Objective. We have previously identified in articular cartilage an abundant pool of the heparin-binding growth factor, fibroblast growth factor 2 (FGF-2), which is bound to the pericellular matrix heparan sulfate proteoglycan, perlecan. This pool of FGF-2 activates chondrocytes upon tissue loading and is released following mechanical injury. In vitro, FGF-2 suppresses interleukin-1-driven aggrecanase activity in human cartilage explants, suggesting a chondroprotective role in vivo. We undertook this study to investigate the in vivo role of FGF-2 in murine cartilage.Methods. Basal characteristics of the articular cartilage of Fgf2 -/-and Fgf2 ؉/؉ mice were determined by histomorphometry, nanoindentation, and quantitative reverse transcriptase-polymerase chain reaction. The articular cartilage was graded histologically in aged mice as well as in mice in which osteoarthritis (OA) had been induced by surgical destabilization of the medial meniscus. RNA was extracted from the joints of Fgf2 -/-and Fgf2 ؉/؉ mice following surgery and quantitatively assessed for key regulatory molecules. The effect of subcutaneous administration of recombinant FGF-2 on OA progression was assessed in Fgf2 -/-mice.Results. Fgf2 -/-mice were morphologically indistinguishable from wild-type (WT) animals up to age 12 weeks; the cartilage thickness and proteoglycan staining were equivalent, as was the mechanical integrity of the matrix. However, Fgf2 -/-mice exhibited accelerated spontaneous and surgically induced OA. Surgically induced OA in Fgf2 -/-mice was suppressed to levels in WT mice by subcutaneous administration of recombinant FGF-2. Increased disease in Fgf2 -/-mice was associated with increased expression of messenger RNA of Adamts5, the key murine aggrecanase. Conclusion. These data identify FGF-2 as a novel endogenous chondroprotective agent in articular cartilage.The structural integrity of articular cartilage is determined principally by homeostasis of the 2 major macromolecules of the extracellular matrix, type II collagen and the chondroitin sulfate-rich proteoglycan aggrecan. In healthy tissue, there is a balance between anabolic (synthetic) and catabolic (degradative) processes that allows matrix turnover. Excessive catabolic activity results in matrix breakdown, a hallmark of osteoarthritis (OA). One key early event in matrix breakdown is loss of aggrecan, which is caused by aggrecanase enzymes, members of the ADAMTS family. In humans, ADAMTS-4 and ADAMTS-5 are thought to be the major aggrecanases in cartilage (1,2). In the mouse, deletion of ADAMTS-5, but not ADAMTS-4, was shown to protect against the development of OA and inflammatory arthritis, suggesting that ADAMTS-5 is the main murine aggrecanase (3,4).We have identified fibroblast growth factor 2 (FGF-2) as a potential regulatory molecule in articular cartilage. It is bound to the heparan sulfate chains of the proteoglycan perlecan in the pericellular matrix of hu-

Objective. Mechanical joint loading is critical for the development of osteoarthritis (OA). Although once regarded as a disease of cartilage attrition, OA is now known to be controlled by the expression and activity of key proteases, such as ADAMTS-5, that drive matrix degradation. This study was undertaken to investigate the link between protease expression and mechanical joint loading in vivo.Methods. We performed a microarray analysis of genes expressed in the whole joint following surgical induction of murine OA (by cutting the medial meniscotibial ligament). Gene expression changes were validated by reverse transcriptase-polymerase chain reaction in whole joints and microdissected tissues of the joint, including the articular cartilage, meniscus, and epiphysis. Following surgery, mouse joints were immobilized, either by prolonged anesthesia or by sciatic neurectomy.Results. Many genes were regulated in the whole joint within 6 hours of surgical induction of OA in the mouse. These included Arg1, Ccl2, Il6, Tsg6, Mmp3, Il1b, Adamts5, Adamts4, and Adamts1. All of these were significantly regulated in the articular cartilage. When joints were immobilized by prolonged anesthesia, regulation of the vast majority of genes was abrogated. When joints were immobilized by sciatic neurectomy, regulation of selected genes was abrogated, and OA was prevented up to 12 weeks postsurgery.Conclusion. These findings indicate that gene expression in the mouse joint following the induction of OA is rapid and highly mechanosensitive. Regulated genes include the known pathogenic protease ADAMTS-5. Targeting the mechanosensing

The origin of pain in osteoarthritis is poorly understood, but it is generally thought to arise from inflammation within the innervated structures of the joint, such as the synovium, capsule and bone. We investigated the role of nerve growth factor (NGF) in pain development in murine OA, and the analgesic efficacy of the soluble NGF receptor, TrkAD5. OA was induced in mice by destabilisation of the medial meniscus and pain was assessed by measuring hind-limb weight distribution. RNA was extracted from joints, and NGF and TNF expressions were quantified. The effect of tumour necrosis factor (TNF) and neutrophil blockade on NGF expression and pain were also assessed. NGF was induced in the joints during both post-operative (day 3) and OA (16weeks) pain, but not in the non-painful stage of disease (8weeks post-surgery). TrkAd5 was highly effective at suppressing pain in both phases. Induction of NGF in the post-operative phase of pain was TNF-dependent as anti-TNF reduced NGF expression in the joint and abrogated pain. However, TNF was not regulated in the late OA joints, and pain was not affected by anti-TNF therapy. Fucoidan, by suppressing cellular infiltration into the joint, was able to suppress post-operative, but not late OA pain. These results indicate that NGF is an important mediator of OA pain and that TrkAd5 represents a potent novel analgesic in this condition. They also suggest that, unlike post-operative pain, induction of pain in OA may not necessarily be driven by classical inflammatory processes.

SummaryObjectiveThe role of inflammation in structural and symptomatic osteoarthritis (OA) remains unclear. One key mediator of inflammation is the chemokine CCL2, primarily responsible for attracting monocytes to sites of injury. We investigated the role of CCL2 and its receptor CCR2 in experimental OA.DesignOA was induced in 10 weeks old male wild type (WT), Ccl2−/− and Ccr2−/− mice, by destabilisation of the medial meniscus (DMM). RNA was extracted from whole joints at 6 h and 7 days post-surgery and examined by reverse transcription polymerase chain reaction (RT-PCR). Gene expression changes between naïve and DMM-operated mice were compared. Chondropathy scores, from mice at 8, 12, 16 and 20 weeks post DMM were calculated using modified Osteoarthritis Research Society International (OARSI) grading systems. Changes in hind paw weight distribution, as a measure of pain, were assessed by Linton incapacitance.ResultsAbsence of CCL2 strongly suppressed (>90%) selective inflammatory response genes in the joint 6 h post DMM, including arginase 1, prostaglandin synthase 2, nitric oxide synthase 2 and inhibin A. IL6, MMP3 and tissue inhibitor of metalloproteinase 1 were also significantly suppressed. Similar trends were also observed in the absence of CCR2. A lower average chondropathy score was observed in both Ccl2−/− and Ccr2−/− mice at 12, 16 and 20 weeks post DMM compared with WT mice, but this was only statistically significant at 20 weeks in Ccr2−/− mice. Pain-related behaviour in Ccl2−/− and Ccr2−/− mice post DMM was delayed in onset.ConclusionThe CCL2/CCR2 axis plays an important role in the development of pain in murine OA, but contributes little to cartilage damage.

SummaryObjectiveIdentify gene changes in articular cartilage of the medial tibial plateau (MTP) at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice. Compare our data with previously published datasets to ascertain dysregulated pathways and genes in osteoarthritis (OA).DesignRNA was extracted from the ipsilateral and contralateral MTP cartilage, amplified, labelled and hybridized on Illumina WGv2 microarrays. Results were confirmed by real-time polymerase chain reaction (PCR) for selected genes.ResultsTranscriptional analysis and network reconstruction revealed changes in extracellular matrix and cytoskeletal genes induced by DMM. TGFβ signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (Fn1) is a hub in a reconstructed network at 2 weeks. Regulated genes decrease over time. By 8 weeks fibromodulin (Fmod) and tenascin N (Tnn) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets identified genes overlapping between our array and eight other studies.ConclusionsCartilage contributes a minute percentage to the RNA extracted from the whole joint (<0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time dissipating by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGFβ. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify may warrant further investigation as biomarkers or modulators of OA.

ObjectiveTo explore whether aberrant transient chondrocyte behaviors occur in the joints of STR/Ort mice (which spontaneously develop osteoarthritis [OA]) and whether they are attributable to an endochondral growth defect.MethodsKnee joints from STR/Ort mice with advanced OA and age‐matched CBA (control) mice were examined by Affymetrix microarray profiling, multiplex polymerase chain reaction (PCR) analysis, and immunohistochemical labeling of endochondral markers, including sclerostin and MEPE. The endochondral phenotype of STR/Ort mice was analyzed by histologic examination, micro–computed tomography, and ex vivo organ culture. A novel protocol for quantifying bony bridges across the murine epiphysis (growth plate fusion) using synchrotron x‐ray computed microtomography was developed and applied.ResultsMeta‐analysis of transcription profiles showed significant elevation in functions linked with endochondral ossification in STR/Ort mice (compared to CBA mice; P < 0.05). Consistent with this, immunolabeling revealed increased matrix metalloproteinase 13 (MMP‐13) and type X collagen expression in STR/Ort mouse joints, and multiplex quantitative reverse transcriptase–PCR showed differential expression of known mineralization regulators, suggesting an inherent chondrocyte defect. Support for the notion of an endochondral defect included accelerated growth, increased zone of growth plate proliferative chondrocytes (P < 0.05), and widespread type X collagen/MMP‐13 labeling beyond the expected hypertrophic zone distribution. OA development involved concomitant focal suppression of sclerostin/MEPE in STR/Ort mice. Our novel synchrotron radiation microtomography method showed increased numbers (P < 0.001) and mean areal growth plate bridge densities (P < 0.01) in young and aged STR/Ort mice compared to age‐matched CBA mice.ConclusionTaken together, our data support the notion of an inherent endochondral defect that is linked to growth dynamics and subject to regulation by the MEPE/sclerostin axis and may represent an underlying mechanism of pathologic ossification in OA.

ObjectiveThe articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury.MethodsFemoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2−/− mice. Reverse transcription–polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis.ResultsMurine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor–stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo.ConclusionFGF-2–dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.

Objective Female C57BL/6 mice exhibit less severe chondropathy than male mice. This study was undertaken to test the robustness of this observation and explore underlying mechanisms. Methods Osteoarthritis was induced in male and female C57BL/6 or DBA/1 mice (n = 6–15 per group) by destabilization of the medial meniscus (DMM) or partial meniscectomy (PMX). Some mice were ovariectomized (OVX) (n = 30). In vivo repair after focal cartilage defect or joint immobilization (sciatic neurectomy) following DMM was assessed. Histologic analysis, evaluation of gene expression in whole knees, and behavioral analysis using Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS) and Linton incapacitance testing (n = 7–10 mice per group) were performed. Results Female mice displayed less severe chondropathy (20–75% reduction) across both strains and after both surgeries. Activity levels after PMX were similar for male and female mice. Some repair‐associated genes were increased in female mouse joints after surgery, but no repair differences were evident in vivo. Despite reduced chondropathy, female mice developed pain‐like behavior at the same time as male mice. At the time of established pain‐like behavior (10 weeks after PMX), pain‐associated genes were significantly up‐regulated in female mice, including Gdnf (mean ± SEM fold change 2.54 ± 0.30), Nrtn (6.71 ± 1.24), Ntf3 (1.92 ± 0.27), and Ntf5 (2.89 ± 0.48) (P < 0.01, P < 0.01, P < 0.05, and P < 0.001, respectively, versus male mice). Inflammatory genes were not regulated in painful joints in mice of either sex. Conclusion We confirm strong structural joint protection in female mice that is not due to activity or intrinsic repair differences. Female mice develop pain at the same time as males, but induce a distinct set of neurotrophins. We speculate that heightened pain sensitivity in female mice protects the joint by preventing overuse.