- Email: [email protected]
Synovial fluid fetuin-A levels in patients affected by osteoarthritis with or without evidence of calcium crystals
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
concentration of all MMPs / ADAMTS-5 for macrophage/fibroblast cultures compared to T cell/fibroblast cultures, with the highest enhancement for MMP-9 concentration (p <0.05). Examining the T cell/chondrocyte co-cultures we detected a significant reduction for MMP-1, MMP-9 and ADAMTS-5 concentration in the Treg/ chondrocyte co-cultures (p <0.05) in comparison to T effector/chondrocyte co-cultures, while the in native SM most enriched MMP-3 showed no significant differences between the two groups. Interestingly, MMP-13 and ADAMTS-4 were not detectable in both experiments. Conclusions: The significantly higher concentration of MMP-1 / -3 / -9 and ADAMTS-5 in SM after T cell depletion indicates a higher influence of macrophages on the induction of ECM-degradation in the cellularenzymatic junction in end-stage OA. Treg reduce the concentration of MMP-1, MMP-9 and ADAMTS-5 significantly compared to co-cultures of chondrocytes with proinflammatory T effector cells and thus may maintain the physiological balance between anabolic and catabolic mechanisms in ECM metabolism. Our data suggests that MNCs play an important role in the process of enzymatic cartilage degradation during disease progression. We show here that polarization of the mononuclear cell infiltration may be highly influential on the production of cartilage-degrading enzymes. These results underline the relevance of a proinflammatory MNC infiltration in the process of joint destruction and show the possible therapeutic aspect of Treg activation for keeping a balanced equilibrium of catabolic and anabolic processes in ECM metabolism. 542 REGULATION OF INDUCIBLE PROSTAGLANDIN E SYNTHASE-1 (MPGES-1) EXPRESSION IN HUMAN OA CHONDROCYTES L. Tuure y, M. H€ am€ al€ ainen y, R. Nieminen y, T. Moilanen z, E. Moilanen y. y The Immunopharmacology Res. Group, Univ. of Tampere Sch. of Med. and Tampere Univ. Hosp., Tampere, Finland; z Coxa Hosp. for Joint Replacement, Tampere, Finland Purpose: Microsomal PGE synthase-1 (mPGES-1) is a terminal enzyme in the production of prostaglandin E2 (PGE2), and its expression is upregulated during inflammation. mPGES-1 is considered as a potential drug target for the treatment of (osteo)arthritis and related diseases, in order to reduce adverse effects related to the current non-steroidal antiinflammatory drugs (NSAIDs): Compounds which inhibit mPGES-1 expression or activity would have similar therapeutic effect as NSAIDs through decreasing inflammation-associated PGE2 synthesis. Whereas they would have no effect on the physiological PGE2 synthesis or on the balance of prostacyclin and thromboxane synthesis, which mechanisms have been linked to gastrointestinal and cardiovascular adverse effects of NSAIDS, respectively. In the present study, our primary objective was to study the expression of mPGES-1 in primary human chondrocytes, and to investigate the effects of glucocorticoids and clinically used antirheumatic drugs (DMARDs) on it. In addition, we aimed to study the regulatory mechanisms concerning the expression of mPGES-1 in chondrocytes because they remain mostly unknown. Based on our promising preliminary results, we focused on the role of mitogen-activated protein kinase phosphatase 1 (MKP-1) in the regulation of mPGES-1 expression. MKP1 is an endogenous anti-inflammatory mediator able to inactivate through dephosphorylation the mitogen-activated protein kinases p38 and JNK, which are essential signaling pathways in inflammation. From pharmacological viewpoint, MKP-1 is a highly interesting molecule, as it is known to mediate many of the anti-inflammatory effects of glucocorticoids and some other anti-inflammatory drugs. Moreover, we investigated the effects of MAP kinases JNK or p38 on mPGES-1 expression and tested the hypothesis that glucocorticoids downregulate the expression of mPGES-1 through increased MKP-1 expression and decreased MAP kinase phosphorylation. Methods: Primary human chondrocytes were isolated by enzymatic digestion from cartilage samples obtained from patients undergoing total knee replacement surgery. In addition, J774 macrophage cell line, and cartilage tissue and peritoneal macrophages from MKP-1 deficient (knock-out, KO) and corresponding wild type (WT) mice were used. Expression of mPGES-1 and MKP-1 was investigated by quantitative real-time PCR and Western blot analysis; and MAP kinase activation, namely phosphorylation, by Western blotting by using antibodies against phosphorylated and total p38 and JNK kinases. Prostaglandin E2 levels were measured by enzyme-linked immunosorbent assay.
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Results: Primary human chondrocytes expressed mPGES-1 at low level and its expression was enhanced when the cells were exposed to interleukin-1 (IL-1): mPGES-1 protein levels continued to increase up to the 96 hours’ follow-up when stimulated with IL-1. Interestingly, aurothiomalate inhibited mPGES-1 expression and PGE2 production in a dosedependent manner as did the anti-inflammatory steroid dexamethasone. Other disease-modifying antirheumatic drugs (DMARDs) studied, i.e. sulfasalazine, methotrexate and hydroxychloroquine did not alter mPGES-1 expression. Dexamethasone and aurothiomalate also enhanced MKP-1 expression in chondrocytes; and interestingly, mPGES-1 expression was increased in IL-1 stimulated articular cartilage from MKP-1 deficient mice as compered to the tissue from WT animals. Studies with peritoneal macrophages from WT and MKP-1 deficient mice also directly showed that MKP-1 mediates the inhibitory effect of dexamethasone on mPGES-1 expression. Further, MKP-1 was found to induce dephosphorylation on both p38 and JNK MAP kinases but only the selective JNK inhibitor SP600125 downregulated mPGES-1 expression. Conclusions: The results introduce glucocorticoid dexamethasone and DMARD aurothiomalate as the first and so far the only drugs found to be able to inhibit mPGES-1 expression in human OA chondrocytes and that effect is likely involved in the mechanisms of action of these antiinflammatory compounds. Based on the studies with cells and tissues from MKP-1 deficient mice, these drug effects on mPGES-1 expression are likely mediated through increased MKP-1 expression and decreased JNK activation. In general, these results advance our understanding on the regulatory mechanisms of mPGES-1 expression that are under intensive research in order to develop new anti-inflammatory treatments for arthritis. 543 SYNOVIAL FLUID FETUIN-A LEVELS IN PATIENTS AFFECTED BY OSTEOARTHRITIS WITH OR WITHOUT EVIDENCE OF CALCIUM CRYSTALS M. Favero y, z, E. Belluzzi y, P. Frallonardo y, L. Peruzzo x, L. Tauro k, F. Oliviero y, R. Ramonda y, L. Punzi y. y Rheumatology Unit, Dept. of Med. - DIMED, Univ. Hosp. of Padova, Padova, Italy; z RAMSES Lab., RIT Dept., Rizzoli Orthopedic Res. Inst., Bologna, Italy; x Inst. for GeoSci.s and Earth Resources IGG-CNR, Padova, Italy; k Dept. of GeoSci.s - Padova Univ., Padova, Italy Purpose: Osteoarthritis (OA) is the most common joint disorder worldwide and the major cause of pain and disability in older adults. Crystal’s deposition has been detected in at least 60% of OA patients. Two types of calcium crystals (CC) seem to be linked to OA: calcium pyrophosphate crystals (CPP) and basic calcium phosphate crystals (BCP). The role of CPP and BCP in OA as well as the mechanism responsible of its formation are not fully understood. However, experimental evidence supports that crystals might exacerbate the disease increasing cytokine production and enhancing the inflammatory state. Fetuin-A (FA) is a secreted protein that inhibits ectopic mineralization. FA acts binding matrix Gla protein (MGP) and forming the soluble fetuin-mineral complex (FMC). An impairment of FMC formation has been recently demonstrated in human OA chondrocytes, suggesting a potential role of FA in calcium crystals formation in OA patients. Synovial fluid (SF) FA levels in patients with OA have been measured in two previous studies, without obtaining clear results. The aim of the present study was to measure SF FA levels in the following subgroups of patients affected by symptomatic (pain, effusion) knee OA (KOA): patients without crystals, patients with CPP, and patients with BCP. Moreover, interleukin-6 (IL-6) and total protein concentration have been detected in SF. Methods: SF samples were collected from patients affected by symptomatic KOA and undergoing arthrocentesis. All the patients gave written informed consent. SF total white blood cell (WBC) count was determined using a Bürker counting chamber, while differential cell count was performed with pre-stained slides for SF cell morphology (Testsimplets®). CC were identified by scanning electron microscopy. The following clinical data were collected: age, sex, body max index (BMI), and disease duration. FA levels were detected by ELISA assay kit (Biovendor) according to the manufacturing instructions. SF IL-6 protein concentration was measured by ELISA assay kit (eBioscience) and total protein concentration by BCA assay (Thermoscientific). The differences between subgroups were evaluated by Mann-Whitney test and correlations were assessed by Spearman test using Graph-Pad Prism software, version 5.0.
S334
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Results: SF were obtained from 24 patients affected by symptomatic KOA (5 male and 19 female) (median age 75 years, interquartile range [IQR] (80.75-69.00): 8 patients without CC (median age 66.5 years), 8 patients with CPP (median age 76.5 years), and 8 patients with BCP (median age 80.0 years). Patients with SF CPP and BCP were statistically older than patients without (p¼0.0221 and p¼0.0009). SF WBCs were found higher in patients with CC compared with patients without. SF FA levels were statistically higher in patients with CC compared with patients without (no CC vs CPP p¼0.0011; no CC vs BCP group p¼0.0104). SF FA levels were correlated with age (r ¼0.4927, P¼0.0144). Patients with CPP and BCP had increased SF total protein concentration compared with patients without crystals (no CC vs CPP p¼0.0148; no CC vs OA BCP p¼0.0047). Moreover, a significant increasing in SF IL-6 levels was found in patients with CCP e BCP compared with patients without (no CC vs CPP p¼0.0379; no CC vs BCP p¼0.0047). Conclusions: In the present study, increased SF FA levels in patients affected by symptomatic KOA with presence of calcium crystals (both CPP and BCP) compared with patients without have been detected, suggesting a potential role of FA in calcium crystals formation. In addition, increased IL-6 and total protein concentration in patients with CC compared with patients without has been found. These findings confirmed literature data reporting a potential role of crystals in inducing inflammation. 544 EXPRESSION OF INTEGRIN CD11C IN PERIPROSTHETIC TISSUES FROM FAILED TOTAL HIP ARTHROPLASTIES AND ITS REGULATION BY WEAR PARTICLES IN CELL CULTURE K. Chamaon, F. Awiszus, C.H. Lohmann. Univ. Magdeburg, Magdeburg, Germany Purpose: A growing number of osteoarthritis patients require the supply of an joint replacement. Friction mechanisms acting on each contact surface of artificial joint leads to the formation of abrasive particles. The occurring wear may be an origin of inflammatory processes leading to the loss of endoprostheses. Different cellular responses have been described in the presence of foreign body wear material in the periprosthetic tissues such as increased occurence of immune cell populations, enhancement of inflammatory parameters, or the formation of foreign body giant cells (FBGC). The aim of the presented study was to analyze the expression of integrin CD11c (alphaXbeta2) in periprosthetic tissue and human cell lines incubated with wear particles. The objective is to better understand cellular reactions to foreign body material formed during endoprosthesis movement. Methods: CD11c immunohistochemistry was performed on periprosthetic tissues from 23 ceramic-on-ultra-high molecular weight polyethylene (UHMWPE), 20 metal-on-UHMWPE, and 2 metal-on-metal total hip arthroplasties (THAs). The inter- and intra-observer reliabilities of scoring for CD11c positive cells and intracellular wear were examined using the k-coefficient. Association analyses of CD11c expression pattern with intracellular wear were performed by determination of Exact 2sided Significance p<0.05 in Fisher's Exact Test using SPSS 22.0.CD11c expression of two human monocytic cell lines MUTZ-3 and MonoMac-6 after incubation with wear particles (UHMWPE, 150-10.000 nm) was determind by fluorescence microscopy and flow cytometry. Metabolic activity of cell lines was measured by WST-1 assay in light microscopy. Results: We established a new scoring system by immunohistochemical staining of CD11c in periprosthetic tissues. We classified CD11c positive cells into four different expression patterns. First, mono- and multinuclear cells with up to three nuclei and cytoplasmic expression of CD11c (MMC-C, inter-rater k¼0.738, intra-rater k¼0.692), second, mono- and multinuclear cells with up to three nuclei with CD11c expression on the cell membrane (MMC-M, inter-rater k¼0.761, intra-rater k¼0.815), third CD11c positive cells, larger in size, with more than six nuclei and expressing CD11c on the cell membrane, called multinucleated giant cells (MGC, inter-rater k¼0.622, intra-rater k¼0.613) and fourth, larger cells with more than 6 nuclei and visible wear inclusion, called foreign body giant cells (FBGC, inter-rater k¼0.585, intra-rater k¼0.640). MMC-M and MMC-C possess a strong association with UHMWPE (MMC-M, p¼0.000152 and MMC-C, p¼0.006, respectively) and FBGC are significantly associated with UHMWPE (p¼0.000014). In contrast, in the case of MGC the association with UHMWPE is not proven, however has a noticeable trend (p¼0.063). Further, using human monocytic cell lines preliminary data indicate an increase of CD11c expression after UHMWPE particle incubation using flow cytometry and fluorescence microscopy.
Conclusions: Our work documents a simultaneous cellular diversity as a response to debris material in the tissue of orthopaedic endoprostheses and describes the UHMWPE response as a multicellular dynamic process. Further, preliminary data show that incubation with PE particles leads to an upregulation of CD11c in monocytic cells. Further investigations are needed to clarify the functional changes on basis of CD11c upregulation e.g. signaling cascades, cell adhesion, and foreign body giant cell generation. 545 TARGETING SIALIC ACID MODIFIED RECEPTORS AS A POTENTIAL THERAPY FOR OSTEOARTHRITIS P. Carpintero-Fernandez y, R. Gago-Fuentes y, M. Varela-Eirin y, B. Acea y, E. Fonseca y, G. Golberg z, F.J. Blanco x, M.D. Mayan x. y ~ a (CHUACCellCOM-SB Res. Group, INIBIC, Hosp. Univ.rio A Corun ~ a., A Corun ~ a, Spain; z Sch. of XXIAC) SERGAS, Univ. of A Corun x ~ a, NJ, Spain; Rheumatology Osteopathic Med., Rowan Univ., A Corun ~ a (CHUAC-XXIAC) SERGAS, Univ. of A Div., INIBIC, Hosp. Univ.rio A Corun ~ a, A Corun ~ a, Spain Corun Purpose: Glycosylated proteins are essential components of the cartilage extracellular matrix and contribute to maintain the cartilage structure and function. A notable shift from glycoproteins containing a2,6-linked sialic acids to those containing a-2,3-linked sialic acids has been associated with the onset of disorders such as rheumatoid arthritis (RA) and OA that concur with progressive cartilage degeneration. Here we studied the effects of the Maackia amurensis seed lectin (MASL) on chondrocytes and cartilage integrity from healthy donors, OA patients and animal models of arthritis Methods: Chondrocytes were isolated from articular cartilage and cultured in DMEM containing 15% FCS. For immunofluorescence and immunohistochemistry assays, in situ cartilage was fixed and frozen using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Cell viability, cell adhesion and growth assay were performed with using commercial kits. Reactive oxygen species (ROS) levels were measured by DCFH-DA and by Flow Cytometry. Oligomycin and LPS were used to induce cartilage degeneration in vitro and in vivo (mouse model, Mus musculus BALC/c). Results: The treatment of primary human chondrocytes with 400 and 720nM of the lectin MASL did not affect cell viability, adhesion or growth. To mimic pathological conditions, cells and cartilage explants were treated with 5 mg/ml of oligomycin without affecting cell viability. The treatment of primary cells with MASL effectively protects chondrocytes from ROS production and expression of inflammatory cytokines when incubated in the presence of oligomycin. The treatment of cartilage punches with 5 mg/ml of oligomycin for 7 days decreased safranin uptake and disrupted the ECM structure. However, the presence of 400nM of MASL prevented the cartilage destruction and inhibited the expression of COX2 and MMP3 induced by oligomycin. Finally, the oral administration of 1 mg of MASL per week protected articular cartilage from the degeneration induced by LPS intra-articular injection. Conclusions: This study demonstrates that nanomolar concentrations of MASL protect chondrocytes and prevent cartilage breakdown initiated by ROS, inflammatory cytokines, and metalloproteinases. These findings reveal that specific lectins that target sialylated transmembrane receptors on human chondrocytes may be effective in inhibiting cartilage destruction in the face of arthitic insults. These findings shed mechanistic light on how a specific lectin could be used as novel avenues to treat inflammatory and degenerative conditions of the joints such as OA and RA. 546 OBESITY DOES NOT AFFECT THE SIZE OF INFRAPATELLAR FAT PAD ADIPOCYTES: IMPLICATIONS FOR THE PATHOGENESIS OF KNEE OSTEOARTHRITIS J. Garcia y, W. Wei z, J. Runhaar z, K. Wright y, C. Mennan y, S. Roberts y, G. Van Osch z, Y. Bastiaansen-Jenniskens z. y The Robert Jones and Agnes Hunt Orthopaedic Hosp. NHS Fndn. Trust, Oswestry, United Kingdom; z Erasmus MC Univ. Med. Ctr., Rotterdam, Netherlands Purpose: Obesity is known to be a risk factor for the development of osteoarthritis (OA). Obesity is accompanied by adipocyte hypertrophy and elevated inflammation in subcutaneous and visceral adipose tissue. The infrapatellar fat pad (IPFP) is an adipose tissue that contains adipocytes. The IPFP is believed to be influential in determining the
concentration of all MMPs / ADAMTS-5 for macrophage/fibroblast cultures compared to T cell/fibroblast cultures, with the highest enhancement for MMP-9 concentration (p <0.05). Examining the T cell/chondrocyte co-cultures we detected a significant reduction for MMP-1, MMP-9 and ADAMTS-5 concentration in the Treg/ chondrocyte co-cultures (p <0.05) in comparison to T effector/chondrocyte co-cultures, while the in native SM most enriched MMP-3 showed no significant differences between the two groups. Interestingly, MMP-13 and ADAMTS-4 were not detectable in both experiments. Conclusions: The significantly higher concentration of MMP-1 / -3 / -9 and ADAMTS-5 in SM after T cell depletion indicates a higher influence of macrophages on the induction of ECM-degradation in the cellularenzymatic junction in end-stage OA. Treg reduce the concentration of MMP-1, MMP-9 and ADAMTS-5 significantly compared to co-cultures of chondrocytes with proinflammatory T effector cells and thus may maintain the physiological balance between anabolic and catabolic mechanisms in ECM metabolism. Our data suggests that MNCs play an important role in the process of enzymatic cartilage degradation during disease progression. We show here that polarization of the mononuclear cell infiltration may be highly influential on the production of cartilage-degrading enzymes. These results underline the relevance of a proinflammatory MNC infiltration in the process of joint destruction and show the possible therapeutic aspect of Treg activation for keeping a balanced equilibrium of catabolic and anabolic processes in ECM metabolism. 542 REGULATION OF INDUCIBLE PROSTAGLANDIN E SYNTHASE-1 (MPGES-1) EXPRESSION IN HUMAN OA CHONDROCYTES L. Tuure y, M. H€ am€ al€ ainen y, R. Nieminen y, T. Moilanen z, E. Moilanen y. y The Immunopharmacology Res. Group, Univ. of Tampere Sch. of Med. and Tampere Univ. Hosp., Tampere, Finland; z Coxa Hosp. for Joint Replacement, Tampere, Finland Purpose: Microsomal PGE synthase-1 (mPGES-1) is a terminal enzyme in the production of prostaglandin E2 (PGE2), and its expression is upregulated during inflammation. mPGES-1 is considered as a potential drug target for the treatment of (osteo)arthritis and related diseases, in order to reduce adverse effects related to the current non-steroidal antiinflammatory drugs (NSAIDs): Compounds which inhibit mPGES-1 expression or activity would have similar therapeutic effect as NSAIDs through decreasing inflammation-associated PGE2 synthesis. Whereas they would have no effect on the physiological PGE2 synthesis or on the balance of prostacyclin and thromboxane synthesis, which mechanisms have been linked to gastrointestinal and cardiovascular adverse effects of NSAIDS, respectively. In the present study, our primary objective was to study the expression of mPGES-1 in primary human chondrocytes, and to investigate the effects of glucocorticoids and clinically used antirheumatic drugs (DMARDs) on it. In addition, we aimed to study the regulatory mechanisms concerning the expression of mPGES-1 in chondrocytes because they remain mostly unknown. Based on our promising preliminary results, we focused on the role of mitogen-activated protein kinase phosphatase 1 (MKP-1) in the regulation of mPGES-1 expression. MKP1 is an endogenous anti-inflammatory mediator able to inactivate through dephosphorylation the mitogen-activated protein kinases p38 and JNK, which are essential signaling pathways in inflammation. From pharmacological viewpoint, MKP-1 is a highly interesting molecule, as it is known to mediate many of the anti-inflammatory effects of glucocorticoids and some other anti-inflammatory drugs. Moreover, we investigated the effects of MAP kinases JNK or p38 on mPGES-1 expression and tested the hypothesis that glucocorticoids downregulate the expression of mPGES-1 through increased MKP-1 expression and decreased MAP kinase phosphorylation. Methods: Primary human chondrocytes were isolated by enzymatic digestion from cartilage samples obtained from patients undergoing total knee replacement surgery. In addition, J774 macrophage cell line, and cartilage tissue and peritoneal macrophages from MKP-1 deficient (knock-out, KO) and corresponding wild type (WT) mice were used. Expression of mPGES-1 and MKP-1 was investigated by quantitative real-time PCR and Western blot analysis; and MAP kinase activation, namely phosphorylation, by Western blotting by using antibodies against phosphorylated and total p38 and JNK kinases. Prostaglandin E2 levels were measured by enzyme-linked immunosorbent assay.
S333
Results: Primary human chondrocytes expressed mPGES-1 at low level and its expression was enhanced when the cells were exposed to interleukin-1 (IL-1): mPGES-1 protein levels continued to increase up to the 96 hours’ follow-up when stimulated with IL-1. Interestingly, aurothiomalate inhibited mPGES-1 expression and PGE2 production in a dosedependent manner as did the anti-inflammatory steroid dexamethasone. Other disease-modifying antirheumatic drugs (DMARDs) studied, i.e. sulfasalazine, methotrexate and hydroxychloroquine did not alter mPGES-1 expression. Dexamethasone and aurothiomalate also enhanced MKP-1 expression in chondrocytes; and interestingly, mPGES-1 expression was increased in IL-1 stimulated articular cartilage from MKP-1 deficient mice as compered to the tissue from WT animals. Studies with peritoneal macrophages from WT and MKP-1 deficient mice also directly showed that MKP-1 mediates the inhibitory effect of dexamethasone on mPGES-1 expression. Further, MKP-1 was found to induce dephosphorylation on both p38 and JNK MAP kinases but only the selective JNK inhibitor SP600125 downregulated mPGES-1 expression. Conclusions: The results introduce glucocorticoid dexamethasone and DMARD aurothiomalate as the first and so far the only drugs found to be able to inhibit mPGES-1 expression in human OA chondrocytes and that effect is likely involved in the mechanisms of action of these antiinflammatory compounds. Based on the studies with cells and tissues from MKP-1 deficient mice, these drug effects on mPGES-1 expression are likely mediated through increased MKP-1 expression and decreased JNK activation. In general, these results advance our understanding on the regulatory mechanisms of mPGES-1 expression that are under intensive research in order to develop new anti-inflammatory treatments for arthritis. 543 SYNOVIAL FLUID FETUIN-A LEVELS IN PATIENTS AFFECTED BY OSTEOARTHRITIS WITH OR WITHOUT EVIDENCE OF CALCIUM CRYSTALS M. Favero y, z, E. Belluzzi y, P. Frallonardo y, L. Peruzzo x, L. Tauro k, F. Oliviero y, R. Ramonda y, L. Punzi y. y Rheumatology Unit, Dept. of Med. - DIMED, Univ. Hosp. of Padova, Padova, Italy; z RAMSES Lab., RIT Dept., Rizzoli Orthopedic Res. Inst., Bologna, Italy; x Inst. for GeoSci.s and Earth Resources IGG-CNR, Padova, Italy; k Dept. of GeoSci.s - Padova Univ., Padova, Italy Purpose: Osteoarthritis (OA) is the most common joint disorder worldwide and the major cause of pain and disability in older adults. Crystal’s deposition has been detected in at least 60% of OA patients. Two types of calcium crystals (CC) seem to be linked to OA: calcium pyrophosphate crystals (CPP) and basic calcium phosphate crystals (BCP). The role of CPP and BCP in OA as well as the mechanism responsible of its formation are not fully understood. However, experimental evidence supports that crystals might exacerbate the disease increasing cytokine production and enhancing the inflammatory state. Fetuin-A (FA) is a secreted protein that inhibits ectopic mineralization. FA acts binding matrix Gla protein (MGP) and forming the soluble fetuin-mineral complex (FMC). An impairment of FMC formation has been recently demonstrated in human OA chondrocytes, suggesting a potential role of FA in calcium crystals formation in OA patients. Synovial fluid (SF) FA levels in patients with OA have been measured in two previous studies, without obtaining clear results. The aim of the present study was to measure SF FA levels in the following subgroups of patients affected by symptomatic (pain, effusion) knee OA (KOA): patients without crystals, patients with CPP, and patients with BCP. Moreover, interleukin-6 (IL-6) and total protein concentration have been detected in SF. Methods: SF samples were collected from patients affected by symptomatic KOA and undergoing arthrocentesis. All the patients gave written informed consent. SF total white blood cell (WBC) count was determined using a Bürker counting chamber, while differential cell count was performed with pre-stained slides for SF cell morphology (Testsimplets®). CC were identified by scanning electron microscopy. The following clinical data were collected: age, sex, body max index (BMI), and disease duration. FA levels were detected by ELISA assay kit (Biovendor) according to the manufacturing instructions. SF IL-6 protein concentration was measured by ELISA assay kit (eBioscience) and total protein concentration by BCA assay (Thermoscientific). The differences between subgroups were evaluated by Mann-Whitney test and correlations were assessed by Spearman test using Graph-Pad Prism software, version 5.0.
S334
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
Results: SF were obtained from 24 patients affected by symptomatic KOA (5 male and 19 female) (median age 75 years, interquartile range [IQR] (80.75-69.00): 8 patients without CC (median age 66.5 years), 8 patients with CPP (median age 76.5 years), and 8 patients with BCP (median age 80.0 years). Patients with SF CPP and BCP were statistically older than patients without (p¼0.0221 and p¼0.0009). SF WBCs were found higher in patients with CC compared with patients without. SF FA levels were statistically higher in patients with CC compared with patients without (no CC vs CPP p¼0.0011; no CC vs BCP group p¼0.0104). SF FA levels were correlated with age (r ¼0.4927, P¼0.0144). Patients with CPP and BCP had increased SF total protein concentration compared with patients without crystals (no CC vs CPP p¼0.0148; no CC vs OA BCP p¼0.0047). Moreover, a significant increasing in SF IL-6 levels was found in patients with CCP e BCP compared with patients without (no CC vs CPP p¼0.0379; no CC vs BCP p¼0.0047). Conclusions: In the present study, increased SF FA levels in patients affected by symptomatic KOA with presence of calcium crystals (both CPP and BCP) compared with patients without have been detected, suggesting a potential role of FA in calcium crystals formation. In addition, increased IL-6 and total protein concentration in patients with CC compared with patients without has been found. These findings confirmed literature data reporting a potential role of crystals in inducing inflammation. 544 EXPRESSION OF INTEGRIN CD11C IN PERIPROSTHETIC TISSUES FROM FAILED TOTAL HIP ARTHROPLASTIES AND ITS REGULATION BY WEAR PARTICLES IN CELL CULTURE K. Chamaon, F. Awiszus, C.H. Lohmann. Univ. Magdeburg, Magdeburg, Germany Purpose: A growing number of osteoarthritis patients require the supply of an joint replacement. Friction mechanisms acting on each contact surface of artificial joint leads to the formation of abrasive particles. The occurring wear may be an origin of inflammatory processes leading to the loss of endoprostheses. Different cellular responses have been described in the presence of foreign body wear material in the periprosthetic tissues such as increased occurence of immune cell populations, enhancement of inflammatory parameters, or the formation of foreign body giant cells (FBGC). The aim of the presented study was to analyze the expression of integrin CD11c (alphaXbeta2) in periprosthetic tissue and human cell lines incubated with wear particles. The objective is to better understand cellular reactions to foreign body material formed during endoprosthesis movement. Methods: CD11c immunohistochemistry was performed on periprosthetic tissues from 23 ceramic-on-ultra-high molecular weight polyethylene (UHMWPE), 20 metal-on-UHMWPE, and 2 metal-on-metal total hip arthroplasties (THAs). The inter- and intra-observer reliabilities of scoring for CD11c positive cells and intracellular wear were examined using the k-coefficient. Association analyses of CD11c expression pattern with intracellular wear were performed by determination of Exact 2sided Significance p<0.05 in Fisher's Exact Test using SPSS 22.0.CD11c expression of two human monocytic cell lines MUTZ-3 and MonoMac-6 after incubation with wear particles (UHMWPE, 150-10.000 nm) was determind by fluorescence microscopy and flow cytometry. Metabolic activity of cell lines was measured by WST-1 assay in light microscopy. Results: We established a new scoring system by immunohistochemical staining of CD11c in periprosthetic tissues. We classified CD11c positive cells into four different expression patterns. First, mono- and multinuclear cells with up to three nuclei and cytoplasmic expression of CD11c (MMC-C, inter-rater k¼0.738, intra-rater k¼0.692), second, mono- and multinuclear cells with up to three nuclei with CD11c expression on the cell membrane (MMC-M, inter-rater k¼0.761, intra-rater k¼0.815), third CD11c positive cells, larger in size, with more than six nuclei and expressing CD11c on the cell membrane, called multinucleated giant cells (MGC, inter-rater k¼0.622, intra-rater k¼0.613) and fourth, larger cells with more than 6 nuclei and visible wear inclusion, called foreign body giant cells (FBGC, inter-rater k¼0.585, intra-rater k¼0.640). MMC-M and MMC-C possess a strong association with UHMWPE (MMC-M, p¼0.000152 and MMC-C, p¼0.006, respectively) and FBGC are significantly associated with UHMWPE (p¼0.000014). In contrast, in the case of MGC the association with UHMWPE is not proven, however has a noticeable trend (p¼0.063). Further, using human monocytic cell lines preliminary data indicate an increase of CD11c expression after UHMWPE particle incubation using flow cytometry and fluorescence microscopy.
Conclusions: Our work documents a simultaneous cellular diversity as a response to debris material in the tissue of orthopaedic endoprostheses and describes the UHMWPE response as a multicellular dynamic process. Further, preliminary data show that incubation with PE particles leads to an upregulation of CD11c in monocytic cells. Further investigations are needed to clarify the functional changes on basis of CD11c upregulation e.g. signaling cascades, cell adhesion, and foreign body giant cell generation. 545 TARGETING SIALIC ACID MODIFIED RECEPTORS AS A POTENTIAL THERAPY FOR OSTEOARTHRITIS P. Carpintero-Fernandez y, R. Gago-Fuentes y, M. Varela-Eirin y, B. Acea y, E. Fonseca y, G. Golberg z, F.J. Blanco x, M.D. Mayan x. y ~ a (CHUACCellCOM-SB Res. Group, INIBIC, Hosp. Univ.rio A Corun ~ a., A Corun ~ a, Spain; z Sch. of XXIAC) SERGAS, Univ. of A Corun x ~ a, NJ, Spain; Rheumatology Osteopathic Med., Rowan Univ., A Corun ~ a (CHUAC-XXIAC) SERGAS, Univ. of A Div., INIBIC, Hosp. Univ.rio A Corun ~ a, A Corun ~ a, Spain Corun Purpose: Glycosylated proteins are essential components of the cartilage extracellular matrix and contribute to maintain the cartilage structure and function. A notable shift from glycoproteins containing a2,6-linked sialic acids to those containing a-2,3-linked sialic acids has been associated with the onset of disorders such as rheumatoid arthritis (RA) and OA that concur with progressive cartilage degeneration. Here we studied the effects of the Maackia amurensis seed lectin (MASL) on chondrocytes and cartilage integrity from healthy donors, OA patients and animal models of arthritis Methods: Chondrocytes were isolated from articular cartilage and cultured in DMEM containing 15% FCS. For immunofluorescence and immunohistochemistry assays, in situ cartilage was fixed and frozen using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Cell viability, cell adhesion and growth assay were performed with using commercial kits. Reactive oxygen species (ROS) levels were measured by DCFH-DA and by Flow Cytometry. Oligomycin and LPS were used to induce cartilage degeneration in vitro and in vivo (mouse model, Mus musculus BALC/c). Results: The treatment of primary human chondrocytes with 400 and 720nM of the lectin MASL did not affect cell viability, adhesion or growth. To mimic pathological conditions, cells and cartilage explants were treated with 5 mg/ml of oligomycin without affecting cell viability. The treatment of primary cells with MASL effectively protects chondrocytes from ROS production and expression of inflammatory cytokines when incubated in the presence of oligomycin. The treatment of cartilage punches with 5 mg/ml of oligomycin for 7 days decreased safranin uptake and disrupted the ECM structure. However, the presence of 400nM of MASL prevented the cartilage destruction and inhibited the expression of COX2 and MMP3 induced by oligomycin. Finally, the oral administration of 1 mg of MASL per week protected articular cartilage from the degeneration induced by LPS intra-articular injection. Conclusions: This study demonstrates that nanomolar concentrations of MASL protect chondrocytes and prevent cartilage breakdown initiated by ROS, inflammatory cytokines, and metalloproteinases. These findings reveal that specific lectins that target sialylated transmembrane receptors on human chondrocytes may be effective in inhibiting cartilage destruction in the face of arthitic insults. These findings shed mechanistic light on how a specific lectin could be used as novel avenues to treat inflammatory and degenerative conditions of the joints such as OA and RA. 546 OBESITY DOES NOT AFFECT THE SIZE OF INFRAPATELLAR FAT PAD ADIPOCYTES: IMPLICATIONS FOR THE PATHOGENESIS OF KNEE OSTEOARTHRITIS J. Garcia y, W. Wei z, J. Runhaar z, K. Wright y, C. Mennan y, S. Roberts y, G. Van Osch z, Y. Bastiaansen-Jenniskens z. y The Robert Jones and Agnes Hunt Orthopaedic Hosp. NHS Fndn. Trust, Oswestry, United Kingdom; z Erasmus MC Univ. Med. Ctr., Rotterdam, Netherlands Purpose: Obesity is known to be a risk factor for the development of osteoarthritis (OA). Obesity is accompanied by adipocyte hypertrophy and elevated inflammation in subcutaneous and visceral adipose tissue. The infrapatellar fat pad (IPFP) is an adipose tissue that contains adipocytes. The IPFP is believed to be influential in determining the