Subchondral Bone Sclerosis Research Articles

Resveratrol, a natural antioxidant, protects monosodium iodoacetate-induced osteoarthritic pain in rats

Osteoarthritis (OA) is a chronic progressive joint disease characterized by advanced joint pain, subchondral bone sclerosis and articular cartilage degeneration. Resveratrol has been shown to have anti-inflammatory, cardioprotective and antioxidant properties and to inhibit platelet aggregation and coagulation. However, the effects of resveratrol on OA have not been examined. In this study, we investigate the protective effects of resveratrol on monosodium iodoacetate (MIA)-induced OA through inhibition of cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) signaling pathway in a rat model. A single intra-articular injection of MIA was injected into rats for the induction of OA. The mechanical, heat and cold hyperalgesia were measured at days 0, 7 and 14. The serum and synovial fluid levels of IL-1β, IL-10 and TNF-α and osteocalcin were measured by enzyme-linked immunosorbent assay. The mRNA and protein expressions of IL-1β, IL-10, TNF-α, Il-6, MMP-13 and COX-2 and iNOS were determined by RT-PCR and western blot, respectively. Osteoarthritic lesion in the knee joint was evaluated by histological analysis. MIA-injected rats treated with resveratrol at a dose of either 5 or 10mg/kg body weight were significantly reduced hyperalgesia of mechanical, heat and cold and increased the vertical and horizontal movements. Subsequently, MIA-injected rats increased serum and synovial fluid levels of IL-1β, IL-10, IL-6, TNF-α, MMP-13 and osteoclastic activity marker, osteocalcin and its articular cartilage mRNA and protein expressions. Further, MIA-injected rats increased COX-2 and iNOS mRNA and protein expressions were decreased by resveratrol. The protective effect of resveratrol was comparable to a reference drug, etoricoxib. The cartilage damage induced by MIA were attenuated by resveratrol. Taken together, resveratrol has the potential to improve MIA-induced cartilage damage by inhibiting the levels and expressions of inflammatory mediators suggesting that resveratrol may be a potential therapeutic agent for OA.

Subchondral bone sclerosis and cancellous bone loss following OA induction depend on the underlying bone phenotype

ObjectivesOsteoarthritis (OA) is increasingly considered a disease of the whole joint, yet the interplay between the articular cartilage and the subchondral bone remains obscure. We here set out to investigate the impact of bone mass on the progression of surgically induced knee OA in the mouse. MethodsOA was induced in the right knees of female C57BL/6 (low bone mass) and STR/ort (high bone mass) mice via anterior cruciate ligament transection and destabilization of the medial meniscus. At 36 weeks of age, left and right knee joints were histologically compared for cartilage degeneration and via microCT analysis for subchondral bone plate thickness. In addition, femora were analyzed for bone mass at diaphysis and distal meta- and epiphysis. ResultsThe severity of cartilage deterioration did not differ under high and low bone mass conditions. However, the extent of bone sclerosis differed and was proportional to the baseline subchondral bone plate thickness. Moreover, the cancellous bone loss following OA progression was inversely related to the bone mass: high bone mass restricted the loss to the epiphysis, whereas low bone mass allowed for a more widespread loss extending into the metaphysis. ConclusionsOur results suggest that cartilage degeneration is independent of the underlying bone mass. In contrast, subchondral bone remodeling associated with OA progression seem to correlate with the initial bone mass and suggest an enhanced crosstalk between the deteriorating cartilage and the subchondral bone under low bone mass conditions.

Histopathological Features of Facet Osteoarthritis in Patients with Lumbar Spinal Stenosis

Introduction Lumbar spinal stenosis (LSS) is a degenerative, age-related narrowing of the lower spinal canal that causes pressure on the nerves, leading to pain and reduced mobility. Hypertrophy of ligament flava and facet joints combined with disc protrusions are a causative for LSS. Osteoarthritic changes to the facet joint, including joint space narrowing, subchondral cysts and osteophyte formation are commonly detected using magnetic resonance imaging (MRI) and computed tomography (CT) scanning. However, the pathomechanisms of facet joint osteoarthritis (OA) at a cellular and molecular level are poorly understood and have been scarcely studied. In this study we sought to investigate the histological features and to uncover cellular pathomechanisms of facet joint OA. Material and Methods Fifteen patients undergoing surgical decompression due to degenerative LSS were included in this study (9 female/6 male, median age 66, range 49–85). Routine preoperative X-ray and MRI scanning of the lumbar spine was performed in all subjects. Severity of facet joint OA was assessed in MRI images using the Weishaupt grading system for the lumbar spine. The medial portion of facet joints was collected during surgical decompression of the spinal canal and processed for tissue histology. Tissue morphology was evaluated using haematoxylin and eosin (HE), Safranin-O and van Gieson's stains. The presence of macrophages, blood vessels and nerve fibers was investigated using immunohistological staining for their respective markers CD68, CD34 and PGP9.5. Functional osteoclasts were visualized using tartrate-resistant acid phosphatase (TRAP) staining. Subchondral bone area fraction (B.Ar/T.Ar) was determined using the ImageJ-plugin BoneJ. Results OA was evident in MRI images characterized by joint space narrowing, bone edema and cysts and hypertrophy of articular processes. The severe OA phenotype in facet joints from LSS patients was confirmed at a histological level by complete loss of proteoglycan staining, CD34+ vessel penetration, and fissuring of cartilage tissue. Subchondral B.Ar/T.Ar ranged between 0.6 and 0.8. In all samples, subchondral marrow spaces contained CD34+ blood vessels and CD68+ mononuclear macrophages. CD68+ multinucleated osteoclasts were detected in resorption pits at the bone surface in 80 percent of the patients. Functionality of osteoclasts was confirmed by positive staining of multinucleated bone cells for TRAP in serial sections. Osteoblast activity was demonstrated in 60 percent of the patients and predominantly characterized by large areas of intramembranous bone formation near the osteochondral junction. Formation of an osteoclast-rich pannus-like tissue was seen in one sample. Innervation of subchondral marrow spaces by PGP9.5-positive nerve fibers was scarce and exclusively found in the vicinity of arterioles. Conclusion Facet joints in LSS patients display radiological and histopathological features of OA. Two major OA phenotypes can be distinguished based upon cellular pathomechanisms in subchondral bone tissue: 1) osteoblast-rich intramembranous bone formation and 2) osteoclast/macrophage rich remodeling. Osteoimmunological mechanisms, specifically the interaction of CD68+ macrophages with bone-resident cells, play a – previously unknown – role in regulating subchondral bone sclerosis in progressive osteoarthritis. Targeting osteoimmunology might hold potential as a disease-modifying treatment for osteoarthritis.

Crystallographic orientation of the c-axis of biological apatite as a new index of the quality of subchondral bone in knee joint osteoarthritis.

The aim of the present study was to investigate the preferred orientation of biological apatite (BAp) as a new index of the quality of subchondral bone (SB) in knee joint osteoarthritis (OA). Ten OA and five normal knee joints were obtained. Thickness, quantity and bone mineral density (BMD) of SB were analyzed at the medial condyle of the femur in dry conditions by peripheral quantitative computed tomography. In addition, the preferred crystallographic orientation of the c-axis of BAp was evaluated as bone quality parameter using a microbeam X-ray diffractometer technique. BMD and thickness of SB were significantly increased in OA specimens compared to normal knee specimens (P<0.01), and the preferred orientation of the c-axis of BAp along the normal direction of SB surface was significantly higher in OA specimens (P<0.01), reflecting the change in stress of concentration in the pathological portion without cartilage. SB sclerosis in OA results in both proliferation of bone tissues and enhanced degree of preferential alignment of the c-axis of BAp. Our findings could have major implications for the diagnosis of clinical studies, including pathologic elucidation in OA.

Ablation of Perlecan Domain 1 Heparan Sulfate Reduces Progressive Cartilage Degradation, Synovitis, and Osteophyte Size in a Preclinical Model of Posttraumatic Osteoarthritis.

To investigate the role of the heparan sulfate (HS) proteoglycan perlecan (HSPG-2) in regulating fibroblast growth factor (FGF) activity, bone and joint growth, and the onset and progression of posttraumatic osteoarthritis (OA) in a mouse gene-knockout model. Maturational changes were evaluated histologically in the knees of 3-, 6-, and 12-week-old wild-type (WT) mice and Hspg2(Δ3-/Δ3-) mice (Hspg2 lacking domain 1 HS, generated by ablation of exon 3 of perlecan). Cartilage damage, subchondral bone sclerosis, osteophytosis, and synovial inflammation were scored at 4 and 8 weeks after surgical induction of OA in WT and Hspg2(Δ3-/Δ3-) mice. Changes in cartilage expression of FGF-2, FGF-18, HSPG-2, FGF receptor 1 (FGFR-1), and FGFR-3 were examined immunohistochemically. Femoral head cartilage from both mouse genotypes was cultured in the presence or absence of interleukin-1α (IL-1α), FGF-2, and FGF-18, and the content and release of glycosaminoglycan (GAG) and expression of messenger RNA (mRNA) for key matrix molecules, enzymes, and inhibitors were quantified. No effect of perlecan HS ablation on growth plate or joint development was detected. After induction of OA, Hspg2(Δ3-/Δ3-) mice had significantly reduced cartilage erosion, osteophytosis, and synovitis. OA-induced loss of chondrocyte expression of FGF-2, FGF-18, and HSPG-2 occurred in both genotypes. Expression of FGFR-1 after OA induction was maintained in WT mice, while FGFR-3 loss after OA induction was significantly reduced in Hspg2(Δ3-/Δ3-) mice. There were no genotypic differences in GAG content or release between unstimulated control cartilage and IL-1α-stimulated cartilage. However, IL-1α-induced cartilage expression of Mmp3 mRNA was significantly reduced in Hspg2(Δ3-/Δ3-) mice. Cartilage GAG release in either the presence or absence of IL-1α was unaltered by FGF-2 in both genotypes. In cartilage cultures with FGF-18, IL-1α-stimulated GAG loss was significantly reduced only in Hspg2(Δ3-/Δ3-) mice, and this was associated with maintained expression of Fgfr3 mRNA and reduced expression of Mmp2/Mmp3 mRNA. Perlecan HS has significant roles in directing the development of posttraumatic OA, potentially via the alteration of FGF/HS/FGFR signaling. These data suggest that the chondroprotection conferred by perlecan HS ablation could be attributed, at least in part, to the preservation of FGFR-3 and increased FGF signaling.

Atsttrin, an engineered protein derived from progranulin growth factor, is therapeutic in osteoarthritis

Purpose: Progranulin (PGRN) is a multifunctional growth factor which is composed of seven-and-a-half repeats of a cysteine-rich motif in the order P-G-F-B-A-C-D-E. PGRN directly binds to TNF-α receptors (TNFR), and inhibits TNF-α activity. Atsttrin(A50), an engineered protein composed of three PGRN fragments (1/2F-1/2A-1/2C), exhibited selective TNFR binding, and potently prevented inflammation in multiple arthritis mouse models (Tang, et al, Science, 2011). In addition, recent studies demonstrated that local injection of PGRN significantly prevented OA progression (Zhao et al, Annals of the Rheumatic Diseases, 2015). These previous data promoted us to determine whether Atsttrin also has therapeutic effects in preventing OA, and if so what is the molecular mechanism involved. Methods: Mice models were established to determine the role A50 in OA pathogenesis. Primary human cartilage and chondrocyte were used to determine the anti-catabolic and anabolic effect of A50. Results: 1. Atsttrin effectively prevents osteoarthritis in surgically-induced mice model in vivo. ACL (anterior cruciate ligament) transection model in wild type mice was established to determine the preventative effect of A50. 6μg A50 was intra-articular injected weekly for 4 weeks once the model was established. As shown in Fig.1A, Safranin O staining indicated A50 effectively prevents cartilage loss in OA progression. Additionally, as demonstrated in Fig.1B&C, histological grading analysis showed significant improvement of OA score and attenuation of proteoglycan loss in cartilage with treatment of A50. Furthermore, as shown in Fig.1D, immunochemistry staining demonstrated that the A50 decreased the degradation of cartilage matrix molecules (e.g. COMP), and reduced the hypertrophic markers of chondrocyte (e.g. Col X), and suppressed the expression of matrix-degrading enzymes (e.g. MMP13), slowing down OA progression. Furthermore, OA is primarily characterized by cartilage destruction but also involves other pathological changes, including synovial inflammation and subchondral bone sclerosis, in all tissues of joints. As indicated in Fig.1E, H&E staining revealed that A50 effectively prevented subchondral bone sclerosis and synovial inflammation. 2. Atsttrin inhibits TNF-α-induced catabolic metabolism and promotes anabolic metabolism. It is well established that inflammatory cytokine TNF-α plays an important role in cartilage degeneration in OA. The finding that A50 prevented cartilage destruction in inflammatory arthritis mediated by TNF-α (Tang, et al, Science, 2011) prompted us to investigate the potential association between A50 and TNF-α in OA. Cartilage samples from patients with OA were isolated and cultured with 10 ng/mL TNF-α in presence or absence of 200 ng/mL A50 for 7 days. As revealed in Fig.2A&B, Safranin O staining of the cartilage samples indicated that TNF-α markedly enhanced loss of proteoglycan, while the additional use of A50 largely rescued this effect of TNF-α. Moreover, primary human chondrocytes were treated in with 10 ng/mL TNF-α in presence or absence of 200 ng/mL A50 for indicated time points. As shown in Fig.2C, A50 dramatically inhibited TNF-α-mediated pro-inflammatory signaling(P38, JNK pathways). In addition, chondrocytes were cultured for 72 hours, as demonstrated in Fig.2D, A50 dose-dependently inhibited expression of TNF-α-induced matrix-degrading enzymes (e.g. MMP-3, MMP-13, ADAMTS-4) and inflammatory severity marker, NOS-2. More importantly, as revealed in Fig.2E, A50 remarkably promoted anabolism in primary human cartilage. As indicated in Fig.2F&G, anabolic effect of A50 was further confirmed by the increased gene expression of aggrecan and Col II in human primary chondrocytes. Conclusions: These findings not only provide novel insights into the role of Atsttrin in cartilage homeostasis and arthritis in vivo, but may also lead to the development of novel therapeutic intervention strategies for osteoarthritis.Fig.2 Atsttrin inhibits TNF-α-induced catabolic metabolism and promotes anabolic metabolism. (A) Safranin O staining of primary human cartilage in presence or absence of 200ng/ml Atsttrin with or without 10ng/ml TNF-α.(B) Statistic analysis of proteoglycan area based on Safranin O staining. (C) Primary human chondrocytes were incubated with TNF-α in the presence or absence of Atsttrin, and phosphorylation and expression of the indicated signaling molecules at various time points were determined by immunoblotting. Tubulin is shown as a loading control. (D) The protein level of MMP-3, MMP-13, ADAMTS-4 and NOS-2 in primary human chondrocytes with 10ng/ml TNF-α in absence or presence of Atsttrin. (E) Atsttrin promoted anabolism in primary human cartilage, assayed by Safranin O staining. (F-G) Levels of anabolic biomarkers in chondrocyte, including Acn and Col II, as measured by real-time PCR. Values are the normalised mean±SEM; *p<0.05, **p<0.01 versus control group. Six cartilages were used in each group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Targeting VEGF and Its Receptors for the Treatment of Osteoarthritis and Associated Pain.

Increased vascular endothelial growth factor (VEGF) levels are associated with osteoarthritis (OA) progression. Indeed, VEGF appears to be involved in OA-specific pathologies including cartilage degeneration, osteophyte formation, subchondral bone cysts and sclerosis, synovitis, and pain. Moreover, a wide range of studies suggest that inhibition of VEGF signaling reduces OA progression. This review highlights both the potential significance of VEGF in OA pathology and pain, as well as potential benefits of inhibition of VEGF and its receptors as an OA treatment. With the emergence of the clinical use of anti-VEGF therapy outside of OA, both as high-dose systemic treatments and low-dose local treatments, these particular therapies are now more widely understood. Currently, there is no established disease-modifying drug available for patients with OA, which warrants continued study of the inhibition of VEGF signaling in OA, as stand-alone or adjuvant therapy. © 2016 American Society for Bone and Mineral Research.

Attenuation of cartilage degeneration by calcitonin gene-related paptide receptor antagonist via inhibition of subchondral bone sclerosis in osteoarthritis mice.

Osteoarthritis (OA) is a progressive joint disorder which affects cartilage and subchondral bone. Calcitonin gene-related peptide (CGRP) plays a role in bone metabolism. The purpose of this study is to examine the therapeutic effect of the blocking CGRP on OA progression in mice by inhibition of subchondral bone sclerosis. OA was induced by the resection of the medial meniscotibial ligament of the knee in C57/BL6 mice. An intraperitoneal injection of the CGRP receptor antagonist (BIBN4096) was administered after OA surgery. At 1, 4, and 8 weeks after injection, histological analysis were performed. In vitro, the effect of CGRP and BIBN4096 on osteogenesis and osteoclastogenesis was analyzed. BIBN4096 could prevent cartilage degeneration and subchondral bone sclerosis. The OARSI score in the BIBN4096 group was significantly lower than that in the control. In vitro, CGRP up regulated osteocalcin expression, but its expression was down regulated by BIBN4096. CGRP inhibited osteoclastogenesis of raw 267.4 cells, but its effect was reduced by the addition of BIBN4096.The current study showed that subchondral bone sclerosis and increasing expression of CGRP occurs in the early phase of OA in relation to cartilage degeneration, and that BIBN4096 could effectively attenuate OA progression. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1177-1184, 2016.

Effectiveness of Ozone in Osteoarthritis of the Knee

Osteoarthritis (OA) is a degenerative disease that is very prevalent, and it usually increases exponentially with age [1]. OA affects weight bearing articulations as the knee, and is very common in women [1]. Knee OA affects negatively the function and autonomy of older people [2], decreasing patient’s quality of life [3]. OA produces a great use of limited health resources [3]. The normal knee is composed by cartilage, subchondral bone, synovial tissue and articular capsule. Knee OA produces destruction of articular cartilage and sclerosis of subchondral bone [2]. Knee OA diminishes the articular space, and it favours the formation of osteophyte and subchondral cysts [2]. To date, there is neither effective treatment nor cure for OA. The objectives on treating Knee OA are to ameliorate pain, rigidity, inflammation and to avoid articular damage and joint destruction [2]. Recently, OA is related to chronic inflammation, besides mechanical and biological factors. In that scenario, future OA treatments must regulate inflammation to avoid OA progression. Fernandez-Cuadros et al state that Ozone acts on the modulation of inflammation [2], based on several studies and years of clinical experience. It is believed that Ozone acts on the articulation as a stimulator, repairing the fibroblastic tissue. Moreover, Ozone could reduce inflammation and promote growth of new cartilage [2]. We present the case of an 83 years-old female, with Kellgren-Lawrence 3rd degree, and with joint narrowing space and subchondral cyst, and moderate-severe pain on the knee. After a two series of ozone therapy (20 cc of a mixture of OxygenOzone 95-5%, at 20 ug concentration, 4 weekly sessions) separated by six months, the recovering of the joint space and of the subchondral cyst and a decrease in pain is been observed (Figures 1-3). This case report and many years of experience demonstrate that Ozone is capable of diminishing pain and inflammation. Ozone acts on the repair of articular cartilage and subchondral bone, because it increases vascularisation of the articular cartilage [2].

Non-invasive techniques for studying macrophages in joint inflammation

Folate-based radiotracers have been used in patients with cancer and inflammatory diseases to visualize folate receptor expressing cells using PET or SPECT techniques. Activated macrophages express folate receptor beta (FR-β) and this allows specific imaging of these cells in-vivo. From previous work using SPECT imaging to visualize folate receptor expressing macrophages in both animal models and in patients with OA we know that macrophages are present in OA affected joints. However, it remains unclear what role these macrophages play in the different stages of OA and whether their role can be influenced by specific targeting. In Wistar rats osteoarthritis was induced using a low dose of intra-articular papain injections in one knee joint combined with exposure to a moderate exercise protocol. After six weeks and twelve an in vivo folate SPECT/CT scan and micro-CT analyses were performed. Macrophages from human peripheral blood monocytes were cultured (7 days) in the presence of GM-CSF (M1 proinflammatory phenotype) or M-CSF (M2 anti-inflammatory phenotype). Subsequently the macrophages were treated with LPS, cytokines (IL-4, IL-10, IFN-y) or a corticoid steroid (triamcinolone acetonide, 1µg/ml). Folate receptor beta (FRβ) as well as other macrophage marker expressions were measured using FACS. Intra-articular injections with triamcinolone strongly enhanced FRβ+ macrophage activation and fully prevented osteophyte formation. There were no beneficial effects of the corticoid steroid against cartilage degradation or subchondral bone sclerosis. In in-vitro cultures triamcinolone strongly induced the monocyte-macrophages differentiation towards CD163+ and FRβ+ cells, specifically in GM-CSF stimulated (M1) cultures. Addition of triamcinolone to M-CSF stimulated (M2) monocytes showed enhanced IL10 expression on mRNA level. In conclusion triamcinolone enhanced FRβ expression in monocytes that were induced to macrophage differentiation. The triamcinolone injections stimulate synovial macrophage activation and triggers the macrophages towards a more anti-inflammatory subtype. Figure 1 Macrophage activation determined after injection of 111In-DTPA-folate using SPECT/CT in Papain induced osteoarthritis (OA) combined with moderate exercise. Representative sagittal SPECT/CT images of knee joints from representative animals per experimental ...

Triamcinolone acetonide activates an anti-inflammatory and folate receptor-positive macrophage that prevents osteophytosis in vivo.

IntroductionTriamcinolone acetonide (TA) is used for osteoarthritis management to reduce pain, and pre-clinical studies have shown that TA limits osteophyte formation. Osteophyte formation is known to be facilitated by synovial macrophage activation. TA injections might influence macrophage activation and subsequently reduce osteophytosis. Although widely applied in clinical care, the mechanism through which TA exerts this effect remains unknown. In this animal study, we investigated the in vivo effects of TA injections on macrophage activation, osteophyte development and joint degeneration. Furthermore, in vitro macrophage differentiation experiments were conducted to further explain working mechanisms of TA effects found in vivo.MethodsOsteoarthritis was induced in rat knees using papain injections and a running protocol. Untreated and TA-treated animals were longitudinally monitored for 12 weeks with in vivo micro–computed tomography (μCT) to measure subchondral bone changes. Synovial macrophage activation was measured in vivo using folate receptor β (FRβ)-targeted single-photon emission computed tomography/computed tomography. Articular cartilage was analyzed at 6 and 12 weeks with ex vivo contrast-enhanced μCT and histology. To further explain the outcomes of our in vivo study, TA on macrophages was also studied in vitro. These cultured macrophages were either M1- or M2-activated, and they were analyzed using fluorescence-activated cell sorting for CD163 and FRβ expression as well as for messenger RNA (mRNA) expression of interleukin (IL)-10.ResultsOur in vivo study showed that intra-articular injections with TA strongly enhanced FRβ+ macrophage activation. Despite stimulated macrophage activation, osteophyte formation was fully prevented. There was no beneficial effect of TA against cartilage degradation or subchondral bone sclerosis. In vitro macrophage cultures showed that TA strongly induced monocyte differentiation towards CD163+ and FRβ+ macrophages. Furthermore, TA-stimulated M2 macrophages showed enhanced IL-10 expression at the mRNA level.ConclusionsTA injections potently induce a CD163+- and FRβ+-activated macrophage with anti-inflammatory characteristics such as reduced IL-10 production in vitro and lack of osteophytosis in vivo.

Insulin-Like Growth Factor II (IGF-II) Inhibits IL-1β-Induced Cartilage Matrix Loss and Promotes Cartilage Integrity in Experimental Osteoarthritis.

Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1β-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1β, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro. In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA.

Therapeutic effects of an anti-ADAMTS-5 antibody on joint damage and mechanical allodynia in a murine model of osteoarthritis

The primary goal of this study was to test the disease-modifying effect of blocking a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 with a neutralizing monoclonal antibody (mAb) starting 4 weeks after destabilization of the medial meniscus (DMM) in the mouse. We also investigated whether ADAMTS-5 blockade reversed mechanical allodynia and decreased monocyte chemoattractant protein (MCP)-1 production by dorsal root ganglia (DRG) cells. Ten-week old male C57BL/6 mice underwent DMM surgery and were either left untreated or treated with anti-ADAMTS-5 mAb or IgG2c isotype control mAb starting 4 weeks after surgery. Knees were collected for histopathology 4 or 12 weeks later. Mechanical allodynia was monitored biweekly in the ipsilateral hind paw through 16 weeks. DRG were collected and cultured 8 weeks after DMM for analysis of MCP-1 production. By 4 weeks after DMM, mild cartilage degeneration was evident in the medial compartment, small osteophytes were present, and subchondral bone sclerosis was established. By 16 weeks after surgery, significant cartilage deterioration was apparent on the medial tibial plateaux and medial femoral condyles, osteophyte size had increased, and subchondral bone sclerosis was maintained. Treatment with ADAMTS-5 mAb from week 4 to 16 after surgery slowed cartilage degeneration and osteophyte growth but did not affect subchondral bone sclerosis. Moreover, ADAMTS-5 blockade resulted in temporary reversal of mechanical allodynia, which correlated with decreased MCP-1 production by cultured DRG cells. This study suggests therapeutic efficacy of an ADAMTS-5 mAb in the DMM model, when therapy starts early in disease.

Brief Review of Genomics in Osteoarthritis

Osteoarthritis (OA), the most common form of arthritis around the world, is a multifactorial degenerative joint disorder characterized by articular cartilage degradation, subchondral bone sclerosis, osteophyte formation, synovial membrane inflammation and afection of extraarticular tissue as tendon, ligaments and articular capsule. Although various factors have been recognized as essential in OA development, there is a strong hereditary component and is considered to be a polygenic disease. The identification of genes associated with osteoarthritis can help reveal biological mechanisms that may lead to development of new therapeutic targets or biomarkers for early detection and risk stratification. The goal of this brief review is to offer an overview of the current status of genetics of osteoarthritis.

Acute loading injuries and their relation to post-traumatic osteoarthritis in a preclinical model

Purpose: Up to 50% of knee injuries that result in tears of the ACL progress to post-traumatic OA (ptOA) within 10-15 years of the initial trauma. This represents an almost 5-fold increase in risk compared to non-injured joints, with current interventions (including surgical reconstruction) not significantly altering OA risk. Factors other than altered biomechanics may therefore play a significant role in disease development. Risk, rate of onset, and progression of ptOAmay be linked to the trauma's impact severity and the joint structures affected both acutely and chronically. A better understanding of the interaction of these variables may improve prognostic capability and enable the development of specific treatments for injury management and disease prevention. In the present study we investigated the relationships between age, gender, body mass, joint load, ACL rupture, and ptOA pathology using a murine model of non-surgical ACL rupture. Methods: ACL injury was induced by a single, axial compressive load to the flexed knee of themouse. At the peak load,10-15 seconds of sustained compression was applied before release. An initial cadaveric study assessed ACL failure loads over a range of mouse biometrics (9-52 week old, male and female C57BL-6, n1⁄4164). The failure load was determined from force-displacement curves and the mode of failure by microscopic evaluation. The potential predictors of these outcomes were assessed using mixed model linear regression and mixed model multinomial logistic regression. A subsequent in vivo study loaded the knees of anaesthetisedmice (10-12 week, male C57Bl-6) to either failure or 55% of the mean failure. Joint pathology was assessed histologically at 2 or 4 weeks post-injury (n1⁄47) in both the medial and lateral compartments. Results: In the cadaveric study, damage induced by loading was largely restricted to the ACL with little evidence of injury to other joint tissues. The ACL was consistently ruptured but the mode of failure varied between specimens. Ruptures occurred towards the femoral insertion with 16% of tears entirely midsubstance, 35% total avulsion fractures and 49% demonstrating a mixture of the two. Logistic regression determined that there was no effect of age, mass, rupture load or joint stiffness on the mode of failure. However, when comparing males and females with avulsion fractures, the relative risk for females was 7.11 (95% CI [1.36 37.07]). The ACL rupture load itself was demonstrated to have a positive association with mass (p < 0.001) but not age or gender when correcting for other covariates. In the in vivo study, complete ACL rupture resulted in significant femorotibial OApathology, particularly towards theposterior region and greatest in the medial compartment. At 2 weeks substantial synovitis, tibio-femoral cartilage erosion and structural damage to themedialmeniscuswere evident. By 4 weeks cartilage erosion progressed and subchondral bone sclerosis, osteophytes and enthesophytes developed. In contrast, subfailure loading of the knee resulted in only mild OA pathology. In these knees the ACL remained intact and there was no evidence of synovitis or osteophyte development. There was mild cartilage damage (unlike ACL failure this was similar in medial and lateral compartments) with significant progression from2 to 4weeks. Avery localised subchondral bone remodelling response was also evident posteriorly in the tibia. Conclusions: In this model ACL failure loads have been established for a range of mouse biometrics and were shown to be independent of the age and sex of the mouse. In vivo, complete ACL failure results in the rapid onset of pathology with a clinically relevant pattern of OA change in all other joint tissues. At the early time points examined in this study, mechanical loading alone (no ACL rupture) induces mild changes largely restricted to the cartilage, although this may progress further given more time. Comparisons with other loading regimes and surgical models of ptOA may further illuminate the complex interactions that occur between different joint tissues and risk factors for ptOA.

Elevated hepatocyte growth factor levels in osteoarthritis osteoblasts contribute to their altered response to bone morphogenetic protein-2 and reduced mineralization capacity

PurposeClinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts is involved in the progression of osteoarthritis (OA). Human osteoblasts isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/ß-catenin pathway, and a reduced mineralization in vitro as well as in vivo. These alterations were linked with an abnormal response to BMP-2. OA osteoblasts release factors such as the hepatocyte growth factor (HGF) that contribute to cartilage loss whereas chondrocytes do not express HGF. HGF can stimulate BMP-2 expression in human osteoblasts, however, the role of HGF and its effect in OA osteoblasts remains unknown. Here we investigated whether elevated endogenous HGF levels in OA osteoblasts are responsible for their altered response to BMP-2. MethodsWe prepared primary human subchondral osteoblasts using the sclerotic medial portion of the tibial plateaus of OA patients undergoing total knee arthroplasty, or from tibial plateaus of normal individuals obtained at autopsy. The expression of HGF was evaluated by qRT-PCR and the protein production by western blot analysis. HGF expression was reduced with siRNA technique whereas its activity was inhibited using the selective inhibitor PHA665752. Alkaline phosphatase activity (ALPase) and osteocalcin release were measured by substrate hydrolysis and EIA respectively. Canonical Wnt/β-catenin signaling (cWnt) was evaluated both by target gene expression using the TOPflash TCF/lef luciferase reporter assay and western blot analysis of β-catenin levels in response to Wnt3a stimulation. Mineralization in response to BMP-2 was evaluated by alizarin red staining. ResultsThe expression of HGF was increased in OA osteoblasts compared to normal osteoblasts and was maintained during their in vitro differentiation. OA osteoblasts released more HGF than normal osteoblasts as assessed by western blot analysis. HGF stimulated the expression of TGF-β1. BMP-2 dose-dependently (1 to 100ng/ml) stimulated both ALPase and osteocalcin in normal osteoblasts whereas, it inhibited them in OA osteoblasts. HGF–siRNA treatments reversed this response in OA osteoblasts and restored the BMP-2 response. cWnt is reduced in OA osteoblasts compared to normal, and HGF–siRNA treatments increased cWnt in OA osteoblasts almost to normal. Smad1/5/8 phosphorylation in response to BMP-2, which is reduced in OA osteoblasts, was corrected when these cells were treated with PHA665752. The BMP-2-dependent mineralization of OA osteoblasts, which is also reduced compared to normal, was only partially restored by PHA665752 treatment whereas 28days treatment with HGF reduced the mineralization of normal osteoblasts. ConclusionOA osteoblasts expressed more HGF than normal osteoblasts. Increased endogenous HGF production in OA osteoblasts stimulated the expression of TGF-β1 and reduced their response to BMP-2. Inhibiting HGF expression or HGF signaling restored the response to BMP-2 and Smad1/5/8 signaling. In addition, decreased HGF signaling partly corrects the abnormal mineralization of OA osteoblasts while increased HGF prevents the normal mineralization of normal osteoblasts. In summary, we hypothesize that sustained elevated HGF levels in OA osteoblasts drive their abnormal phenotype and is implicated in OA pathophysiology.

Deficiency of G-protein coupled receptor 40, a lipid-activated receptor, heightens in vitro- and in vivo-induced murine osteoarthritis.

Osteoarthritis (OA) is an age-related degenerative joint disease. To date, its management is focused on symptoms (pain and inflammation). Studies suggest that fatty acids can reduce the expression of inflammatory and catalytic mediators, and improve invivo joint function. Free fatty acid receptors (FFARs) such as G-protein coupled receptor 40 (GPR40) are proposed as attractive therapeutic targets to counteract inflammation and cartilage degradation observed in OA. This study aims to elucidate the involvement of GPR40 in OA. In this study, we used an invitro model of OA, and surgically induced OA by ligament transection and partial meniscectomy in wild-type and GPR40 deficient mice. OA phenotype was investigated invivo by histology and genes expression. We demonstrate that IL-1β-treated GPR40(-/-) chondrocytes secret more inflammatory mediators (nitric oxide, interleukin-6, prostaglandin E2) and active catabolic enzymes (metalloproteinase-2, -9 [MMP-2, MMP-9]), and show decreased anabolism (glycoaminoglycan) compared to GPR40(+/+) cells. In accordance with these results, we show that GPR40(-/-) mice exhibit an aggravated OA-induced phenotype characterized by higher tidemark exposure, frequency of osteophyte formation and subchondral bone sclerosis. Altogether our results demonstrate that GPR40 deficiency leads to an extended OA phenotype, providing evidence that increasing GPR40 activity, by natural or synthetic ligands, could be a new strategy in the management of OA.

Diacerein protects against iodoacetate-induced osteoarthritis in the femorotibial joints of rats.

The present study was undertaken to investigate the effect of diacerein on the histopathology of articular cartilage and subchondral bone of the femorotibial joint in rats. Osteoarthritis was induced in rats after single intra-articular injection of sodium iodoacetate. Rats were sacrificed 1, 2, 4, and 8 weeks post intra-articular injection to evaluate the progression of histopathogenesis of osteoarthritis. Diacerein was orally administered (15 mg/kg) once daily post 1 and 2 weeks of iodoacetate injection in two groups, respectively, for up to 12 weeks. Articular cartilage and subchondral bone of the rats of both groups were examined after 8 and 12 weeks, respectively. Quantitative histological analyses were performed by scoring these sections as per the OARSI system. Chondroitin sulfate was also estimated in articular cartilage by decrease in absorbance of methylene blue on complexation with chondroitin sulfate using a spectrophotometer. Intra-articular injection of iodoacetate induced loss of articular cartilage with progressive subchondral bone sclerosis and degeneration. Based on histopathological and biochemical findings, diacerein treatment showed chondroprotective effect. Furthermore, the chondroprotective effect of diacerein was found to be more pronounced after 12 weeks as compared to 8 weeks in both cases (i.e., post 1 and 2 weeks of iodoacetate injection). Similar results were observed by investigation of chondroitin sulfate during biochemical study, showing the chondroprotective effect. In conclusion, diacerein exhibits chondroprotective effect in rats with late onset of action.

Obesity and osteoarthritis, more than just wear and tear: pivotal roles for inflamed adipose tissue and dyslipidaemia in obesity-induced osteoarthritis.

OA is a degenerative joint disease characterized by articular cartilage degradation, osteophyte formation, synovitis, and subchondral bone sclerosis. One of OAs main risk factors is obesity. To date, it is not fully understood how obesity results in OA. Historically, this link was ascribed to excessive joint loading as a result of increased body weight. However, the association between obesity and OA in non-weight-bearing joints suggests a more complex aetiology for obesity-induced OA. In the present review, the link between obesity and OA is discussed. First, the historical view of altered joint loading leading to wear and tear of the joint is addressed. Subsequently, the effects of a disturbed lipid metabolism, low-grade inflammation, and adipokines on joint tissues are discussed and linked to OA. Taken together, inflamed adipose tissue and dyslipidaemia play pivotal roles in obesity-induced OA. It becomes increasingly clear that the link between obesity and OA transcends excessive loading.

Iodoacetate and allogenous cartilage particles as models for arthritis induction in equine

Experimental models of osteoarthritis (OA) have been widely developed in different animal species, because of the high incidence of osteoarthritis diseases in humans and animals. To date, no ideal OA animal model has been reported. The present study compare different osteoarthritis models to determine which one is suitable for inducing experimental equine OA. Fifteen donkeys were divided into three equal groups (n=5). The radio carpal joints of the right forelimb of 15 donkeys were injected with 25mg monoiodoacetate (MIA) (group A), 50mg allogenous cartilage particles (ACP) (group B), or vehicle solution (group C) over a period of 70days. Osteoarthritis induction was evaluated weekly through lameness score, carpal circumference, joint flexion angel, synovial fluid analysis (total protein and WBC count), and radiology. Animal were euthanized and joints histopathology were performed at 70days. Lameness score and joint circumference was increased in both group A and B however joint flexion angel was decreased compared to group C (p<0.05). Osteophytes were observed in MIA injected joints only accompanied with subchondral bone sclerosis. Cartilage damage was observed grossly and histologically in Group A together with synovial membrane fibrosis. Group B had on cartilage damage grossly however histological examination revealed some cartilage surface discontinuity with synovial membrane edema. Injection of monoiodoacetate in the donkey is a successful model to create the acute clinical signs of joint disease as well as cartilage damage. However, allogenous cartilage particles injection need more investigation to be applied.