Human Osteoarthritic Chondrocytes Express Nineteen Different TRP-Genes-TRPA1 and TRPM8 as Potential Drug Targets.

Transient receptor potential (TRP) ion channels are expressed in neuronal and some non-neuronal cells and are involved particularly in pain and thermosensation. We previously showed that TRPA1 is functionally expressed in human osteoarthritic (OA) chondrocytes and mediates inflammation, cartilage degradation, and pain in monosodium-iodoacetate-induced experimental OA. In the present study, we explored the expression of TRP-channels in primary human OA chondrocytes and investigated whether drugs used in the treatment of OA, ibuprofen and glucocorticoids, have effects on TRP-channel expression. OA cartilage was obtained from knee replacement surgery and chondrocytes were isolated with enzyme digestion. NGS analysis showed the expression of 19 TRP-genes in OA chondrocytes, with TRPM7, TRPV4, TRPC1, and TRPM8 having the highest counts in unstimulated cells. These results were verified with RT-PCR in samples from a different group of patients. Interleukin-1ß (IL-1ß) significantly increased TRPA1 expression, while TRPM8 and TRPC1 expression was decreased, and TRPM7 and TRPV4 expression remained unaffected. Furthermore, dexamethasone attenuated the effect of IL-1ß on TRPA1 and TRPM8 expression. The TRPM8 and TRPA1 agonist menthol increased the expression of the cartilage-degrading enzymes MMP-1, MMP-3, and MMP-13 and the inflammatory factors iNOS and IL-6 in OA chondrocytes. In conclusion, human OA chondrocytes express 19 different TRP-genes, of which the significant TRPM8 expression is a novel finding. Dexamethasone attenuated IL-1ß-induced TRPA1 expression. Interestingly, the TRPM8 and TRPA1 agonist menthol increased MMP expression. These results support the concept of TRPA1 and TRMP8 as potential novel drug targets in arthritis.

Dexamethasone Attenuates the Expression of MMP-13 in Chondrocytes through MKP-1.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) is upregulated in inflammation and reduces the activity of proinflammatory mitogen-activated protein kinases (MAP kinases) by dephosphorylation. MAP kinases are intracellular signaling pathways that mediate the cellular effects of proinflammatory cytokines. In the present study, we investigated the effects of the glucocorticoid dexamethasone on the expression of catabolic enzymes in chondrocytes and tested the hypothesis that these effects are mediated through MKP-1. Dexamethasone was found to significantly attenuate the expression of matrix metalloproteinase (MMP)-13 in human OA chondrocytes as well as in chondrocytes from MKP-1 WT mice, but not in chondrocytes from MKP-1 KO mice. Dexamethasone also increased the expression of MKP-1 in murine and human OA chondrocytes. Furthermore, p38 MAP kinase inhibitors significantly attenuated MMP-13 expression in human OA chondrocytes, while JNK MAP kinase inhibitors had no effect. The results indicate that the effect of dexamethasone on MMP-13 expression in chondrocytes was mediated by an MKP-1 and p38 MAP kinase-dependent manner. These findings, together with previous results, support the concept of MKP-1 as a protective factor in articular chondrocytes in inflammatory conditions and as a potential drug target to treat OA.

Effects of ibuprofen on gene expression in chondrocytes from patients with osteoarthritis as determined by RNA-Seq.

Non-steroidal anti-inflammatory drugs are a widely used symptomatic treatment in osteoarthritis (OA), but their effects on cartilage remain controversial. We studied the effects of ibuprofen on gene expression in chondrocytes from patients with OA using RNA-Seq. Chondrocytes were isolated from cartilage samples of patients with OA undergoing knee replacement surgery, cultured with ibuprofen, and total mRNA was sequenced. Differentially expressed genes were identified with edgeR using pairwise comparisons. Functional analysis was performed using ingenuity pathway analysis (IPA). Ibuprofen did not induce statistically significant changes in chondrocyte transcriptome when the cells were cultured in the absence of added cytokines. In inflammatory conditions (when the cells were exposed to the OA-related cytokine interleukin (IL)-1ß), 51 genes were upregulated and 42 downregulated by ibuprofen with fold change >1.5 in either direction. The upregulated genes included anti-inflammatory factors and genes associated with cell adhesion, while several mediators of inflammation were among the downregulated genes. IPA analysis revealed ibuprofen having modulating effects on inflammation-related pathways such as integrin, IL-8, ERK/MAPK and cAMP-mediated signalling pathways. In conclusion, the effects of ibuprofen on primary OA chondrocyte transcriptome appear to be neutral in normal conditions, but ibuprofen may shift chondrocyte transcriptome towards anti-inflammatory phenotype in inflammatory environments.

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines.

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1ß, IL-4 and IL-17 are associated with different phenotypes of T helper (TH) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to TH cells and macrophages. We studied the effects of IFNγ, IL-1ß, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1ß [in the C(IL-1ß) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1ß) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1ß) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFß signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to TH cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.

Is the Preoperative Use of Antidepressants and Benzodiazepines Associated with Opioid and Other Analgesic Use After Hip and Knee Arthroplasty?

BACKGROUND: Mental health disorders can occur in patients with pain conditions, and there have been reports of an increased risk of persistent pain after THA and TKA among patients who have psychological distress. Persistent pain may result in the prolonged consumption of opioids and other analgesics, which may expose patients to adverse drug events and narcotic habituation or addiction. However, the degree to which preoperative use of antidepressants or benzodiazepines is associated with prolonged analgesic use after surgery is not well quantified. QUESTION/PURPOSES: (1) Is the preoperative use of antidepressants or benzodiazepine medications associated with a greater postoperative use of opioids, NSAIDs, or acetaminophen? (2) Is the proportion of patients still using opioid analgesics 1 year after arthroplasty higher among patients who were taking antidepressants or benzodiazepine medications before surgery, after controlling for relevant confounding variables? (3) Does analgesic drug use decrease after surgery in patients with a history of antidepressant or benzodiazepine use? (4) Does the proportion of patients using antidepressants or benzodiazepines change after joint arthroplasty compared with before? METHODS: Of the 10,138 patients who underwent hip arthroplasty and the 9930 patients who underwent knee arthroplasty at Coxa Hospital for Joint Replacement, Tampere, Finland, between 2002 and 2013, those who had primary joint arthroplasty for primary osteoarthritis (64% [6502 of 10,138] of patients with hip surgery and 82% [8099 of 9930] who had knee surgery) were considered potentially eligible. After exclusion of another 8% (845 of 10,138) and 13% (1308 of 9930) of patients because they had revision or another joint arthroplasty within 2 years of the index surgery, 56% (5657 of 10,138) of patients with hip arthroplasty and 68% (6791 of 9930) of patients with knee arthroplasty were included in this retrospective registry study. Patients who filled prescriptions for antidepressants or benzodiazepines were identified from a nationwide drug prescription register, and information on the filled prescriptions for opioids (mild and strong), NSAIDs, and acetaminophen were extracted from the same database. For the analyses, subgroups were created according to the status of benzodiazepine and antidepressant use during the 6 months before surgery. First, the proportions of patients who used opioids and any analgesics (that is, opioids, NSAIDs, or acetaminophen) were calculated. Then, multivariable logistic regression adjusted with age, gender, joint, Charlson Comorbidity Index, BMI, laterality (unilateral/same-day bilateral), and preoperative analgesic use was performed to calculate odds ratios for any analgesic use and opioid use 1 year postoperatively. Additionally, the proportion of patients who used antidepressants and benzodiazepines was calculated for 2 years before and 2 years after surgery. RESULTS: At 1 year postoperatively, patients with a history of antidepressant or benzodiazepine use were more likely to fill prescriptions for any analgesics than were patients without a history of antidepressant or benzodiazepine use (adjusted odds ratios 1.9 [95% confidence interval 1.6 to 2.2]; p < 0.001 and 1.8 [95% CI 1.6 to 2.0]; p < 0.001, respectively). Similarly, patients with a history of antidepressant or benzodiazepine use were more likely to fill prescriptions for opioids than patients without a history of antidepressant or benzodiazepine use (adjusted ORs 2.1 [95% CI 1.7 to 2.7]; p < 0.001 and 2.0 [95% CI 1.6 to 2.4]; p < 0.001, respectively). Nevertheless, the proportion of patients who filled any analgesic prescription was smaller 1 year after surgery than preoperatively in patients with a history of antidepressant (42% [439 of 1038] versus 55% [568 of 1038]; p < 0.001) and/or benzodiazepine use (40% [801 of 2008] versus 55% [1098 of 2008]; p < 0.001). The proportion of patients who used antidepressants and/or benzodiazepines was essentially stable during the observation period. CONCLUSION: Surgeons should be aware of the increased risk of prolonged opioid and other analgesic use after surgery among patients who were on preoperative antidepressant and/or benzodiazepine therapy, and they should have candid discussions with patients referred for elective joint arthroplasty about this possibility. Further studies are needed to identify the most effective methods to reduce prolonged postoperative opioid use among these patients. LEVEL OF EVIDENCE: Level III, therapeutic study.

Transient Receptor Potential Ankyrin 1 (TRPA1) Is Involved in Upregulating Interleukin-6 Expression in Osteoarthritic Chondrocyte Models.

Transient receptor potential ankyrin 1 (TRPA1) is a membrane-bound ion channel found in neurons, where it mediates nociception and neurogenic inflammation. Recently, we have discovered that TRPA1 is also expressed in human osteoarthritic (OA) chondrocytes and downregulated by the anti-inflammatory drugs aurothiomalate and dexamethasone. We have also shown TRPA1 to mediate inflammation, pain, and cartilage degeneration in experimental osteoarthritis. In this study, we investigated the role of TRPA1 in joint inflammation, focusing on the pro-inflammatory cytokine interleukin-6 (IL-6). We utilized cartilage/chondrocytes from wild-type (WT) and TRPA1 knockout (KO) mice, along with primary chondrocytes from OA patients. The results show that TRPA1 regulates the synthesis of the OA-driving inflammatory cytokine IL-6 in chondrocytes. IL-6 was highly expressed in WT chondrocytes, and its expression, along with the expression of IL-6 family cytokines leukemia inhibitory factor (LIF) and IL-11, were significantly downregulated by TRPA1 deficiency. Furthermore, treatment with the TRPA1 antagonist significantly downregulated the expression of IL-6 in chondrocytes from WT mice and OA patients. The results suggest that TRPA1 is involved in the upregulation of IL-6 production in chondrocytes. These findings together with previous results on the expression and functions of TRPA1 in cellular and animal models point to the role of TRPA1 as a potential mediator and novel drug target in osteoarthritis.

Widespread regulation of gene expression by glucocorticoids in chondrocytes from patients with osteoarthritis as determined by RNA-Seq.

BACKGROUND: Intra-articular glucocorticoid (GC) injections are widely used as a symptomatic treatment for osteoarthritis (OA). However, there are also concerns about their potentially harmful effects, and their detailed effects on chondrocyte phenotype remain poorly understood. METHODS: We studied the effects of dexamethasone on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were isolated from the cartilage from OA patients undergoing knee replacement surgery and cultured with or without dexamethasone for 24 h. Total RNA was isolated and sequenced, and functional analysis was performed against the Gene Ontology (GO) database. Results for selected genes were confirmed with RT-PCR. We also investigated genes linked to OA in recent genome-wide expression analysis (GWEA) studies. RESULTS: Dexamethasone increased the expression of 480 and reduced that of 755 genes with a fold change (FC) 2.0 or greater. Several genes associated with inflammation and cartilage anabolism/catabolism as well as lipid and carbohydrate metabolism were among the most strongly affected genes. In the GO analysis, genes involved in the extracellular matrix organization, cell proliferation and adhesion, inflammation, and collagen synthesis were enriched among the significantly affected genes. In network analysis, NGF, PI3KR1, and VCAM1 were identified as central genes among those most strongly affected by dexamethasone. CONCLUSIONS: This is the first study investigating the genome-wide effects of GCs on the gene expression in OA chondrocytes. In addition to clear anti-inflammatory and anticatabolic effects, GCs affect lipid and glucose metabolism in chondrocytes, an observation that might be particularly important in the metabolic phenotype of OA.

Regulation of gene expression by MF63, a selective inhibitor of microsomal PGE synthase 1 (mPGES1) in human osteoarthritic chondrocytes.

BACKGROUND AND PURPOSE: mPGES1 catalyses the production of PGE2 , the most abundant prostanoid related to inflammation and pain in arthritis. mPGES1 is suggested to be a safer and more selective drug target in inflammatory conditions compared to the COX enzymes inhibited by NSAIDs. In the present study, we investigated the effects of the selective mPGES1 inhibitor MF63 on gene expression in primary human chondrocytes from patients with osteoarthritis (OA). EXPERIMENTAL APPROACH: Chondrocytes were isolated from articular cartilage obtained from osteoarthritis patients undergoing knee replacement surgery. The effects of MF63 were studied in the primary chondrocytes with RNA-sequencing based genome-wide expression analysis. The main results were confirmed with qRT-PCR and compared with the effects of the NSAID ibuprofen. Functional analysis was performed with the GO database and interactions between the genes were studied with STRING. KEY RESULTS: MF63 enhanced the expression of multiple metallothionein 1 (MT1) isoforms as well as endogenous antagonists of IL-1 and IL-36. The expression of IL-6, by contrast, was down-regulated. These genes were also essential in functional and interaction network analyses. The effects of MF63 were consistent in qRT-PCR analysis, whereas the effects of ibuprofen overlapped only partly with MF63. There were no evident findings of catabolic effects by MF63. CONCLUSION AND IMPLICATIONS: Metallothionein 1 has been suggested to have anti-inflammatory and protective effects in cartilage. Up-regulation of the antagonists of IL-1 superfamily and down-regulation of the pro-inflammatory cytokine IL-6 also support novel anti-inflammatory and possibly disease-modifying effects of mPGES1 inhibitors in arthritis.

Predictors of the use of analgesic drugs 1 year after joint replacement: a single-center analysis of 13,000 hip and knee replacements.

BACKGROUND: Pain persists in a moderate number of patients following hip or knee replacement surgery. Persistent pain may subsequently lead to the prolonged consumption of analgesics after surgery and expose patients to the adverse drug events of opioids and NSAIDs, especially in older patients and patients with comorbidities. This study aimed to identify risk factors for the increased use of opioids and other analgesics 1 year after surgery and focused on comorbidities and surgery-related factors. METHODS: All patients who underwent a primary hip or knee replacement for osteoarthritis from 2002 to 2013 were identified. Redeemed prescriptions for acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), and opioids (mild and strong) were collected from a nationwide Drug Prescription Register. The user rates of analgesics and the adjusted risks ratios for analgesic use 1 year after joint replacement were calculated. RESULTS: Of the 6238 hip replacement and 7501 knee replacement recipients, 3591 (26.1%) were still using analgesics 1 year after surgery. Significant predictors of overall analgesic use (acetaminophen, NSAID, or opioid) were (risk ratio (95% CI)) age 65-74.9 years (reference < 65), 1.1 (1.03-1.2); age > 75 years, 1.2 (1.1-1.3); female gender, 1.2 (1.1-1.3); BMI 30-34.9 kg/m2 (reference < 25 kg/m2), 1.1 (1.04-1.2); BMI > 35 kg/m2, 1.4 (1.3-1.6); and a higher number of comorbidities (according to the modified Charlson Comorbidity Index score), 1.2 (1.1-1.4). Diabetes and other comorbidities were not significant independent predictors. Of the other clinical factors, the preoperative use of analgesics, 2.6 (2.5-2.8), and knee surgery, 1.2 (1.1-1.3), predicted the use of analgesics, whereas simultaneous bilateral knee replacement (compared to unilateral procedure) was a protective factor, 0.86 (0.77-0.96). Opioid use was associated with obesity, higher CCI score, epilepsy, knee vs hip surgery, unilateral vs bilateral knee operation, total vs unicompartmental knee replacement, and the preoperative use of analgesics/opioids. CONCLUSIONS: Obesity (especially BMI > 35 kg/m2) and the preoperative use of analgesics were the strongest predictors of an increased postoperative use of analgesics. It is remarkable that also older age and higher number of comorbidities predicted analgesic use despite these patients being the most vulnerable to adverse drug events.

Novel adipokine associated with OA: retinol binding protein 4 (RBP4) is produced by cartilage and is correlated with MMPs in osteoarthritis patients.

OBJECTIVE: Retinol binding protein 4 (RBP4) is a member of the lipocalin family and a vitamin A carrier in the blood. More recently, RBP4 has been described as an adipokine that is involved in insulin resistance and metabolic syndrome (MetS). As obesity, MetS and some adipokines contribute to the pathogenesis of osteoarthritis (OA), we investigated RBP4 in patients with OA. MATERIALS AND METHODS: Cartilage, synovial fluid and blood samples were collected from 100 OA patients undergoing total knee replacement surgery. Primary chondrocytes and cartilage tissue were cultured to measure the RBP4 expression. The concentrations of RBP4, other adipokines (adipsin, adiponectin, leptin and resistin) and biomarkers of OA (COMP, MMP-1, MMP-3 and YKL-40) were measured by immunoassay, and gene expression was measured by next-generation RNA sequencing. RESULTS: The OA cartilage samples released RBP4 into the culture medium, and the levels correlated positively with the expression of the adipokines adipsin, adiponectin, leptin and resistin. RBP4 was the most prominently expressed of these adipokines in the OA chondrocytes, and the expression of the RBP4 receptors STRA6 (stimulated by retinoic acid gene homologue 6) and TLR4 (Toll-like receptor 4) was also detected. Within the cartilage culture medium, RBP4 showed a positive correlation with MMP-1, MMP-3 and YKL-40. RBP4 was also present in the synovial fluid from the OA patients and correlated positively with the concentrations of RBP4 found in the plasma and the cartilage culture medium. Plasma RBP4 concentrations also showed a positive correlation with MMP-3 and adipsin. CONCLUSIONS: We show here, for the first time, that RBP4 is produced within OA joints and that it is associated with increased levels of adipokines and MMPs. The results suggest a role for RBP4 in the pathogenesis of OA and as a possible target for the disease-modifying drugs for the treatment of OA.

Use of prescription analgesic drugs before and after hip or knee replacement in patients with osteoarthritis.

BACKGROUND: Analgesic drugs are recommended to treat pain caused by osteoarthritis, and joint replacement should decrease the need for them. We aimed to determine the user rates of analgesic drugs before and after joint replacement. METHODS: All patients who underwent a primary hip or knee replacement for osteoarthritis from 2002 to 2013 in a region of 0.5 million people were identified. Patients with revision or other joint replacements during the study period (operation date +/- two years) were excluded, leaving 6238 hip replacements (5657 patients) and 7501 knee replacements (6791 patients) for analyses. Medication data were collected from a nationwide Drug Prescription Register and the prevalence (with its 95% confidence intervals) of acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), mild opioids, strong opioids, and medications used for neuropathic pain was calculated in three-month periods two years before and after surgery. RESULTS: Between two years and three months preoperatively, the proportion of patients who redeemed at least one type of analgesic drug increased from 28% (95% CI, 27-30%) to 48% (47-50%) on hip replacement patients and from 33% (32-34%) to 41% (40-42%) on knee replacement patients. Postoperatively, the proportions decreased to 23% (22-24%) on hip and to 30% (29-31%) on knee patients. Hip replacement patients used more NSAIDs (34% (32-35%) hip vs 26% (25-27%) knee, p < 0.001), acetaminophen (14% (13-15%) vs 12% (11-13%), p < 0.001), and mild opioids (14% (13-15%) vs 9% (8-9%), p < 0.001) than knee patients preoperatively, but postoperatively hip patients used less NSAIDs (12% (11-13%) vs 16% (15-16%), p < 0.001), acetaminophen (9% (8-10%) vs 11% (11-12%), p < 0.001), and mild opioids (5% (5-6%) vs 8% (7-8%), p < 0.001). CONCLUSION: Use of analgesic drugs increases prior to joint replacement, and is reduced following surgery. However, a considerable proportion of patients continue to use analgesics in two-year follow-up.

Downregulation of microsomal prostaglandin E synthase-1 (mPGES-1) expression in chondrocytes is regulated by MAP kinase phosphatase-1 (MKP-1).

OBJECTIVES: Microsomal prostaglandin E synthase-1 (mPGES-1) catalyses the formation of PGE2 in inflammatory tissues. It is considered a potential drug target in inflammatory conditions to achieve clinical benefits comparable to NSAIDs with a better tolerability. Inhibitors of mPGES-1 are under development but the pharmacological regulation of mPGES-1 expression remains poorly known. MAP kinase phosphatase-1 (MKP-1) is an enzyme that limits the activity of pro-inflammatory MAP kinases p38 and JNK. In the present study, we discovered that dexamethasone down-regulates mPGES-1 expression in articular chondrocytes in an MKP-1 and p38 kinase dependent manner. METHODS: Primary human chondrocytes were isolated from cartilage samples obtained from osteoarthritis (OA) patients undergoing knee replacement surgery. Primary mouse chondrocytes were isolated from cartilage samples of MKP-1 deficient (knock-out, KO) and corresponding wild type (WT) mice. Expression of mPGES-1 and MKP-1 were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot, and MAP kinase phosphorylation by Western blot. RESULTS: Dexamethasone inhibited the expression of mPGES-1 in primary human chondrocytes and in chondrocytes from wild type but not from MKP-1 deficient mice. Dexamethasone enhanced MKP-1 expression in chondrocytes from wild type mice as well as in primary human OA chondrocytes. Dexamethasone induced the dephosphorylation of both p38 and JNK, whereas mPGES-1 expression was downregulated by selective inhibitors of p38 only. CONCLUSIONS: The results show that MKP-1 is a crucial mediator of pharmacological control of inflammatory mPGES-1 expression by glucocorticoids, and underline MKP-1 as a potential anti-inflammatory drug target.

Gene expression in adverse reaction to metal debris around metal-on-metal arthroplasty: An RNA-Seq-based study.

Joint replacement surgery is a standard treatment of advanced osteoarthritis (OA). Since 2000, cobalt-chromium (CoCr) metal-on-metal (MoM) implants were widely used in hip arthroplasties. Some patients developed "adverse reaction to metal debris" (ARMD) around the prosthesis, resulting in a need for revision surgery. In the present study, we addressed the pathogenesis of ARMD by genome-wide expression analysis. Pseudosynovial ARMD tissue was obtained from revision surgery of Articular Surface Replacement (ASR, DePuy, Warsaw, IN, USA) hip arthroplasties. Control tissue was 1) OA synovium from primary hip arthroplasties and 2) inflammatory pseudosynovial tissue from metal-on-plastic (MoP) implant revisions. In ARMD tissue, the expression of 1446 genes was significantly increased and that of 1881 decreased as compared to OA synovium. Genes associated with immune response, tissue development and certain leukocyte signaling pathways were enriched in the differently (FC > 2) expressed genes. The network analysis proposed PRKACB, CD2, CD52 and CD53 as the central regulators of the greatest (FC > 10) differences. When ARMD tissue was compared to MoP tissue, the expression of 16 genes was significantly higher and that of 21 lower. Many of these genes were associated with redox homeostasis, metal ion binding and transport, macrophage activation and apoptosis. Interestingly, genes central to myofibroblast (AEBP1 and DES) and osteoclast (CCL21, TREM2 and CKB) development were upregulated in the MoP tissue. In network analysis, IL8, NQO1, GSTT1 and HMOX1 were identified as potential central regulators of the changes. In conclusion, excessive amounts of CoCr debris produced by MoM hip implants induces in a group of patients a unique adverse reaction characterized with enhanced expression of genes associated with inflammation, redox homeostasis, metal ion binding and transport, macrophage activation and apoptosis.

IL-6 in Osteoarthritis: Effects of Pine Stilbenoids.

Interleukin-6 (IL-6) is involved in the pathogenesis of various inflammatory diseases, like rheumatoid arthritis (RA). In the present study, we investigated the role of IL-6 in osteoarthritis (OA) patients and the effects of the stilbenoids monomethyl pinosylvin and pinosylvin on the expression of the cartilage matrix components aggrecan and collagen II and the inflammatory cytokine IL-6 in human OA chondrocytes. Synovial fluid and plasma samples were obtained from 100 patients with severe OA [BMI 29.7 (8.3) kg/m², age 72 (14) years, median (IQR); 62/38 females/males] undergoing total knee replacement surgery. IL-6 and matrix metalloproteinase (MMP) concentrations in synovial fluid and plasma were measured by immunoassay. The effects of pinosylvin on the expression of IL-6, aggrecan, and collagen II were studied in primary cultures of human OA chondrocytes. IL-6 levels in synovial fluid from OA patients [119.8 (193.5) pg/mL, median (IQR)] were significantly increased as compared to the plasma levels [3.1 (2.7) pg/mL, median (IQR)] and IL-6 levels in synovial fluid were associated with MMPs and radiographic disease severity. Natural stilbenoids monomethyl pinosylvin and pinosylvin increased aggrecan expression and suppressed IL-6 production in OA chondrocytes. The results propose that IL-6 is produced within OA joints and has an important role in the pathogenesis of OA. Stilbenoid compounds monomethyl pinosylvin and pinosylvin appeared to have disease-modifying potential in OA chondrocytes.

Factors Associated With Trunnionosis in the Metal-on-Metal Pinnacle Hip.

BACKGROUND: Trunnionosis of the tapered head-stem junction of total hip arthroplasties, either through corrosion or mechanical wear, has been implicated in early implant failure. Retrieval analysis of large numbers of failed implants can help us better understand the factors that influence damage at this interface. METHODS: In this study, we examined 120 retrieved total hip arthroplasties of one bearing design, the 36-mm diameter metal-on-metal, DePuy Pinnacle, that had been paired with 3 different stems. We measured material loss of the bearing and head-trunnion taper surfaces and collected clinical and component data for each case. We then used multiple linear regression analysis to determine which factors influenced the rate of taper material loss. RESULTS: We found 4 significant variables: (1) longer time to revision (P = .004), (2) the use of a 12/14 taper for the head-trunnion junction (P < .001), (3) decreased bearing surface wear (P = .003), and (4) vertical femoral offset (P = .05). These together explained 29% of the variability in taper material loss. CONCLUSION: Our most important finding is the effect of trunnion design. Of the 3 types studied, we found that S-ROM design was the most successful at minimizing trunnionosis.

Catabolic and proinflammatory effects of leptin in chondrocytes are regulated by suppressor of cytokine signaling-3.

BACKGROUND: Previous studies provide evidence that adipokine leptin increases production of catabolic and proinflammatory factors in chondrocytes and serves as a link between obesity and osteoarthritis (OA). However, the magnitude of the response to leptin treatment varies greatly between chondrocytes from different donor patients. In the present study, we investigated the regulatory role of suppressor of cytokine signaling-3 (SOCS-3) in the leptin-induced responses in OA cartilage. METHODS: Cartilage and synovial fluid samples from 97 patients with OA undergoing knee replacement surgery were collected. Cartilage samples were cultured with leptin (10 µg/ml), and the levels of proinflammatory and catabolic factors in synovial fluid and in the cartilage culture media, and SOCS-3 expression in the cartilage were measured. The role of SOCS-3 in leptin signaling was further studied in H4 murine chondrocytes by downregulating SOCS-3 with siRNA. RESULTS: Leptin-induced expression of matrix metalloproteinases MMP-1, MMP-3, MMP-13, interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were higher in the cartilage samples with low SOCS-3 expression. Accordingly, downregulation of SOCS-3 by siRNA in H4 chondrocytes led to enhanced leptin-induced expression of MMP-3, MMP-13, IL-6 and iNOS. Synovial fluid leptin was associated positively, and cartilage SOCS-3 negatively with synovial fluid levels of MMPs in a multivariate model in obese (body mass index (BMI) >30 kg/m2) but not in non-obese (BMI <30 kg/m2) patients. CONCLUSIONS: Our results show, for the first time, that SOCS-3 regulates leptin-induced responses in cartilage, and could thus be a future drug target in the treatment or prevention of OA, especially in obese patients.

Transient receptor potential ankyrin 1 (TRPA1) is functionally expressed in primary human osteoarthritic chondrocytes.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) is a membrane-associated cation channel, widely expressed in neuronal cells and involved in nociception and neurogenic inflammation. We showed recently that TRPA1 mediates cartilage degradation and joint pain in the MIA-model of osteoarthritis (OA) suggesting a hitherto unknown role for TRPA1 in OA. Therefore, we aimed to investigate whether TRPA1 is expressed and functional in human OA chondrocytes. METHODS: Expression of TRPA1 in primary human OA chondrocytes was assessed by qRT-PCR and Western blot. The functionality of the TRPA1 channel was assessed by Ca(2+)-influx measurements. Production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 subsequent to TRPA1 activation was measured by immunoassay. RESULTS: We show here for the first time that TRPA1 is expressed in primary human OA chondrocytes and its expression is increased following stimulation with inflammatory factors IL-1ß, IL-17, LPS, and resistin. Further, the TRPA1 channel was found to be functional, as stimulation with the TRPA1 agonist AITC caused an increase in Ca(2+) influx, which was attenuated by the TRPA1 antagonist HC-030031. Genetic depletion and pharmacological inhibition of TRPA1 downregulated the production of MMP-1, MMP-3, MMP-13, IL-6, and PGE2 in osteoarthritic chondrocytes and murine cartilage, respectively. CONCLUSIONS: The TRPA1 cation channel was found to be functionally expressed in primary human OA chondrocytes, which is an original finding. The presence and inflammatory and catabolic effects of TRPA1 in human OA chondrocytes propose a highly intriguing role for TRPA1 as a pathogenic factor and drug target in OA.

The Inflammatory Phenotype in Failed Metal-On-Metal Hip Arthroplasty Correlates with Blood Metal Concentrations.

INTRODUCTION: Hip arthroplasty is the standard treatment of a painful hip destruction. The use of modern metal-on-metal (MOM) bearing surfaces gained popularity in total hip arthroplasties during the last decade. Recently, worrisome failures due to adverse reaction to metal debris (ARMD), including pseudotumor response, have been widely reported. However, the pathogenesis of this reaction remains poorly understood. The aim of the present study was to investigate the ARMD response by flow cytometry approach. METHODS: Sixteen patients with a failed Articular Surface Replacement (ASR) hip prosthesis were included in the study. Samples of pseudotumor tissues collected during revision surgery were degraded by enzyme digestion and cells were typed by flow cytometry. Whole blood chromium and cobalt concentrations were analyzed with mass spectrometry before revision surgery. RESULTS: Flow cytometry analysis showed that the peri-implant pseudotumor tissue expressed two principal phenotypes, namely macrophage-dominated and T-lymphocyte-dominated response; the average portions being 54% (macrophages) and 25% (T-lymphocytes) in macrophage-dominated inflammation and 20% (macrophages) and 54% (T-lymphocytes) in T-lymphocyte-dominated response. The percentages of B-lymphocytes and granulocytes were lower in both phenotypes. Interestingly, the levels of blood chromium and cobalt were significantly higher in patients with macrophage-dominated response. CONCLUSIONS: The results suggest that the adverse tissue reactions induced by MOM wear particles contain heterogeneous pathogeneses and that the metal levels are an important factor in the determination of the inflammatory phenotype. The present results support the hypothesis that higher metal levels cause cytotoxicity and tissue injury and macrophages are recruited to clear the necrotic debris. On the other hand, the adverse response developed in association with lower metal levels is T-lymphocyte-dominated and is likely to reflect hypersensitivity reaction.

Higher Blood Cobalt and Chromium Levels in Patients With Unilateral Metal-on-Metal Total Hip Arthroplasties Compared to Hip Resurfacings.

BACKGROUND: Adverse soft tissue reactions in metal-on-metal (MoM) hip replacements are associated with cobalt (Co) and chromium (Cr) ions in blood. We report the prevalence and risk factors for elevated blood Co and Cr levels in patients with a unilateral MoM hip. METHODS: From a single institution, blood Co and Cr levels were analyzed in 1748 patients (692 hip resurfacings and 1056 total hip arthroplasties [THAs]). Concentrations exceeding 7 ppb were considered elevated, and the risk factors for elevated levels were calculated with binary logistic regression. RESULTS: Elevated blood metal ion levels were more common in MoM THA than in resurfacing patients (17.4% vs 5.9%, P < .001), and in 5 of the 7 THA brands, more than 20% of patients had elevated metal ion concentrations, whereas the proportion was less than 10% in all hip resurfacings. In resurfacings, small femoral head (odds ratio [OR] 1.30 per millimeter decrease [CI, 1.12-1.49]), high acetabular inclination (OR 1.15 per degree increase [CI 1.09-1.22]), and young age (OR 1.05 per year decrease [1.02-1.10]) were independent risk factors for elevated ions. In the THA group, female gender (OR 2.04 [CI 1.35-3.06]), longer time between surgery and ion measurement (OR 1.19 per year increase [CI 1.05-1.34]), and large headsize (OR 1.07 per millimeter increase [CI 1.01-1.13]) were risk factors for elevated ions. CONCLUSION: Given the high percentage of elevated levels, the systematic surveillance of especially large diameter MoM THAs seems justified.