Background Adipose-derived mesenchymal stem cells (ASC) might act as a cellular source of soluble factors exerting anti-inflammatory or trophic effects on cells. Osteoarthritis (OA) is characterized by the progressive loss of structure and functionality of articular cartilage. Objectives In the present study we explored the effect of conditioned medium from adipose-derived mesenchymal stem cells (ASC-CM) on the metabolism of OA chondrocytes in primary culture. Methods ADC were isolated from adipose tissue of patients subjected to abdominal lipectomy surgery, by collagenase treatment. Cells were incubated in DMEM/F12 + 15% human serum. Cell phenotype was analysed by flow cytometry with specific antibodies anti-CD105-PE, anti-CD90PerCP-eFluo 710, anti-CD34APC, and anti-CD45-PE (International Society of Cellular Therapy), and cellular viability with propidium iodide. ASC-CM was collected after 48h of culture, centrifuged and stored at -80°C in sterile conditions. Protocols were approved by the Institutional Ethical Committee. Chondrocytes were isolated from knee cartilage samples from patients with diagnosis of advanced OA. OA chondrocytes in primary culture were stimulated with 10 ng/ml interleukin (IL)-1β for 1 or 5 days. We have evaluated the senescence marker β-galactosidase by histochemical staining of cells. Collagen II production was analyzed by immunofluorescence, gene expression by real-time qPCR and protein expression by ELISA. Results The percentage of senescent chondrocytes was increased by IL-1β treatment after 1 or 5 days whereas ASC-CM significantly counteracted this effect. When OA chondrocytes were treated with IL-1β and/or ASC-CM for 5 days, we observed a significant maintenance of collagen II expression in chondrocytes cultured with ASC-CM either in basal conditions or in the presence of IL-1β stimulation. Cytokine stimulation of chondrocytes augmented gene expression and protein release into the culture medium of matrix metalloproteinases (MMPs). Our data indicate that ASC-CM significantly decreased the production of MMP-13. In addition, AC-CM reduced gene expression and protein levels of the pro-inflammatory cytokine IL-6 and enhanced the production of the anti-inflammatory cytokine IL-10. Conclusions We have shown that ASC-CM treatment of OA chondrocytes prevents the appearance of senescence features and the production of degradative mediators. These findings suggest a protective role for ASC-CM in OA chondrocytes. Acknowledgements This work was funded by grants SAF2010-22048 (MINECO), RETICEF RD06/0013/2001 (MINECO, ISCIII, FEDER), and Prometeo2010-047 (Generalitat Valenciana). Disclosure of Interest None Declared

Purpose: Articular chondrocytes are adapted to live in conditions of low O2. Such hypoxic conditions in the avascular cartilage play an important role in extracellular matrix synthesis and survival of chondrocytes. In previous work we have shown anti-inflammatory and chondroprotective effects of heme oxygenase-1 (HO-1) on osteoarthritic (OA) condrocytes in primary cultures in 20% O2. However, the influence of O2 tension on HO-1 function in healthy and OA chondrocytes remains unknown. Methods: Human chondrocytes were obtained from healthy donors and patients with diagnosis of advanced OA undergoing total knee joint replacement. The chondrocytes were isolated by digestion with collagenase and used in primary culture. Healthy and OA chondrocyes were cultured in 20% or 1% O2 tension, and in 5% CO2. Cells were stimulated with IL-1β (10 ng/ml) for 48h. HO-1 was induced by incubation with 10 μM cobalt protoporphyrin IX (CoPP). Protein expression was assessed by Western blot, ELISA and immunocytochemistry. Nitrite production and matrix metalloproteinase (MMP) activity were evaluated by fluorometric methods. HO-1 gene silencing was achieved by using a gene-specific siRNA. Results: HO-1 protein was expressed in both healthy and OA chondrocytes in hypoxia (1% O2) and normoxia (20% O2). IL-1β down-regulated HO-1 expression in all conditions whereas CoPP treatment counteracted this effect. CoPP treatment was able to reduce the levels of TNFα and MMP activity after IL-1β stimulation. CoPP also decreased the production of nitrite induced by IL-1β in healthy and OA cells at both O2 concentrations. This effect was accompanied by a reduction in iNOS expression at 24h. In hypoxic conditions HIF-2α and SOX9 expression was decreased by IL-1β in both healthy and OA chondrocytes. However, HO-1 induction was able to reverse this effect and prevented the decrease in type collagen II. In addition, IL-1β induced HIF-1α expression irrespective of O2 tension whereas HO-1 induction by CoPP down-regulated HIF-1α expression in OA chondrocytes only. Conclusions: HO-1 induction in primary chondrocytes cultured in hypoxic conditions resulted in stronger anti-inflammatory and chondroprotective effects compared with normoxia. These results suggest that HO-1 could be a physiologically important chondroprotective factor.

Presentations / Osteoarthritis and Cartilage 18, Supplement 2 (2010) S9-S44 S31 sera (m±SD: 789±211 vs 501±98 OD ×1000; p<0.0001).At the same time kOA patients had significantly higher serum EM-IgG levels: 71% AII (m±SD: 409±111 vs 274±84; p<0.0005), 59% VEGF (m±SD: 669±241 vs 471±189; p<0.0003), 35% bFGF (m±SD: 689±245 vs 455±115; p<0.009).Serum BK-IgG overexpression in kOA patients were positively associated with destructive changes (K&L>4; r=0.75; p<0.005).kOA patients in whom K&L scores progress rapidly tend to have higher serum BK-IgG levels at all time point.Elevated serum BK-IgG level was significantly correlated with enhanced pain (r=0.85;p<0.0001) and loss of functions (r=0.69;p<0.0003) in kOA patients.At the same time kOA patients had significantly lower serum EM-IgM levels relative to normal sera: 66% AII (m±SD: 289±99 vs 455±101; p<0.0001), 79% VEGF (m±SD: 366±181 vs 559±198; p<0.005), 61% bFGF (m±SD: 521±222 vs 705±239; p<0.007).No significant difference in binding of serum EM-IgA levels in kOA patients in comparison with that in control.No correlations were found to conventional parameters for pain and function with expression of EM-IgA, EM-IgM.Conclusions: We showed that our ELISA was able to demonstrate immune responses to each of the 4 type specific self-antigens in kOA patients.EM represent a group of novel selfantigens which are targeted by NA from kOA patients.Serum NA profiling is a promising approach for early detection and diagnosis of kOA.This results show a specific dysbalance of Ig content in kOA patients.Circulating BK-IgG in the sera has been proposed as a sensitive and specific marker of diagnosing kOA at early stages of the disease.Our results have potential applications for controlling unwanted angiogenesis, inflammation, infection, pain and future response to therapy in kOA patients.

Purpose: Interleukin 1β (IL-1β) is considered one of the key players in osteoarthritis (OA) pathogenesis. It increases the transcription of several mediators and enzymes, amongst which the inducible cycloxygenase isoform (COX2). Bradykinin (BK, H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) is a proinflammatory and algogenic peptide which is enzymatically generated from kininogen precursors circulating in plasma and interstitial fluids. BK, also detected into the synovial fluid, activates B2 receptors present on the membrane of fibroblast-like synoviocytes to release prostaglandins, cytokines and chemokines. Aim of this study was to investigate the interaction of BK with IL-1β, at concentrations resembling those measured in the OA synovial fluid, and to evaluate the effect of BK B2 receptor blockade, by using the highly selective and potent B2 receptor antagonist MEN16132 (m.w. 873.16). Methods: Human synoviocytes (ECACC, 408-05a) were cultured in synoviocyte growth medium (ECACC, 06091516) supplemented with Gln 2mM, penicillin (50 μg/ml), streptomycin (50 μg/ml), amphotericin B (0.75 μg/ml). Experiments were performed with cells at confluence plated onto 12-well plates. Cells were incubated at the indicated compound concentration in F12 medium supplemented with foetal bovine serum (1%), Gln (2 mM), and captopril (1 μM). At the end of the experiments, surnatants were stored at -80°C and used for PGE2 content detection (Cayman, 514010), whereas cells were collected for RNA extraction and following qPCR COX2 gene expression. Data are expressed as -fold increase versus untreated cells (basal) and represent the mean ± s.e.m. of 3-4 experiments, each in duplicate. Results: A prolonged incubation (24h) of synoviocytes with BK (1 μM) induced a significant production of PGE2 (3.2±0.8 -fold of basal) and COX2 gene expression (1.9±0.3 -fold of basal). However, PGE2 production and COX2 gene expression were reduced by the pretreatment (30 min) with the B2 receptor antagonist MEN16132 (1 μM) (PGE2: 1.6±0.3; COX2: 0.7±0.1 -fold of basal), which per se did not affect the basal release of PGE2 (1.1±0.2 -fold of basal) or the COX2 gene expression (0.9±0.2 -fold of basal). In contrast, the effect of IL-1β (15 pg/ml) was significantly more pronounced, since it augmented the PGE2 release by 44.7±13.3 -fold and the COX2 gene expression by 11.2±2.1 -fold in respect to basal values obtained in control cells. The combined treatment of cells with BK and IL-1β induced an even increase, both in terms of released PGE2 (148.0±35.4 -fold of basal) and COX2 gene expression (28.2±4.9 -fold of basal), which indicated a synergistic rather than an additive effect. These potentiating effects of BK on both the PGE2 production and increased COX2 gene expression produced by IL-1β were B2 receptor mediated such as the pretreatment with the the B2 receptor antagonist could prevent it (PGE2: 54.6±7.3; COX2: 14.0±2.5 -fold of basal). Conclusions: These results indicate that BK has potentiating effect on the COX2 gene expression and consequent prostanoid production induced by IL-1β, and suggest that B2 receptor blockade by MEN16132 may represent a potential symptomatic therapy for OA.

437 –Table 1. Components of Factors retained by PCA using data from the medial tibial plateaus Factor 1 Factor 2 Factor 3 Factor 4 Factor 5 Parameters Parameters Parameters Parameters Parameters Articular Cartilage (AC) Area (0.94) CCD Area (0.86) Subchondral Bone (SCB) Area (0.71) Area of Weight-Bearing Meniscus (0.87) Axial Osteophyte (OP) Size

Pro‐inflammatory cytokines, matrix metalloproteinases (MMPs) and other catabolic factors participate in the pathogenesis of cartilage damage in osteoarthritis (OA). Pro‐inflammatory cytokines such as interleukin‐1β (IL‐1β) mediate cartilage degradation and might be involved in the progression of OA. Previously, we found that haem oxygenase‐1 (HO‐1) is down‐regulated by pro‐inflammatory cytokines and up‐regulated by IL‐10 in OA chondrocytes. The aim of this study was to determine whether HO‐1 can modify the catabolic effects of IL‐1β in OA cartilage and chondrocytes. Up‐regulation of HO‐1 by cobalt protoporphyrin IX significantly reduced glycosaminoglycan degradation elicited by IL‐1β in OA cartilage explants but increased glycosaminoglycan synthesis and the expression of collagen II in OA chondrocytes in primary culture, as determined by radiometric procedures, immunoblotting and immunocytochemistry. HO‐1 decreased the activation of extracellular signal‐regulated kinase 1/2. This was accompanied by a significant inhibition in MMP activity and expression of collagenases MMP‐1 and MMP‐13 at the protein and mRNA levels. In addition, HO‐1 induction caused a significant increase in the production of insulin‐like growth factor‐1 and a reduction in the levels of insulin‐like growth factor binding protein‐3. We have shown in primary culture of chondrocytes and articular explants from OA patients that HO‐1 counteracts the catabolic and anti‐anabolic effects of IL‐1β. Our data thus suggest that HO‐1 may be a factor regulating the degradation and synthesis of extracellular matrix components in OA. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

S86Osteoarthritis and Cartilage Vol.16 Supplement 4 subsequently treated with tumour necrosis factor-a (TNF-a) in combination with Actinomycin D to induce apoptosis.Apoptotic chondrocytes in cartilage sections were identified using an indirect immunohistochemical staining technique to detect expression of active caspase-3.Haematoxylin and eosin/safranin-o stained sections were used to score cellularity and structural differences between samples.Results: Prior to culture, (mean±standard deviation) chondrocyte viability was 80.7% (3.5).The extent of chondrocyte apoptosis induced by TNF-a/Actinomycin D varied markedly according to the joint type that the cartilage was sampled from.For MCP joints, the extent of overall chondrocyte apoptosis was significantly higher (P < 0.001) in TNFa/Actinomycin D-stimulated explants (26.7%, 10.3) than that observed in unstimulated control samples (9.6%, 7.5).Chondrocytes from PIP and DIP joint cartilage did not respond significantly to apoptotic stimulation (P > 0.05); apoptosis in both control and stimulated explants was virtually identical.Significant variations in cellularity and thickness were evident between cartilages of different joint types.Cartilage from DIP and PIP joints was significantly thicker than that of the MCP joint (P < 0.001 and P < 0.05 respectively).Moreover, MCP joint AC was significantly more cellular than both PIP and DIP joints (P < 0.001).Conclusions: Data in this study demonstrate that chondrocytes from three equine joint types with varying prevalence's of OA differ significantly in terms of susceptibility to apoptosis induction.This may provide a possible explanation for the joint-specific nature of the disease.Jointtype dependent differences in cartilage thickness and cellularity may also have contributory roles.

Objectives. Previous work has shown that the CO-releasing molecule CORM-2 protects against cartilage degradation. The aim of this study was to examine whether CORM-2 can control the production of inflammatory mediators in osteoarthritic chondrocytes and determine the mechanisms involved.Methods. Primary cultures of chondrocytes from OA patients were stimulated with IL-1 beta. The production of reactive oxygen species, nitrite, PGE(2), TNF-alpha and IL-1 receptor antagonist (IL-1Ra) were measured in the presence or absence of CORM-2. The expression of nitric oxide synthase-2 (NOS-2), cyclo-oxygenase-2 (COX-2) and microsomal PG E synthase-1 (mPGES-1) was followed by western blot and real-time PCR. Activation of nuclear factor-kappa B (NF-kappa B) and hypoxia inducible factor-1 alpha (HIF-1 alpha), and phosphorylation of NF-kappa B inhibitory protein alpha (I kappa B alpha) were determined by ELISA.Results. CORM-2 decreased the production of oxidative stress, nitrite and PGE(2). In addition, CORM-2 inhibited IL-1 beta-induced TNF-alpha but enhanced IL-1Ra production. Treatment of chondrocytes with CORM-2 strongly down-regulated NOS-2 and mPGES-1 protein expression, whereas COX-2 was reduced to a lesser extent. These changes were accompanied by a significant decrease in mRNA expression for NOS-2 and mPGES-1. CORM-2 showed a concentration-dependent inhibition of DNA-binding activity for p65 NF-kappa B and HIF-1 alpha. I kappa B alpha phosphorylation was also reduced by CORM-2 treatment.Conclusions. These data have opened new mechanisms of action for CORM-2, raising the prospect that CO-releasing molecules are an interesting strategy for the development of new treatments in articular conditions.

Poster Presentationsincreased to around 50% after 72 h.Histology confirmed that slow, severe loading caused considerably more damage than fast, impact load with the presence of fissuring and uneven GAG distribution particularly in the deeper zones of the tissue.Conclusions: Although producing the same strain in the explant, fast, impact loading was less damaging to cartilage than severe loading that took almost 30 times longer to reach peak stress.Induction of apoptosis was more rapid with severe loading although similar levels were reached in both methods after 3 days in culture.The higher modulus, a measure of resistance to deformation, in the fast, impact loading may explain the increased ability of the cartilage to protect itself from damaging stress levels.The peak stresses reached in the fast, impact loading were more than 20 times that of the slow, severe load suggesting that stress alone is not a good indicator of damage.

ITB (6-(p-bromophenyl)amino-7-(p-chlorophenyl)indazolo[2′,3′:1,5]-1,2,4-triazolo[4,3-a]-1,3,5-benzotriazepine) is a novel inhibitor of cyclo-oxygenase-2 (COX-2) with antiinflammatory activity in animal models. In the present study, we investigated the effect of this compound on the production of catabolic or antiinflammatory mediators in osteoarthritis (OA) cartilage. In OA cartilage explants, ITB inhibited the production of prostaglandin E2 (PGE2), tumour necrosis factor-α (TNF-α) and matrix metalloproteinase-13 (MMP-13) in a concentration-dependent manner, whereas nitrite was partially reduced. On the contrary, ITB increased the production of interleukin (IL)-10 and the expression of heme oxygenase-1 (HO-1). ITB inhibited the production of catabolic mediators at concentrations able to increase IL-10 and HO-1 in OA cartilage, suggesting that this compound may be useful in the prevention of cartilage degradation.

Heme oxygenase-1 (HO-1) is implicated in the protection against tissue injury. We investigated the expression of this protein in cartilage sections and chondrocytes obtained from osteoarthritic patients. HO-1 was immunodetected in preparations from cartilage and also in chondrocytes cultured in the absence of stimulation. We found that HO-1 can be modulated by cytokines since the pro-inflammatory cytokines interleukin (IL)-1β, IL-17 and tumour necrosis factor-α (TNF-α) down-regulated this protein, whereas the anti-inflammatory cytokine IL-10 exerted the opposite effect. Our results suggest a role for HO-1 as part of protective mechanisms against tissue injury in human cartilage.