Murine Model Of Osteoarthritis Research Articles

Intra-articular Injection of Urinary Bladder Matrix Reduces Osteoarthritis Development.

Micronized porcine urinary bladder matrix (UBM) is an extracellular matrix biomaterial that has immunomodulatory and pro-regenerative properties. The objective of this study was to assess the ability of UBM to alter disease progression in a mouse model of post-traumatic osteoarthritis (OA). Ten-week-old wild-type C57BL/6 male mice underwent anterior cruciate ligament transection (ACLT) to induce OA. Two weeks after ACLT, UBM (50mg/mL) or saline was injected into the mouse joint. At 4 and 8weeks post-ACLT, cartilage integrity was assessed using OARSI scoring of histology, pain was evaluated, and joints were harvested for quantitative RT-PCR analysis of cartilage-specific and inflammatory gene expression. UBM-treated animals showed improved cartilage integrity at 4 and 8weeks and reduced pain at 4weeks compared to saline-injected mice. Animals injected with UBM expressed higher levels of genes encoding structural cartilage proteins, such as collagen2α1 and aggrecan, as well as anti-inflammatory cytokines, including interleukins 10 and 4. UBM decreased cartilage degeneration in the murine ACLT model of OA, which may be due to reduced inflammation in the joint and maintenance of high expression levels of proteoglycans.

Utility of a Mouse Model of Osteoarthritis to Demonstrate Cartilage Protection by IFNγ-Primed Equine Mesenchymal Stem Cells.

Mesenchymal stem cells isolated from adipose tissue (ASC) have been shown to influence the course of osteoarthritis (OA) in different animal models and are promising in veterinary medicine for horses involved in competitive sport. The aim of this study was to characterize equine ASCs (eASCs) and investigate the role of interferon-gamma (IFNγ)-priming on their therapeutic effect in a murine model of OA, which could be relevant to equine OA. ASC were isolated from subcutaneous fat. Expression of specific markers was tested by cytometry and RT-qPCR. Differentiation potential was evaluated by histology and RT-qPCR. For functional assays, naïve or IFNγ-primed eASCs were cocultured with peripheral blood mononuclear cells or articular cartilage explants. Finally, the therapeutic effect of eASCs was tested in the model of collagenase-induced OA (CIOA) in mice. The immunosuppressive function of eASCs on equine T cell proliferation and their chondroprotective effect on equine cartilage explants were demonstrated in vitro. Both cartilage degradation and T cell activation were reduced by naïve and IFNγ-primed eASCs, but IFNγ-priming enhanced these functions. In CIOA, intra-articular injection of eASCs prevented articular cartilage from degradation and IFNγ-primed eASCs were more potent than naïve cells. This effect was related to the modulation of eASC secretome by IFNγ-priming. IFNγ-priming of eASCs potentiated their antiproliferative and chondroprotective functions. We demonstrated that the immunocompetent mouse model of CIOA was relevant to test the therapeutic efficacy of xenogeneic eASCs for OA and confirmed that IFNγ-primed eASCs may have a therapeutic value for musculoskeletal diseases in veterinary medicine.

Can sulforaphane prevent the onset or slow the progression of osteoarthritis?

Osteoarthritis (OA) is a degenerative joint disease characterised in part by destruction of articular cartilage. There are currently no disease-modifying drugs to treat OA, with joint replacement the only treatment offered to patients at end-stage disease. With age the major risk factor for OA, the number of patients is predicted to double by 2030. An understanding of the role of bioactive molecules from the habitual diet on joint health offers a novel way in which to prevent the onset or slow the progression of OA. Our research has indicated that sulforaphane (SFN), gained from the consumption of cruciferous vegetables, particularly broccoli, could impact upon articular cartilage in laboratory models of OA because (1) it decreased the cytokine-induced expression of cartilage-degrading proteinases from chondrocytes (cartilage cells); (2) it prevented the cytokine-induced degradation of cartilage explants; and (3) it attenuated cartilage destruction in a murine model of OA. The major mechanism of action for SFN in human articular chondrocytes was inhibition of NFB, not activation of Nrf2 nor inhibition of histone deacetylases. A proof-of-principle human trial was performed to measure uptake of SFN, or its metabolites, in the human knee joint following a broccoli-rich diet, and the expression or levels of several genes and proteins in cartilage, fat and synovial fluid were also measured. Data from this trial are about to be published. Overall, these findings support the utility of SFN in the prevention or treatment of OA. The proof of this requires an appropriately designed clinical trial of pain and function which we are currently pursuing.

Effect of combined sex hormone replacement on bone/cartilage turnover in a murine model of osteoarthritis

Effect of combined sex hormone replacement on bone/cartilage turnover in a murine model of osteoarthritis

A novel fibroblast growth factor receptor 1 inhibitor protects against cartilage degradation in a murine model of osteoarthritis.

The attenuated degradation of articular cartilage by cartilage-specific deletion of fibroblast growth factor receptor 1 (FGFR1) in adult mice suggests that FGFR1 is a potential target for treating osteoarthritis (OA). The goal of the current study was to investigate the effect of a novel non-ATP-competitive FGFR1 inhibitor, G141, on the catabolic events in human articular chondrocytes and cartilage explants and on the progression of cartilage degradation in a murine model of OA. G141 was screened and identified via cell-free kinase-inhibition assay. In the in vitro study, G141 decreased the mRNA levels of catabolic markers ADAMTS-5 and MMP-13, the phosphorylation of Erk1/2, JNK and p38 MAPK, and the protein level of MMP-13 in human articular chondrocytes. In the ex vivo study, proteoglycan loss was markedly reduced in G141 treated human cartilage explants. For the in vivo study, intra-articular injection of G141 attenuated the surgical destabilization of the medial meniscus (DMM) induced cartilage destruction and chondrocyte hypertrophy and apoptosis in mice. Our data suggest that pharmacologically antagonize FGFR1 using G141 protects articular cartilage from osteoarthritic changes, and intra-articular injection of G141 is potentially an effective therapy to alleviate OA progression.

Treatment of osteoarthritis using a helper-dependent adenoviral vector retargeted to chondrocytes

Osteoarthritis (OA) is a joint disease characterized by degeneration of the articular cartilage, subchondral bone remodeling, and secondary inflammation. It is among the top three causes of chronic disability, and currently there are no treatment options to prevent disease progression. The localized nature of OA makes it an ideal candidate for gene and cell therapy. However, gene and cell therapy of OA is impeded by inefficient gene transduction of chondrocytes. In this study, we developed a broadly applicable system that retargets cell surface receptors by conjugating antibodies to the capsid of helper-dependent adenoviral vectors (HDVs). Specifically, we applied this system to retarget chondrocytes by conjugating an HDV to an α-10 integrin monoclonal antibody (a10mab). We show that a10mab-conjugated HDV (a10mabHDV)-infected chondrocytes efficiently in vitro and in vivo while detargeting other cell types. The therapeutic index of an intra-articular injection of 10mabHDV-expressing proteoglycan 4 (PRG4) into a murine model of post-traumatic OA was 10-fold higher than with standard HDV. Moreover, we show that PRG4 overexpression from articular, superficial zone chondrocytes is effective for chondroprotection in postinjury OA and that α-10 integrin is an effective protein for chondrocyte targeting.

Nerve Growth Factor Regulation by TNF-α and IL-1β in Synovial Macrophages and Fibroblasts in Osteoarthritic Mice.

To investigate the role of macrophages as a regulator and producer of nerve growth factor (NGF) in the synovial tissue (ST) of osteoarthritis (OA) joints, the gene expression profiles of several inflammatory cytokines in the ST, including synovial macrophages and fibroblasts, of OA mice (STR/Ort) were characterized. Specifically, real-time polymerase chain reaction analysis was used to evaluate the expression of tumor necrosis factor- (TNF-) α, interleukin- (IL-) 1β, IL-6, and NGF in CD11b+ and CD11b– cells isolated from the ST of a murine OA model. The effects of TNF-α, IL-1β, and IL-6 on the expression of NGF in cultured synovial cells were also examined. The expression of TNF-α, IL-1β, IL-6, and NGF in the ST of STR/Ort was higher than that in C57/BL6J mice. Compared to the CD11b– cell fraction, higher expression levels of TNF-α, IL-1β, and IL-6 were detected in the CD11b+ cell fraction, whereas no differences in the expression of NGF were detected between the two cell fractions. Notably, TNF-α upregulated NGF expression in synovial fibroblasts and macrophages and IL-1β upregulated NGF expression in synovial fibroblasts. IL-1β and TNF-α may regulate NGF signaling in OA joints and be suitable therapeutic targets for treating OA pain.

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.

A role for CC-chemokine receptor 7 (CCR7) in a murine model of osteoarthritis: Impact on joint structure and function

A role for CC-chemokine receptor 7 (CCR7) in a murine model of osteoarthritis: Impact on joint structure and function

Identification of a novel target, FoxA2, in the onset and development of osteoarthritis

Identification of a novel target, FoxA2, in the onset and development of osteoarthritis

High-bandwidth AFM-based rheology is a sensitive indicator of early cartilage aggrecan degradation relevant to mouse models of osteoarthritis

Murine models of osteoarthritis (OA) and post-traumatic OA have been widely used to study the development and progression of these diseases using genetically engineered mouse strains along with surgical or biochemical interventions. However, due to the small size and thickness of murine cartilage, the relationship between mechanical properties, molecular structure and cartilage composition has not been well studied. We adapted a recently developed AFM-based nano-rheology system to probe the dynamic nanomechanical properties of murine cartilage over a wide frequency range of 1Hz to 10kHz, and studied the role of glycosaminoglycan (GAG) on the dynamic modulus and poroelastic properties of murine femoral cartilage. We showed that poroelastic properties, highlighting fluid–solid interactions, are more sensitive indicators of loss of mechanical function compared to equilibrium properties in which fluid flow is negligible. These fluid-flow-dependent properties include the hydraulic permeability (an indicator of the resistance of matrix to fluid flow) and the high frequency modulus, obtained at high rates of loading relevant to jumping and impact injury in vivo. Utilizing a fibril-reinforced finite element model, we estimated the poroelastic properties of mouse cartilage over a wide range of loading rates for the first time, and show that the hydraulic permeability increased by a factor ~16 from knormal=7.80×10−16±1.3×10−16m4/Ns to kGAG-depleted=1.26×10−14±6.73×10−15m4/Ns after GAG depletion. The high-frequency modulus, which is related to fluid pressurization and the fibrillar network, decreased significantly after GAG depletion. In contrast, the equilibrium modulus, which is fluid-flow independent, did not show a statistically significant alteration following GAG depletion.

Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

IntroductionRecent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.MethodsDestabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).ResultsIntra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.ConclusionThese results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Intra-articular administration of gelatin hydrogels incorporating rapamycin–micelles reduces the development of experimental osteoarthritis in a murine model

Autophagy is a cellular homeostasis mechanism that may have a protective role against osteoarthritis (OA). The present study investigated the therapeutic effect of local administration of rapamycin, a potent activator of autophagy, against OA. To achieve controlled intra-articular administration of rapamycin, gelatin hydrogels incorporating rapamycin-micelles were created and the release profile was evaluated in vitro. The therapeutic effects of gelatin hydrogels incorporating rapamycin-micelles were then tested in a murine OA model. Mice were divided into four groups: Group 1, gelatin hydrogels alone; Group 2, single injection of 1 μg rapamycin; and Groups 3 and 4, gelatin hydrogels incorporating 100 ng or 1 μg rapamycin-micelles, respectively. Immunohistochemical analysis revealed that autophagic marker-positive chondrocytes were increased in the rapamycin-treated mice at 10 weeks after surgery. The histologic score was better in Groups 3 and 4 than in Groups 1 and 2, and Group 2 had a better score than Group 1. Delayed OA progression was maintained even at 16 weeks after surgery in Group 4. Microarray and real-time polymerase chain reaction analysis indicated that OA mediator genes were downregulated in the rapamycin-treated mice. Our novel system for intra-articular administration of rapamycin could be a novel therapeutic approach for treating patients with OA.

Glycogen synthase kinase 3 inhibition stimulates human cartilage destruction and exacerbates murine osteoarthritis.

To assess the role of glycogen synthase kinase 3 (GSK-3) as a regulator of cartilage destruction in human tissue and a murine model of osteoarthritis (OA). Surgical destabilization of the medial meniscus (DMM) was performed to induce experimental murine OA, and joint damage was assessed histologically. Bovine nasal and human OA cartilage samples were incubated with interleukin-1 (IL-1) plus oncostatin M (OSM) and GSK-3 inhibitor. Collagen and proteoglycan release was assessed by hydroxyproline measurement and dye binding assay, collagenase activity was assessed by bioassay, and gene expression was analyzed by real-time polymerase chain reaction. Human articular chondrocytes were isolated by enzymatic digestion and cultured prior to gene silencing and immunoblotting of cell lysates and nuclear fractions. Mice treated with GSK-3 inhibitor exhibited significantly greater cartilage damage compared with sham-operated control mice. GSK-3 inhibition in bovine cartilage dramatically accelerated IL-1 plus OSM-stimulated degradation, concomitant with a profound increase in collagenase activity. GSK-3 inhibitor induced collagen release from human OA cartilage in the presence of IL-1 plus OSM and increased proteoglycan loss. Gene expression profiling of resorbing OA cartilage revealed a marked procatabolic switch in gene expression upon GSK-3 inhibition. This was mirrored in human articular chondrocytes following GSK3 silencing, particularly with the GSK-3β isoform. GSK-3 inhibition or silencing led to enhanced IL-1 plus OSM-stimulated abundance and activity of Jun, and silencing of c-jun ameliorated GSK-3 inhibitor-mediated procatabolic gene expression. GSK-3 is an important regulator of matrix metalloproteinase (MMP)-mediated joint destruction, the inhibition of which by proinflammatory stimuli de-represses catabolic gene expression. Therapeutic strategies that maintain cartilage GSK-3 activity may therefore help curtail aberrant MMP activity during pathologic joint destruction.

Dispensable role of myeloid differentiation primary response gene 88 (MyD88) and MyD88-dependent toll-like receptors (TLRs) in a murine model of osteoarthritis

ObjectivesThe aim of our study was to evaluate the role of cell-membrane expressed TLRs and the signaling molecule MyD88 in a murine model of OA induced by knee menisectomy (surgical partial removal of the medial meniscus [MNX]). MethodsOA was induced in 8–10weeks old C57Bl/6 wild-type (WT) female (n=7) mice and in knockout (KO) TLR-1 (n=7), -2 (n=8), -4 (n=9) -6 (n=5), MyD88 (n=8) mice by medial menisectomy, using the sham-operated contralateral knee as a control. Cartilage destruction and synovial inflammation were evaluated by knee joint histology using the OARSI scoring method. Apoptotic chondrocytes and cartilage metabolism (collagen II synthesis and MMP-mediated aggrecan degradation) were analyzed using immunohistochemistry. ResultsOperated knees exhibited OA features at 8weeks post-surgery compared to sham-operated ones. In menisectomized TLR-1, -2, -4, and -6 deficient mice, cartilage lesions, synovial inflammation and cartilage metabolism were similar to that in operated WT mice. Accordingly, using the same approach, we found no significant protection in MyD88-deficient mice in terms of OA progression as compared to WT littermates. ConclusionsDeficiency of TLRs or their signalling molecule MyD88 did not impact on the severity of experimental OA. Our results demonstrate that MyD88-dependent TLRs are not involved in this murine OA model. Moreover, the dispensable role of MyD88, which is also an adaptor for IL-1 receptor signaling, suggests that IL-1 is not a key mediator in the development of OA. This latter hypothesis is strengthened by the lack of efficiency of IL-1β antagonist in the treatment of OA.

Molecular and functional analysis of a conditional knockout of activin A

Purpose: Osteoarthritis (OA) is the most common degenerative disease of the joint. Knockout mice have been a very powerful tool to elucidate the pathogenesis of OA. However, selectively deleting genes in adult animals in a tissue or time specific manner, still remains challenging. Here we have generated a conditional knockout mouse to study the role of activin A in a murine model of OA. Activin A is strongly regulated in human OA and is essential for normal musculoskeletal development (the constitutive knockout is neonatal lethal). Methods: Activin A floxed mice (Actf/f) were crossed with a line expressing Cre recombinase driven by the human ubiquitin promoter and controlled by a Tamoxifen-sensitive modified estrogen receptor (UbcCreERT2). The resulting mice (UbcCreERT2/Actf/f) were treated with Tamoxifen (TM) on 3 consecutive days at either 4 weeks (for cartilage injury studies), 8 weeks (for OA studies) or 10 weeks (for other studies) to create pan tissue activin A deletion. Activin A messenger RNA production was determined by real time PCR. Activin A protein secretion was measured by enzyme-linked immunosorbent assay (ELISA). Femurs and tibias were collected at 18 weeks for micro CT imaging. Results: In activin A conditional knockout (Act-cKO) mice, less than 10% of activin A mRNA was detected in cartilage, knee joints, spleen, liver and kidney compared with control levels (Act f/f or UbcCreERT2/Actf/f; no TM). ELISA showed that activin A secretion upon cartilage injury in Act-cKO hips was similarly reduced. For functional validation, micro CT analysis showed that 18 week old female Act-cKO had more trabecular bone volume, thickness and trabecular number than controls (p < 0.05). We also observed that Act-cKO showed a much delayed hair re-growth on the dorsal skin after hair removal compared with controls. Conclusions: We have successfully generated an activin A conditional knockout mouse and have validated the deletion at the mRNA and protein level in a number of tissues including cartilage. We have demonstrated functional loss of activin in bone by showing that these mice have increased bone mass, a phenotype that is predicted by other in vivo/vitro studies. The retarded hair re-growth phenotype in Act-cKO mice was in agreement with the fact that constitutive activin A knockout had no whiskers. We are currently awaiting the outcome of the OA experiments to see whether disease is modulated by lack of activin A.

Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo

Purpose: Nutrition impacts directly on ageing and obesity, both of which are major risk factors for osteoarthritis. Sulforaphane (SFN) is an isothiocyanate derived from brassicas, particularly broccoli, and has been reported to regulate signalling pathways relevant to chronic diseases. Our study investigated whether sulforaphane can abrogate cartilage destruction in laboratory models of osteoarthritis and examined mechanism of action in chondrocytes. Methods: The impact of SFN treatment on gene expression, signalling through transcription factors nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor kappaB (NFκB), and histone acetylation were examined in chondrocytes. The intracellular concentrations of SFN and SFN metabolites were quantified in chondrocytes and used to inform transcription factor binding assays. The bovine nasal cartilage explant model (BNC) and destabilisation of medial meniscus (DMM) murine model of osteoarthritis were used to study chondroprotection by SFN. Results: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes (HACs) and in fibroblast-like synovial cells (FLS). SFN can act independently of the Nrf2 transcription factor and histone deacetylase activity in HACs, but does mediate prolonged activation of Jun kinase (JNK) and p38 MAP kinase. SFN attenuates NF-κB signalling through at least inhibition of DNA binding in HACs with attenuation of expression of several NF-κB dependent genes. SFN abrogates cytokine-induced destruction of bovine nasal cartilage at the level of both proteoglycan and collagen breakdown (10μM compared to cytokines alone). It also decreases arthritis score in the DMM murine model of osteoarthritis (3μmol daily dose SFN in diet versus control chow). Conclusions: SFN, at levels which can be obtained through a broccoli-rich diet, inhibits the expression of key metalloproteinases implicated in osteoarthritis independently of Nrf2 and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo. Ongoing studies in man will ascertain the potential of this compound in human osteoarthritis.

PPARdelta promotes the progression of post-traumatic osteoarthritis

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S370 micro computed tomography (mCT, Skyscan 1172, 4.5 um resolution) of the knee at 3, 7, 14, 28, 56, 84, 112 days (n 8 per group). Changes in subchondral bone at 28 days post DMM (n 1⁄4 6) were measured separating the subchondral plate from the subchondral tabecular structures. Results: Osteophytes were identified in 70% of mice by day 7 post DMM and in all mice by day 14. At day 7 most samples had 1 or 2 osteophytes, with somehavingup to 4 osteophytes by day 14. Fromday 28onwards the osteophytes fused and formed into one or two large osteophytes. Osteophytes increased in size and bone content with time, plateauing by day 112. The trabeculae of the subchondral bone at day 28 showed a significant (p<0.03) decrease in trabecular number on the medial side of both theDMMand shamoperatedmicewhen compared to the respective contralateral leg. In the sham samples, the reduction of trabecular number translated to a significant increase in trabecular space, whereas in the DMM mice an increase in trabecular thickness occurred, eliminating the increase in trabecular space. Although bone changes occurred within a week of DMM induction, cartilage damage scores were not significant until day 56 (p<0.05), compared to earlier time points. Conclusions:We herein charachterised differential bone changes in the DMM murine model of OA: there was accelerated osteophyte development within the first week, whereas subchondral bone changes were not evident until day 28. Interestingly sham operated animals showed subchondral trabecular bone loss in the ipsilateral knee, highlighting the need to use the contralateral knee as an internal control. The early bone changes in this OA model significantly preceded cartilage degradation, consistent with altered bone biology being an important driver of OA pathogenesis.Supported By Arthritis Research UK (20199). 651 PPARDELTA PROMOTES THE PROGRESSION OF POST-TRAUMATIC OSTEOARTHRITIS A. Ratneswaran y, E. LeBlanc y, E. Walser y, I. Welch y, J.S. Mort zx, N. Borradaile y, F. Beier yk. yWestern Univ., London, ON, Canada; zMcGill Univ., Montreal, QC, Canada; x Shriner’s Hosp. for Children, Montreal, ON, Canada; k Children’s Hlth.Res. Inst., London, ON, Canada Background: Osteoarthritis (OA) is a degenerative joint disorder, characterized by the breakdown of articular cartilage, subchondral bone thickening, osteophyte formation and synovial inflammation. The human burden of this disease influences both the independence and quality of life of those afflicted. Currently, there are no pharmaceutical treatments to slow, stop or reverse disease progression, resulting in greatly reduced quality of life for OA patients and the need for joint replacement surgeries in many cases. The lack of available treatments for OA is partially due to our incomplete understanding of themolecular mechanisms promoting disease initiation and progression. Recent findings from our laboratory indicate that activation of the transcription factor PPARdelta induces the expression of enzymes involved in proteoglycan breakdown and can lead to cartilage degeneration in OA, prompting us to speculate whether inhibition of PPARdelta, can protect from cartilage breakdown in OA. Purpose:We aimed to characterize the role of PPARdelta in OA through in-vitro, ex-vivo, and in-vivo studies to determine whether it is a feasible target for OA therapy. Hypothesis: We hypothesize that inhibition of PPARdelta will slow the progression of OA in animal models. Methods: Primary mouse chondrocytes and cartilage explants were treated with a pharmacological PPARdelta agonist (GW501516) to evaluate changes in gene expression (qPCR), and histology (Safranin-O, immunohistochemistry) consistent with OA development. In order to examine the role of PPARdelta in-vivo, cartilage-specific knockout mice and wild-type littermate controls were subjected to destabilization of medial meniscus surgery (DMM) at 20 weeks of age. 8 weeks postsurgery mice were compared through classical histological and biochemical measures of OA progression including Safranin-O staining with OARSI scoring, immunohistochemistry for cartilage matrix breakdown products, and picrosirius red staining for collagen fiber structure and orientation. Results: In vitro, PPARdelta agonism (by GW501516) results in the upregulation of expression of proteases implicated in cartilage degeneration (including MMPs, and ADAMTS genes). Activation of PPARdelta also results in proteoglycan breakdown in the cartilage matrix of an explant culture system. Mice with cartilage-specific deletion of PPARdelta do not demonstrate any abnormalities in skeletal growth or development, but do show significant chondroprotection in comparison to wild-type littermate controls after surgical induction of OA. OARSI scoring and immunohistochemistry confirm strong protection of mutant mice against cartilage matrix breakdown, and decreased breakdown products in the cartilage of PPARdelta knockout mice. Conclusion: This study provides strong evidence for catabolic roles of endogenous PPARdelta in post-traumatic OA and suggests that pharmacological inhibition of PPARdelta is a promising therapeutic strategy. 652 DKK3 PREVENTS MATRIX LOSS INDUCED BY PRO-INFLAMMATORY CYTOKINES IN HUMAN CARTILAGE EXPLANTS S.M. Walzer, D. Bieder, B. Simon, R. Windhager, S. Toegel. Karl Chiari Lab for Orthopaedic Biology, Dept. of Orthopaedics, Med. Univ. of Vienna,

Age-related alterations in TGF beta signaling as a causal factor of cartilage degeneration in osteoarthritis.

Age is the most important risk factor for primary osteoarthritis (OA). Members of the TGF-β superfamily play a crucial role in chondrocyte differentiation and maintenance of healthy articular cartilage. We have investigated whether age-related changes in TGF-β superfamily signaling components play a role in the relationship between OA-related cartilage degradation and aging. The relationship between age, OA and TGF-β superfamily signaling was studied using murine experimental OA models, aging mice, bovine articular cartilage and human OA cartilage. The effects of TGF-β on cartilage homeostasis was studied with immunohistochemistry, Q-RT-PCR and signaling pathway analysis with Western blotting and the application of specific TGF-β inhibitors. We have found that TGF-β loses its protective effects in old cartilage. Moreover, we found that on chondrocytes, TGF-β not only signals via the canonical type I receptor ALK5 (TGFBR1) but also via the ALK1 (ACVRL1) receptor. Remarkably, signaling via ALK5 (Smad2/3 route) results in protective while ALK1 signaling (Smad1/5/8 route) results in deleterious responses in articular chondrocytes. In cartilage of aging mice it was detected that the ALK1/ALK5 ratio is significantly increased, favoring TGF-β signaling via the Smad1/5/8 route, inducing changes in chondrocyte differentiation and matrix metalloproteinase-13 (MMP-13) expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. Since in mice aging and OA in often goes hand in hand, we also analyzed age-related expression of TGF-β superfamily related signaling molecules in healthy bovine cartilage in an age range from 6 months to 14 years. In this cohort of aging cartilage, we found that mainly signaling receptors determining the Smad2/3 pathway were decreased with age while Smad1/5/8-related signaling molecules did not alter, confirming our findings in aging mice. Old cartilage appears to be less protected by TGF-β and shows significant alterations in TGF-β signaling pathways. Loss of the protective Smad2/3 pathway during aging can provide an explanation for the relationship between OA and aging.