged insulin signaling within osteoblasts. A fuller understanding of the pathways of NPP1 may inform the development of novel therapeutic strategies for treating insulin resistance.

The suppressor of cytokine signalling (SOCS)-2 knockout mouse (Socs2-/-) is characterised by an overgrowth phenotype due to enhanced growth-hormone (GH) signalling. The objective of this study was to define the Socs2-/- bone phenotype and determine if GH promotes bone mass via insulin-like growth-factor (IGF)-1 dependent mechanisms. Despite no elevation in systemic IGF-1, increased body weight in 4-week-old Socs2-/- mice following GH treatment was associated with increased cortical bone area (Ct.Ar)(p