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Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context-dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well-understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label-free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co-transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin-dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD-mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

Obesity and osteoarthritis (OA) are well-known comorbidities and their precise molecular interactions are still unidentified. Adiponectin, a major adipokine, known to have an anti-inflammatory effect in atherosclerosis or Type 2 Diabetes Mellitus (T2DM), has also been postulated to be pro-inflammatory in OA. This dual role of adiponectin is still not explained. The precise mechanism by which adiponectin affects cartilage and chondrocytes remains to be elucidated. In the present observational study chondrocytes from 30 patients with OA (18 females and 12 males) undergoing total knee replacement (TKR) were isolated. Expression of adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2) was examined both at gene and protein levels in chondrocytes. The difference in adiponectin receptor expression between lean and obese patients with OA and the role of adiponectin in regulating pro-inflammatory genes (MCP-1, IL-6, and VCAM-1, MMP-1, MMP-2, and TIMP-1) has been investigated. We found that ADIPOR1 represented the most abundant adiponectin receptor in primary OA chondrocytes. ADIPOR1 and ADIPOR2 genes and ADIPOR1 protein were differently expressed in OA chondrocytes obtained from obese compared with lean patients with OA. Adiponectin induced gene expression of MCP-1, IL-6, and MMP-1 in all OA patients' chondrocytes. In contrast, VCAM-1 and MMP-2 were differently regulated by adiponectin depending on the patient's body mass index. This study suggests that adiponectin and ADIPOR1 may have important roles in the pathogenesis of cartilage degeneration in OA of obese subjects.

Skeletal muscle wasting represents both a common phenotype of aging and a feature of pathological conditions such as chronic kidney disease (CKD). Although both clinical data and genetic experiments in mice suggest that hyperphosphatemia accelerates muscle wasting, the underlying mechanism remains unclear. Here, we showed that inorganic phosphate (Pi) dose-dependently decreases myotube size, fusion index, and myogenin expression in mouse C2C12 skeletal muscle cells. These changes were accompanied by increases in reactive oxygen species (ROS) production and Nrf2 and p62 expression, and reductions in mitochondrial membrane potential (MMP) and Keap1 expression. Inhibition of Pi entry, cytosolic ROS production, or Nrf2 activation reversed the effects of high Pi on Nrf2, p62, and myogenin expression. Overexpression of Nrf2 respectively increased and decreased the promoter activity of p62-Luc and myogenin-Luc reporters. Analysis of nuclear extracts from gastrocnemius muscles from mice fed a high-Pi (2% Pi) diet showed increased Nrf2 phosphorylation in sham-operated and 5/6 nephrectomized (CKD) mice, and both increased p62 phosphorylation and decreased myogenin expression in CKD mice. These data suggest that high Pi suppresses myogenic differentiation in vitro and promotes muscle atrophy in vivo through oxidative stress-mediated protein degradation and both canonical (ROS-mediated) and non-canonical (p62-mediated) activation of Nrf2 signaling.

Soft tissue tumours are a heterogeneous group of neoplasms with differentiation towards mesenchymal tissue. They may arise anywhere in the body and show similar clinical presentation. Traditional histopathological diagnosis is now complemented by molecular diagnostic techniques that have become firmly established ancillary diagnostic methods. This article provides a short overview of the aetiology and clinical features of soft tissue sarcomas with an update on how molecular genetics is influencing classification and management of these rare tumours.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway in Drosophila. To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss of Ubr5 function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia. Ubr5 loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened (Smo), a key mediator of HH signalling, dramatically enhanced the Ubr5 mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss of Ubr5 stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations between UBR5 expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for the Ubr5 gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway., Author summary Ubiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and in Drosophila is a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

Mammalian Hedgehog (HH) signalling pathway plays an essential role in tissue homeostasis and its deregulation is linked to rheumatological disorders. UBR5 is the mammalian homologue of the E3 ubiquitin-protein ligase Hyd, a negative regulator of the Hh-pathway inDrosophila.To investigate a possible role of UBR5 in regulation of the musculoskeletal system through modulation of mammalian HH signaling, we created a mouse model for specific loss ofUbr5function in limb bud mesenchyme. Our findings revealed a role for UBR5 in maintaining cartilage homeostasis and suppressing metaplasia.Ubr5loss of function resulted in progressive and dramatic articular cartilage degradation, enlarged, abnormally shaped sesamoid bones and extensive heterotopic tissue metaplasia linked to calcification of tendons and ossification of synovium. Genetic suppression of smoothened(Smo), a key mediator of HH signalling, dramatically enhanced theUbr5mutant phenotype. Analysis of HH signalling in both mouse and cell model systems revealed that loss ofUbr5stimulated canonical HH-signalling while also increasing PKA activity. In addition, human osteoarthritic samples revealed similar correlations betweenUBR5expression, canonical HH signalling and PKA activity markers. Our studies identified a crucial function for theUbr5gene in the maintenance of skeletal tissue homeostasis and an unexpected mode of regulation of the HH signalling pathway.Author SummaryUbiquitin ligases modify proteins post-translationally which is essential for a variety of cellular processes. UBR5 is an E3 ubiquitin ligase and inDrosophilais a regulator of Hedgehog signaling. In mammals, the Hedgehog (HH) signalling pathway, among many other roles, plays an essential role in tissue maintenance, a process called homeostasis. A murine genetic system was developed to specifically eliminate UBR5 function from embryonic limb tissue that subsequently forms bone and connective tissue (ligaments and tendons). This approach revealed that UBR5 operates as a potent suppressor of excessive growth of normal cartilage and bone and prevents formation of bone in ectopic sites in connective tissue near the knees and ankle joints. In contrast to abnormal growth, UBR5 inhibits degradation of the articular cartilage that cushions the knee joint leading to extensive exposure of underlying bone. Furthermore, Ubr5 interacts with smoothened, a component of the HH pathway, identifying UBR5 as a regulator of mammalian HH signaling in the postnatal musculoskeletal system. In summary, this work shows that UBR5 interacts with the HH pathway to regulate skeletal homeostasis in and around joints of the legs and identifies targets that may be harnessed for biomedical engineering and clinical applications.

Paget’s sarcomatous transformation is a rare and potentially fatal complication of Paget’s disease. Histologically, it is typically described as a high-grade and extremely aggressive malignancy. We present an unusual radiographic series from a patient diagnosed with a low-grade Paget's osteosarcoma, a very rare and poorly described variant of the disease.

In breast cancer, increased expression of the cytoskeletal adaptor protein Kindlin-1 has been linked to increased risks of lung metastasis, but the functional basis is unknown. Here, we show that in a mouse model of polyomavirus middle T antigen–induced mammary tumorigenesis, loss of Kindlin-1 reduced early pulmonary arrest and later development of lung metastasis. This phenotype relied on the ability of Kindlin-1 to bind and activate β integrin heterodimers. Kindlin-1 loss reduced α4 integrin–mediated adhesion of mammary tumor cells to the adhesion molecule VCAM-1 on endothelial cells. Treating mice with an anti–VCAM-1 blocking antibody prevented early pulmonary arrest. Kindlin-1 loss also resulted in reduced secretion of several factors linked to metastatic spread, including the lung metastasis regulator tenascin-C, showing that Kindlin-1 regulated metastatic dissemination by an additional mechanism in the tumor microenvironment. Overall, our results show that Kindlin-1 contributes functionally to early pulmonary metastasis of breast cancer. Significance: These findings provide a mechanistic proof in mice that Kindin-1, an integrin-binding adaptor protein, is a critical mediator of early lung metastasis of breast cancer. Cancer Res; 78(6); 1484–96. ©2018 AACR.

Objective Obesity is associated with an increased risk of developing osteoarthritis (OA), which is postulated to be secondary to adipose tissue–dependent inflammation. Periarticular adipose tissue depots are present in synovial joints, but the association of this tissue with OA has not been extensively explored. The aim of this study was to investigate differences in local adipose tissue depots in knees with OA and characterize the changes related to class II and class III obesity in patients with end-stage knee OA. Methods Synovium and the infrapatellar fat pad (IPFP) were collected during total knee replacement from 69 patients with end-stage OA. Histologic changes, changes in gene and protein expression of adiponectin, peroxisome proliferator–activated receptor γ (PPARγ), and Toll-like receptor 4 (TLR-4), and immune cell infiltration into the adipose tissue were investigated. Results IPFP and synovium adipose tissue depots differed significantly and were influenced by the patient's body mass index. Compared to adipocytes from the IPFP and synovium of lean patients, adipocytes from the IPFP of obese patients were significantly larger and the synovium of obese patients displayed marked fibrosis, increased macrophage infiltration, and higher levels of TLR4 gene expression. The adipose-related markers PPARγ in the IPFP and adiponectin and PPARγ in the synovium were expressed at lower levels in obese patients compared to lean patients. Furthermore, there were increased numbers of CD45+ hematopoietic cells, CD45+CD14+ total macrophages, and CD14+CD206+ M2-type macrophages in both the IPFP and synovial tissue of obese patients. Conclusion These differences suggest that IPFP and synovium may contain 2 different white adipose tissue depots and support the theory of inflammation-induced OA in patients with class II or III obesity. These findings warrant further investigation as a potentially reversible, or at least suppressible, cause of OA in obese patients.

Magnetic scaffolds have recently attracted significant attention in tissue engineering due to the prospect of improving bone tissue formation by conveying soluble factors such as growth factors, hormones, and polypeptides directly to the site of implantation, as well as to the possibility of improving implant fixation and stability. The objective of this study was to compare bone tissue formation in a preclinical rabbit model of critical femoral defect treated either with a hydroxyapatite (HA)/magnetite (90/10 wt %) or pure HA porous scaffolds at 4 and 12 weeks after implantation. The biocompatibility and osteogenic activity of the novel magnetic constructs was assessed with analysis of the amount of newly formed bone tissue and its nanomechanical properties. The osteoconductive properties of the pure HA were confirmed. The HA/magnetite scaffold was able to induce and support bone tissue formation at both experimental time points without adverse tissue reactions. Biomechanically, similar properties were obtained from nanoindentation analysis of bone formed following implantation of magnetic and control scaffolds. The results indicate that the osteoconductive properties of an HA scaffold are maintained following inclusion of a magnetic component. These provide a basis for future studies investigating the potential benefit in tissue engineering of applying magnetic stimuli to enhance bone formation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 546-554, 2018.

Summary CSPG4/NG2 is a multifunctional transmembrane protein with limited distribution in adult tissues including articular cartilage. The purpose of this study was to investigate the possible roles of CSPG4/NG2 in chondrosarcomas and to establish whether this molecule may have potential for targeted therapy. Stable knock-down of CSPG4/NG2 in the JJ012 chondrosarcoma cell line by shRNA resulted in decreased cell proliferation and migration as well as a decrease in gene expression of the MMP (matrix metalloproteinase) 3 protease and ADAMTS4 (aggrecanase). Chondrosarcoma cells in which CSPG4/NG2 was knocked down were more sensitive to doxorubicin than wild-type cells. The results indicate that CSPG4/NG2 has roles in regulating chondrosarcoma cell function in relation to growth, spread and resistance to chemotherapy and that anti-CSPG4/NG2 therapies may have potential in the treatment of surgically unresectable chondrosarcoma.

The morphological appearances of small cell lung carcinoma (SCLC) are often characteristic however immunohistochemistry (IHC) is frequently employed to support the diagnosis, especially when there is crush artefact (1). In general histopathologists look for expression of cytokeratins, CD56 and TTF1 but it is well recognised that SCLC may not show typical staining reactions. While CD56 is usually expressed TTF1 may be negative and cytokeratin staining may be very focal or even absent (1,2). We report three cases of mediastinal alveolar rhabdomyosarcoma who presented with radiological features of lung cancer where the morphological and immunohistochemical profile mimicked SCLC.

The magnetization of mesenchymal stem cells (MSC) has the potential to aid tissue engineering approaches by allowing tracking, targeting, and local retention of cells at the site of tissue damage. Commonly used methods for magnetizing cells include optimizing uptake and retention of superparamagnetic iron oxide nanoparticles (SPIONs). These appear to have minimal detrimental effects on the use of MSC function as assessed by in vitro assays. The cellular content of magnetic nanoparticles (MNPs) will, however, decrease with cell proliferation and the longer-term effects on MSC function are not entirely clear. An alternative approach to magnetizing MSCs involves genetic modification by transfection with one or more genes derived from Magnetospirillum magneticum AMB-1, a magnetotactic bacterium that synthesizes single-magnetic domain crystals which are incorporated into magnetosomes. MSCs with either or mms6 and mmsF genes are followed by bio-assimilated synthesis of intracytoplasmic magnetic nanoparticles which can be imaged by magnetic resonance (MR) and which have no deleterious effects on MSC proliferation, migration, or differentiation. The stable transfection of magnetosome-associated genes in MSCs promotes assimilation of magnetic nanoparticle synthesis into mammalian cells with the potential to allow MR-based cell tracking and, through external or internal magnetic targeting approaches, enhanced site-specific retention of cells for tissue engineering.

Glucocorticoid therapy, especially at higher doses, is associated with significantadverse side effects including osteoporosis. Leptin, secreted from adipose tissue, has diverse effects on bone tissue regulation. As glucocorticoids stimulate leptin synthesis and secretion directly in adipose tissue we hypothesised that dexamethasone (DEX) induced osteoporosis may, in part, be mediated by an osteoblast dependent leptin-leptin receptor pathway. Human bone cells expressed leptin and leptin receptors (Ob-Ra and Ob-Rb). DEX increased leptin, Ob-Ra and Ob-Rb expression in a dose-dependent manner while decreasing expression of osteocalcin. In the presence of leptin, Cbfa1 and osteonectin expression showed no significant change, whereas osteocalcin expression was decreased. Recombinant human quadruple antagonist leptin suppressed DEX-induced osteocalcin downregulation. The signaling pathway involved up-regulation of JAK2. In conclusion, upregulation of leptin and Ob-Rb in human bone cells by DEX is associated with own-regulation of osteocalcin expression. The down regulation of osteocalcin by DEX was partially through a leptin autocrine/paracrine loop. Adverse effects of DEX on the skeleton may be modified by targeting leptin signaling pathways.

In addition to being the primary organ involved in redox cycling, the liver is one of the most highly innervated tissues in mammals. The interaction between hepatocytes and sympathetic, parasympathetic, and peptidergic nerve fibers through a variety of neurotransmitters and signaling pathways is recognized as being important in the regulation of hepatocyte function, liver regeneration, and hepatic fibrosis. However, less is known regarding the role of the sympathetic nervous system (SNS) in modulating the hepatic response to oxidative stress. Our aim was to investigate the role of the SNS in healthy and oxidatively stressed liver parenchyma. Mice treated with 6-hydroxydopamine hydrobromide were used to realize chemical sympathectomy. Carbon tetrachloride (CCl4) injection was used to induce oxidative liver injury. Sympathectomized animals were protected from CCl4induced hepatic lipid peroxidation-mediated cytotoxicity and genotoxicity as assessed by 4-hydroxy-2-nonenal levels, morphological features of cell damage, and DNA oxidative damage. Furthermore, sympathectomy modulated hepatic inflammatory response induced by CCl4-mediated lipid peroxidation. CCl4induced lipid peroxidation and hepatotoxicity were suppressed by administration of anα-adrenergic antagonist. We conclude that the SNS provides a permissive microenvironment for hepatic oxidative stress indicating the possibility that targeting the hepaticα-adrenergic signaling could be a viable strategy for improving outcomes in patients with acute hepatic injury.

Summary Objective Cannabinoid receptors and their ligands have been implicated in the regulation of various physiological processes but their role in osteoarthritis has not been investigated. The aim of this study was to evaluate the role of the type 2 cannabinoid receptor ( Cnr2 ) in regulating susceptibility to osteoarthritis in mice. Methods We analysed the severity of knee osteoarthritis as assessed by the Osteoarthritis Research Society International (OARSI) scoring system in mice with targeted deletion of Cnr2 ( Cnr2 −/− ) and wild type (WT) littermates. Studies were conducted in mice subjected to surgical destabilisation of the medial meniscus (DMM) and in those with spontaneous age-related osteoarthritis (OA). Results Osteoarthritis was more severe following DMM in the medial compartment of the knee in Cnr2 −/− compared with WT mice (mean ± sem score = 4.9 ± 0.5 vs 3.6 ± 0.3; P = 0.017). Treatment of WT mice with the CB2-selective agonist HU308 following DMM reduced the severity of OA in the whole joint (HU308 = 8.4 ± 0.2 vs vehicle = 10.4 ± 0.6; P = 0.007). Spontaneous age related osteoarthritis was also more severe in the medial compartment of the knee in 12-month old Cnr2 −/− mice compared with WT (5.6 ± 0.5 vs 3.5 ± 0.3, P = 0.008). Cultured articular chondrocytes from Cnr2 −/− mice produced less proteoglycans in vitro than wild type chondrocytes. Conclusion These studies demonstrate that the Cnr2 pathway plays a role in the pathophysiology of osteoarthritis in mice and shows that pharmacological activation of CB2 has a protective effect. Further studies of the role of cannabinoid receptors in the pathogenesis of osteoarthritis in man are warranted.

Background: Proteolytic fragments of fibronectin have catabolic effects on cartilage and menisci. Platelet-rich plasma (PRP) is increasingly being used to treat a range of joint conditions, but it is unknown whether PRP influences fibronectin fragment (FN-f) procatabolic activity. Hypotheses: The procatabolic activity of FN-f on meniscocytes and articular chondrocytes is attenuated by cotreatment with PRP. Study Design: Controlled laboratory study. Methods: Human meniscocytes were treated with FN-f (30 kDa) with or without PRP coincubation, and gene expression was analyzed by complementary DNA microarray analysis. Validation of altered expression of known and novel chemokine and protease genes was undertaken by real-time polymerase chain reaction (RT-PCR) in articular chondrocytes and meniscocytes. Chemokine release was assayed by enzyme-linked immunosorbent assay, and intracellular pathway signaling was evaluated by Western immunoblotting. Results: Microarray analysis and RT-PCR showed increased expression of matrix metalloproteinase ( MMP)1, MMP2, MMP3, MMP9, MMP13, interleukin ( IL)– 6, IL-8 ( CXCL8), CCL5, CCL20, and CXCL10 chemokines in meniscocytes after treatment with FN-f. Upregulation of these genes was significantly attenuated by PRP. Similar results were seen with articular chondrocytes, although no changes in MMP2 or MMP9 levels were identified. PRP-induced suppression of gene expression was associated with activation of Akt and p44/p42. Conclusion: PRP treatment attenuates the 30-kDa FN-f–induced expression of a range of proinflammatory chemokines and MMPs, including IL-8, IL-6, CCL20, CCL5, CXCL10, MMP1, MMP3, and MMP13, by both meniscocytes and articular chondrocytes. Clinical Relevance: These observations provide support for the use and further trials of PRP in management of cartilage and meniscal injuries.

Inactivation of the thiazide-sensitive sodium chloride cotransporter (NCC) due to genetic mutations in Gitelman's syndrome (GS) or pharmacological inhibition with thiazide diuretics causes hypocalciuria and increased bone mineral density (BMD) with unclear extrarenal calcium (Ca(2+) ) regulation. We investigated intestinal Ca(2+) absorption and bone Ca(2+) metabolism in nonsense Ncc Ser707X (S707X) homozygous knockin mice (Ncc(S707X/S707X) mice). Compared to wild-type and heterozygous knockin littermates, Ncc(S707X/S707X) mice had increased intestinal absorption of (45) Ca(2+) and expression of the active Ca(2+) transport machinery (transient receptor potential vanilloid 6, calbindin-D9K , and plasma membrane Ca(2+) ATPase isoform 1b). Ncc(S707X/S707X) mice had also significantly increased Ca(2+) content accompanied by greater mineral apposition rate (MAR) in their femurs and higher trabecular bone volume, cortical bone thickness, and BMD determined by μCT. Their osteoblast differentiation markers, such as bone alkaline phosphatase, procollagen I, osteocalcin, and osterix, were also significantly increased while osteoclast activity was unaffected. Analysis of marrow-derived bone cells, either treated with thiazide or directly cultured from Ncc S707X knockin mice, showed that the differentiation of osteoblasts was associated with increased phosphorylation of mechanical stress-induced focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In conclusion, NCC inhibition stimulates duodenal Ca(2+) absorption as well as osteoblast differentiation and bone Ca(2+) storage, possibly through a FAK/ERK dependent mechanism.

Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9).Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2.Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750.NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.

SummaryC-type natriuretic peptide (CNP) has been demonstrated in human and mouse models to play critical roles in cartilage homeostasis and endochondral bone formation. Indeed, targeted inactivation of the genes encoding CNP results in severe dwarfism and skeletal defects with a reduction in growth plate chondrocytes. Conversely, cartilage-specific overexpression of CNP was observed to rescue the phenotype of CNP deficient mice and significantly enhanced bone growth caused by growth plate expansion. In vitro studies reported that exogenous CNP influenced chondrocyte differentiation, proliferation and matrix synthesis with the response dependent on CNP concentration. The chondroprotective effects were shown to be mediated by natriuretic peptide receptor (Npr)2 and enhanced synthesis of cyclic guanosine-3′,5′-monophosphate (cGMP) production. Recent studies also showed certain homeostatic effects of CNP are mediated by the clearance inactivation receptor, Npr3, highlighting several mechanisms in maintaining tissue homeostasis. However, the CNP signalling systems are complex and influenced by multiple factors that will lead to altered signalling and tissue dysfunction. This review will discuss the differential role of CNP signalling in regulating cartilage and bone homeostasis and how the pathways are influenced by age, inflammation or sex. Evidence indicates that enhanced CNP signalling may prevent growth retardation and protect cartilage in patients with inflammatory joint disease.

Object Increased neurotrophin activity in degenerative intervertebral discs (IVDs) is one potential cause of chronic low-back pain (LBP). The aim of the study was to assess if nerve growth factor (NGF) might alter gene expression of IVD cells and contribute to disc degeneration by enhancing expression or activity of factors that cause breakdown of IVD matrix. Methods Rat-tail IVD cells were stimulated by NGF and subjected to microarray analysis. Real-time polymerase chain reaction, Western blotting, and immunocytochemistry of rat and human IVD cells and tissues treated with NGF in vitro in the absence or presence of the NGF inhibitor Ro 08-2750 were used to confirm findings of the microarray studies. Phosphorylation of mitogen-activated protein kinase (MAPK) was used to identify cell signaling pathways involved in NGF stimulation in the absence or presence of Ro 08-2750. Results Microarray analysis demonstrated increased expression of chitinase 3-like 1 (Chi3l1), lipocalin 2 (Lcn2), and matrix metalloproteinase–3 (Mmp3) following NGF stimulation of rat IVD cells in vitro. Increased gene expression was confirmed by real-time polymerase chain reaction with a relative increase in the Mmp/Timp ratio. Increased expression of Chi3l1, Lcn2, and Mmp3 following NGF stimulation was also demonstrated in rat cells and human tissue in vitro. Effects of NGF on protein expression were blocked by an NGF inhibitor and appear to function through the extracellular-regulation kinase 1/2 (ERK1/2) MAPK pathway. Conclusions Nerve growth factor has potential effects on matrix turnover activity and influences the catabolic/anabolic balance of IVD cells in an adverse way that may potentiate IVD degeneration. Anti-NGF treatment might be beneficial to ameliorate progressive tissue breakdown in IVD degeneration and may lead to pain relief.

Background rheumatoid nodulosis is a rare disease characterised by multiple subcutaneous nodules, a high titre of rheumatoid factor, radiologically detectable cystic bone lesions, but with none or few of the systemic manifestations or joint activity of rheumatoid disease. Histopathologically, nodulosis is the same as the nodules found in rheumatoid arthritis. It is considered to be a benign variant of rheumatoid arthritis. A 69 year old male presents with multiple subcutaneous nodules on the feet. This case study highlights the benefits of ultrasound in establishing a correct diagnosis and management. Although rare, rheumatoid nodulosis is a consideration in the differential diagnoses of soft tissue swellings in the feet.

Case We report a rare case of desmoplastic fibroma (DF) of the midshaft femur presenting as a pathological fracture. This rare benign bone tumor was treated with an acute en bloc excision and femoral shortening over an intramedullary nail. Once union of the acute shortening had been achieved, further surgery was undertaken to lengthen the femur with the use of Intramedullary Skeletal Kinetic Distractors. At 3 years after fracture, our patient had achieved equal leg lengths, had normal knee function, and was disease free. Conclusions DF resulting in pathological fracture of the midshaft femur is extremely rare and has not been reported to occur in the femoral diaphysis. This location is important as preservation of the joint above and below is preferable and en bloc excision is recommended. Restoration of bone stock after en bloc excision is difficult and recurrence needs to be monitored.

Osteoarthritis (OA) is becoming a significant problem worldwide. Pain and loss of joint function have a major impact on an individual's quality of life and through loss of time at work and increasing health care costs has a significant economic burden on society. OA is a complex disease with a number of known risk factors. There is variable involvement of the synovial joints in populations as a result of both environmental and genetic factors. Recent candidate gene studies and genome-wide association studies are beginning to identify known and previously unsuspected genes to be associated with the development of OA. These studies raise the exciting prospect of future stratification of patients into genetically identified sub-groups that may have specific targetable pathways for disease management.

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing. A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague–Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology. The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm-2 (sd 7.63) vs 24.65 Nmm-2 (sd 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (sd 0.75) vs 4.6 mmAl (sd 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007). Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing. Cite this article: Bone Joint J 2013;95-B:1263–8.

Neutrophils act as a first line of defense against bacterial and fungal infections, but they are also important effectors of acute and chronic inflammation. Genome-wide association studies have established that the gene encoding the protein tyrosine phosphatase nonreceptor 22 (PTPN22) makes an important contribution to susceptibility to autoimmune disease, notably rheumatoid arthritis. Although PTPN22 is most highly expressed in neutrophils, its function in these cells remains poorly characterized. We show in this article that neutrophil effector functions, including adhesion, production of reactive oxygen species, and degranulation induced by immobilized immune complexes, were reduced in Ptpn22−/− neutrophils. Tyrosine phosphorylation of Lyn and Syk was altered in Ptpn22−/− neutrophils. On stimulation with immobilized immune complexes, Ptpn22−/− neutrophils manifested reduced activation of key signaling intermediates. Ptpn22−/− mice were protected from immune complex–mediated arthritis, induced by the transfer of arthritogenic serum. In contrast, in vivo neutrophil recruitment following thioglycollate-induced peritonitis and in vitro chemotaxis were not affected by lack of PTPN22. Our data suggest an important role for PTPN22-dependent dephosphorylation events, which are required to enable full FcγR-induced activation, pointing to an important role for this molecule in neutrophil function.

Objective The purpose of this study was to determine if there were variations in chondrocyte viability, matrix glycosaminoglycan (GAG), and water content between different areas of the articular surface of a bovine metatarsophalangeal joint, a common and reliable source of articular cartilage for experimental study, which may compromise the validity of using multiple samples from different sites within the joint. Methods Nine fresh cadaveric bovine metatarsophalangeal joints were obtained. From each joint, 16 osteochondral explants were taken from 4 facets, yielding a total of 144 cartilage specimens for evaluation of chondrocyte viability, matrix GAG, and water content. A less invasive method for harvesting osteochondral explants and for processing the biopsy for the assessment of chondrocyte viability was developed, which maintained maximal viability within each cartilage explant. Results There was no significant difference between the 16 biopsy sites from the different areas of the joint surface with respect to chondrocyte viability, matrix GAG and water content. Pooled data of all samples from each joint established the baseline values of chondrocyte viability to be 89.4% ± 3.8%, 94.4% ± 2.2%, and 77.9% ± 7.8%, in the superficial quarter, central half, and deep quarter (with regard to depth from the articular surface), respectively. The matrix GAG content of bovine articular cartilage was 6.06 ± 0.41 μg/mg cartilage, and the cartilage water content was 72.4% ± 1.5%. There were also no significant differences of these 3 variables between the different joints. Conclusion It is thus reasonable to compare biopsies obtained from different sites, as a biopsy from one site would be considered representative of the whole joint.

The fascinating prospect to direct tissue regeneration by magnetic activation has been recently explored. In this study we investigate the possibility to boost bone regeneration in an experimental defect in rabbit femoral condyle by combining static magnetic fields and magnetic biomaterials. NdFeB permanent magnets are implanted close to biomimetic collagen/hydroxyapatite resorbable scaffolds magnetized according to two different protocols. Permanent magnet only or non-magnetic scaffolds are used as controls. Bone tissue regeneration is evaluated at twelve weeks from surgery from a histological, histomorphometric and biomechanical point of view. The reorganization of the magnetized collagen fibers under the effect of the static magnetic field generated by the permanent magnet produces a highly-peculiar bone pattern, with highly-interconnected trabeculae orthogonally oriented with respect to the magnetic field lines. In contrast, only partial defect healing is achieved within the control groups. We ascribe the peculiar bone regeneration to the transfer of micro-environmental information, mediated by collagen fibrils magnetized by magnetic nanoparticles, under the effect of the static magnetic field. These results open new perspectives on the possibility to improve implant fixation and control the morphology and maturity of regenerated bone providing “in site” forces by synergically combining static magnetic fields and biomaterials.

Recent studies have suggested that polymorphisms in toll-like receptor 9 (TLR-9), an endosomal TLR, are associated with knee osteoarthritis (OA). TLR-3, -7, and -8 are also found on the surface of endosomes and to investigate whether similar associations exist with polymorphisms in these TLR genes we performed a two-stage case–control study and genotyped 11 TLR single nucleotide polymorphisms (SNPs) in 823 OA cases and 594 healthy controls by polymerase chain reaction restriction fragment length polymorphism assays. Real-time PCR was performed to assess the functional expression of an identified promoter polymorphism in TLR-3 following dexamethasone stimulation of articular chondrocytes. An association between TLR-3 SNPs at rs3775296 and rs3775290 and OA was identified in both populations. In males the allelic frequencies of TLR-7 rs179010 and TLR-8 rs5744080 were significantly different between OA cases and healthy controls. The ATCA, CTCA, and CCTA haplotypes of TLR-3 were associated with OA susceptibility. A significant difference in TLR-3 gene expression following dexamethasone treatment was seen among the various genotypes of rs3775296 (p = 0.004). Our findings indicate that a SNP in the promoter region of TLR-3 is associated with elevated TLR-3 gene expression and susceptibility to knee OA in a Chinese Han population. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 680–685, 2013

Objectives Small animal models of fracture repair primarily investigate indirect fracture healing via external callus formation. We present the first described rat model of direct fracture healing. Methods A rat tibial osteotomy was created and fixed with compression plating similar to that used in patients. The procedure was evaluated in 15 cadaver rats and then in vivo in ten Sprague-Dawley rats. Controls had osteotomies stabilised with a uniaxial external fixator that used the same surgical approach and relied on the same number and diameter of screw holes in bone. Results Fracture healing occurred without evidence of external callus on plain radiographs. At six weeks after fracture fixation, the mean stress at failure in a four-point bending test was 24.65 N/mm2 (sd 6.15). Histology revealed ‘cutting-cones’ traversing the fracture site. In controls where a uniaxial external fixator was used, bone healing occurred via external callus formation. Conclusions A simple, reproducible model of direct fracture healing in rat tibia that mimics clinical practice has been developed for use in future studies of direct fracture healing.

BACKGROUND: Osteoarthritis is the most common form of arthritis worldwide and is a major cause of pain and disability in elderly people. The health economic burden of osteoarthritis is increasing commensurate with obesity prevalence and longevity. Osteoarthritis has a strong genetic component but the success of previous genetic studies has been restricted due to insufficient sample sizes and phenotype heterogeneity. METHODS: We undertook a large genome-wide association study (GWAS) in 7410 unrelated and retrospectively and prospectively selected patients with severe osteoarthritis in the arcOGEN study, 80% of whom had undergone total joint replacement, and 11,009 unrelated controls from the UK. We replicated the most promising signals in an independent set of up to 7473 cases and 42,938 controls, from studies in Iceland, Estonia, the Netherlands, and the UK. All patients and controls were of European descent. FINDINGS: We identified five genome-wide significant loci (binomial test p≤5·0×10(-8)) for association with osteoarthritis and three loci just below this threshold. The strongest association was on chromosome 3 with rs6976 (odds ratio 1·12 [95% CI 1·08-1·16]; p=7·24×10(-11)), which is in perfect linkage disequilibrium with rs11177. This SNP encodes a missense polymorphism within the nucleostemin-encoding gene GNL3. Levels of nucleostemin were raised in chondrocytes from patients with osteoarthritis in functional studies. Other significant loci were on chromosome 9 close to ASTN2, chromosome 6 between FILIP1 and SENP6, chromosome 12 close to KLHDC5 and PTHLH, and in another region of chromosome 12 close to CHST11. One of the signals close to genome-wide significance was within the FTO gene, which is involved in regulation of bodyweight-a strong risk factor for osteoarthritis. All risk variants were common in frequency and exerted small effects. INTERPRETATION: Our findings provide insight into the genetics of arthritis and identify new pathways that might be amenable to future therapeutic intervention. FUNDING: arcOGEN was funded by a special purpose grant from Arthritis Research UK.

Glucocorticoids profoundly influence immune responses, and synthetic glucocorticoids are widely used clinically for their potent antiinflammatory effects. Endogenous glucocorticoid action is modulated by the two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD). In vivo, 11β-HSD1 catalyzes the reduction of inactive cortisone or 11-dehydrocorticosterone into active cortisol or corticosterone, respectively, thereby increasing intracellular glucocorticoid levels. 11β-HSD2 catalyzes the reverse reaction, inactivating intracellular glucocorticoids. Both enzymes have been postulated to modulate inflammatory responses. In the K/BxN serum transfer model of arthritis, 11β-HSD1-deficient mice showed earlier onset and slower resolution of inflammation than wild-type controls, with greater exostoses in periarticular bone and, uniquely, ganglion cysts, consistent with greater inflammation. In contrast, K/BxN serum arthritis was unaffected by 11β-HSD2 deficiency. In a distinct model of inflammation, thioglycollate-induced sterile peritonitis, 11β-HSD1-deficient mice had more inflammatory cells in the peritoneum, but again 11β-HSD2-deficient mice did not differ from controls. Additionally, compared with control mice, 11β-HSD1-deficient mice showed greater numbers of inflammatory cells in pleural lavages in carrageenan-induced pleurisy with lung pathology consistent with slower resolution. These data suggest that 11β-HSD1 limits acute inflammation. In contrast, 11β-HSD2 plays no role in acute inflammatory responses in mice. Regulation of local 11β-HSD1 expression and/or delivery of substrate may afford a novel approach for antiinflammatory therapy.

Intracellular protein complexes containing nucleic acids are common targets of autoantibodies in many autoimmune diseases. Central tolerance to these antigens is incomplete, yet nucleosomal DNA is expressed on the surface of cells dying by apoptosis. It is commonly believed that autoimmunity is prevented by the rapid uptake of apoptotic cells (ACs) by neighbors or professional phagocytes to which they deliver anti-inflammatory signals. Self-reactive, innate-like B cells contact and are selected by intracellular antigens expressed on ACs; however, how self-tolerance is maintained is not well understood. Here we report that IL-10 production by B cells, stimulated by contact with ACs, results from the engagement of Toll-like receptor 9 (TLR9) within the B cell after recognition of DNA-containing complexes on the surface of ACs. Until now, TLR9 ligation has been considered an inflammatory signal, but we have confirmed a hitherto unexpected immunoregulatory role by demonstrating the absence of the protective effect of ACs during experimental autoimmune encephalitis (EAE) in TLR9-deficient mice. Human circulating CD27 + B cells also respond to DNA-bearing ACs, but not to DNase-treated cells, by secreting IL-10. Chronic autoimmune disease may arise if this tolerance mechanism is not reimposed after episodes of inflammation, or if the regulatory B-cell response is subverted.

Based on the recent observation that Toll-like receptors (TLRs) may be involved in the pathogenesis of osteoarthritis (OA) we explored the possibility that human TLR gene polymorphisms are associated with OA. Two separate populations were studied in a two-stage case-control study with a total of 503 OA patients and 428 healthy controls. The TLR-2, TLR-4, and TLR-9 genotypes were assessed by real-time polymerase chain reaction. Our data demonstrated a lack of association among TLR-2, TLR-4, and TLR-9 (T-1237C) polymorphisms and the risk of developing OA in both stages of the study. T-1486C was significantly associated with OA in both populations with G1635A of TLR-9 gene was found to be significantly associated with OA when the two populations were combined. Stratifying the samples by K-L score there were significant differences in the genotype of the TLR-9 T-1486C and G1635A between OA of the knee grade 4 and controls. In haplotype analyses, the haplotype TTG and TTA revealed higher risk of OA and TCA confers a lower risk of OA in combined population. The present results demonstrate that TLR-9 polymorphisms, in particular T-1486C is significantly associated with OA. TLR-9 gene polymorphisms may play a role in the etiology of knee OA.

Chondrosarcomas are malignant cartilage tumours. They are poorly responsive to chemotherapy and radiotherapy. Treatment is usually limited to surgical resection; however, survival of patients with high-grade chondrosarcoma is poor, even with wide surgical resection. Induction of apoptosis in chondrosarcoma cells, either directly or by enhancement of the response to chemotherapeutic drugs and radiation, may be a route by which outcome can be improved. In this article, we review potential molecular targets that regulate chondrocyte apoptosis and discuss the experimental evidence for their utility.

A multimodal nonlinear optical microscope that combines coherent anti-Stokes Raman scattering (CARS), two-photon excitation fluorescence (TPEF), second-harmonic generation (SHG) and sum-frequency generation (SFG) was developed and applied to image breast cancer tissue and MCF-7 cells as well as monitoring anticancer drug delivery in live cells. TPEF imaging showed that drugs are preferentially localized in the cytoplasm and the nuclear envelope in resistant cells. Moreover, the extracellular matrix was observed by TPEF signals arising from elastin's autofluorescence and SHG signals from collagen fibrils in breast tissue sections. Additionally, CARS signals arising from proteins and (PO2)− allowed identification of tumors. Label-free imaging with chemical contrast of significant components of cancer cells and tissue suggests the potential of multimodal nonlinear optical microscopy for early detection and diagnosis of cancer. Copyright © 2010 John Wiley & Sons, Ltd.

Determine the effect of IL-1β and dynamic compression on NFκB activation and IκB-α gene expression in chondrocyte/agarose constructs. Constructs were cultured under free-swelling conditions or subjected to dynamic compression for up to 360 min with IL-1β and/or PDTC (inhibits NFκB activation). Nuclear translocation of NFκB-p65 was analysed by immunofluoresence microscopy. Gene expression of IκB-α, iNOS, IL-1β and IL-4 was assessed by real-time qPCR. Nuclear translocation of NFκB-p65 was concomitant with an increase in nuclear fluorescence intensity which reached maximum values at 60 min with IL-1β (p

Mechanical loading of articular cartilage stimulates the metabolism of resident chondrocytes and induces the synthesis of molecules to maintain the integrity of the cartilage. Mechanical signals modulate biochemical activity and changes in cell behavior through mechanotransduction. Compression of cartilage results in complex changes within the tissue including matrix and cell deformation, hydrostatic and osmotic pressure, fluid flow, altered matrix water content, ion concentration and fixed charge density. These changes are detected by mechanoreceptors on the cell surface, which include mechanosensitive ion channels and integrins that on activation initiate intracellular signalling cascades leading to tissue remodelling. Excessive mechanical loading also influences chondrocyte metabolism but unlike physiological stimulation leads to a quantitative imbalance between anabolic and catabolic activity resulting in depletion of matrix components. In this article we focus on the role of mechanical signalling in the maintenance of articular cartilage, and discuss how alterations in normal signalling can lead to pathology.

We report a case of a 27-year-old man who presented with a progressive painful swelling at the base of his left index finger, with radiographs and a computed tomography scan revealing a lytic lesion of the proximal phalanx. Following further investigation, the patient underwent a bone biopsy that revealed a florid noncaseating granulomatous chronic inflammatory infiltrate, compatible with sarcoidosis. Osseous sarcoidosis of the hand is uncommon and, in the absence of significant systemic disease, is rarely the primary presenting feature. Early diagnosis and treatment of such undetermined bone pathology, via referral to a regional musculoskeletal tumor service, can prevent significant future complications.

Summary Objective Classical neuronal signalling molecules such as substance P and glutamate have been identified in cartilage and have roles in regulation of chondrocyte function. This study looks at expression and activity of the ionotropic glutamate NMDA (N-methyl-D-aspartic acid) receptor (NMDAR) in human osteoarthritic (OA) chondrocytes. Method Chondrocytes were obtained from human knee joint arthroplasty specimens. NMDAR subunits and PSD-95 (postsynaptic density protein 95) expression were analysed by reverse transcription-polymerase chain reaction and Western blotting. Activity of NMDAR was assayed by radioactive calcium 45 uptake and changes in membrane potential in the presence and absence of NMDA and NMDAR antagonists and blockade of cell membrane ion channels. Results NMDAR 1, 2A, 2B and PSD-95 were detected in human OA chondrocytes whereas NR2B was absent from normal chondrocytes. NMDA induced calcium flux into OA chondrocytes and cell membrane depolarisation. These responses were blocked by NMDAR antagonists, removal of extracellular calcium, inhibition of nNOS (neuronal nitric oxide synthase) activity and uncoupling of NMDAR from PSD-95. Blockade of sodium channels by tetrodotoxin resulted in NMDA-induced membrane hyperpolarisation which was, in turn inhibited by apamin, a blocker of SK channels. NMDA-induced changes in cell membrane potential were not affected by l-type and stretch activated calcium channel inhibitors. Conclusions Human OA and normal articular chondrocytes differ in the expression of NMDAR subunits. In OA chondrocytes NMDAR signalling requires extracellular calcium, association with PSD-95, and nNOS activity. Downstream signalling results in activation of tetrodotoxin sensitive sodium channels and SK channels, a response that differs from that of normal chondrocytes suggesting altered activity of NMDAR in OA.

A 61-year-old man presented with pain at the left hip and decreased mobility 10 years after total hip replacement. Imaging demonstrated a large destructive expansile mass adjacent to the prosthesis. Histological analysis confirmed the presence of an extra-cranial meningioma. Primary tumours after total hip replacement are rare and include soft tissue sarcomas, bone sarcomas and lymphomas. To our knowledge, no previous cases of primary extracranial meningioma have been identified. The imaging features, histology, pathogenesis and differential diagnosis are discussed.

We have previously demonstrated that the transcription factor termed neuron restrictive silencer factor (NRSF) and the truncated splice variant, NRSF short form (sNRSF) are major modulators of preprotachykinin A (TAC1) gene expression. In this communication we addressed whether TAC1 gene expression would be effected in response to mechanical stimulation of both normal and osteoarthritic (OA) chondrocytes. Chondrocytes were mechanically stimulated for 20 min, and then incubated under normal tissue culture conditions for 1 or 3h. RT-PCR and quantitative PCR (qPCR) were used to investigate expression of TAC1, NRSF and sNRSF mRNA at these time points. Western blotting was used to validate and confirm expression of sNRSF protein in chondrocytes in response to mechanical stimulation. We observed that TAC1 was expressed in normal chondrocytes, with no evidence of NRSF or sNRSF expression. TAC1 mRNA expression did not significantly change following mechanical stimulation in normal cells. OA chondrocytes expressed TAC1 and sNRSF mRNA, though not NRSF, and following mechanical stimulation there was a significant upregulation of both TAC1 and sNRSF mRNA, which returned to baseline levels 3h post-stimulation. sNRSF protein was upregulated at 1 and 2h following stimulation of OA chondrocytes. In summary, differential expression of TAC1 and sNRSF in OA chondrocytes associates their expression with the disease. The change in expression of sNRSF and TAC1 mRNA following mechanical stimulation in OA but not normal chondrocytes suggests that sNRSF may be involved in the regulation of SP production in OA cartilage. These differences between normal and OA mechanotransduction responses may be important in the production of phenotypic changes present in diseased cartilage.

Metal-on-metal resurfacing arthroplasty of the hip has been used increasingly in Europe over the last ten years for the treatment of osteoarthritis of the hip in younger (

To examine whether inhibitors of the MAPK pathways will influence the response of bovine chondrocytes cultured in agarose constructs to IL-1beta and dynamic compression.Dose-response studies were conducted under IL-1beta conditions with either SB203580, SP600125, PDTC or curcumin. In separate experiments, constructs were treated with IL-1beta and an appropriate concentration of inhibitor and subjected to 15% dynamic compression. Nitrite and PGE2 release, 35SO4 and [3H]-thymidine incorporation were subsequently measured using biochemical assays.All inhibitors reduced the IL-1beta induced nitrite and PGE2 release in a dose-dependent manner. The inhibition of [3H]-thymidine incorporation by IL-1beta was partially reversed with SB203580, SP600125 or curcumin, but not PDTC. In most cases, the inhibitors reduced 35SO4 incorporation with IL-1beta. For the mechanical loading studies, the inhibitors reduced the compression-induced inhibition of nitrite and PGE2 release and restored [3H]-thymidine and 35SO4 incorporation.The MAPK, AP-1 and NF-kappaB signalling pathways are involved in the upregulation of NO and PGE2 release by IL-1beta. Dynamic compression stimulates cell proliferation and proteoglycan synthesis in the presence of IL-1beta and/or inhibitors of the MAPKs and NFkappaB and AP-1 signalling pathways. This experimental approach could provide valuable information for the biophysical/pharmacological treatment of OA.

Interleukin-1beta (IL-1beta) induces the release of nitric oxide (.NO) and prostaglandin E2 (PGE2) by chondrocytes and this effect can be reversed with the application of dynamic compression. Previous studies have indicated that integrins may play a role. In addition, IL-1beta upregulates the expression of iNOS and COX-2 mRNA via upstream activation of p38 MAPK. The current study examines the involvement of these pathways in mediating .NO and PGE2 release in IL-1beta stimulated bovine chondrocytes subjected to dynamic compression. Bovine chondrocytes were seeded in agarose constructs and cultured with 0 or 10 ng.ml(-1) IL-1beta with or without the application of 15% dynamic compressive strain at 1 Hz. Selected inhibitors were used to interrogate the role of alpha5beta1 integrin signalling and p38 MAPK activation in mediating the release of .NO and PGE2 in response to both IL-1beta and dynamic compression. The relative expression levels of iNOS and COX-2 were assessed using real-time quantitative PCR. Nitrite, a stable end product of .NO, was measured using the Griess assay and PGE2 release was measured using an enzyme immunoassay. IL-1beta enhanced .NO and PGE2 release and this effect was reversed by the application of dynamic compression. Co-incubation with an integrin binding peptide (GRGDSP) abolished the compression-induced effect. Real-time quantitative PCR analysis revealed that IL-1beta enhanced iNOS and COX-2 mRNA levels, with the maximum expression at 6 or 12 hours. Dynamic compression reduced this effect via a p38 MAPK sensitive pathway. These results suggest that dynamic compression acts to abrogate of .NO and PGE2 release by directly influencing the expression levels of iNOS and COX-2.