Normal Synovial Fluid Research Articles

TH-17 cells in rheumatoid arthritis

IntroductionThe aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-γ.MethodsPeripheral blood (PB) mononuclear cells from normal and RA donors and mononuclear cells from RA SF were examined either without stimulation or after pretreatment with IL-23 followed by stimulation with phorbol myristate acetate (PMA) plus ionomycin (P/I). The abundance of TH-17 cells in RA SF was determined by flow cytometry. IL-17 levels were quantified in synovial tissue from RA, OA and normal individuals by ELISA and IL-23 was identified in SFs by ELISA. RA SF and control in vitro differentiated macrophages were either untreated or treated with the toll-like receptor (TLR) 2 ligand peptidoglycan, and then IL-23, IL-27 and IFN-γ mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR).ResultsTreatment with P/I alone or combined with IL-23 significantly increased the number of TH-17 cells in normal, RA PB and RA SF. With or without P/I plus IL-23, the percentage of TH-17 cells was higher in RA SF compared with normal and RA PB. IL-17 levels were comparable in OA and normal synovial tissues, and these values were significantly increased in RA synovial tissue. Although IL-17 was readily detected in RA SFs, IL-23 was rarely identified in RA SF. However, IL-23 mRNA was significantly increased in RA SF macrophages compared with control macrophages, with or without TLR2 ligation. IL-27 mRNA was also significantly higher in RA SF compared with control macrophages, but there was no difference in IL-27 levels between RA and control macrophages after TLR2 ligation. IFN-γ mRNA was also detectable in RA SF macrophages but not control macrophages and the increase of IFN-γ mRNA following TLR2 ligation was greater in RA SF macrophages compared with control macrophages.ConclusionThese observations support a role for TH-17 cells in RA. Our observations do not strongly support a role for IL-23 in the generation of TH-17 cells in the RA joint, however, they suggest strategies that enhance IL-27 or IFN-γ might modulate the presence of TH-17 cells in RA.

Synovial fluid mesenchymal stem cells in health and early osteoarthritis: Detection and functional evaluation at the single‐cell level

Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit-fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million-fold. These cells were CD271-, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

Aspiration of normal or asymptomatic pathological joints for diagnosis and research: indications, technique and success rate

Although joint aspiration is a basic clinical skill, aspiration of normal joints, or asymptomatic clinically quiescent joints, is only rarely undertaken. There are two main indications for this procedure. Firstly,...

1H NMR investigation of normal and osteoarthritic synovial fluid in the horse

Proton magnetic resonance spectroscopy (1H NMR) has been successfully used in the study of many biological fluids. The data presented here report on the metabolic profiles of normal equine synovial fluids compared with osteoarthritic (OA) fluids. Twenty-five OA synovial fluid samples and eight normal ones were collected from the forelimb fetlock joint in 22 horses, aged between five and 24 years. 1H NMR spectroscopy was carried out with a Bruker Avance DRX 500 equiped with a cryo-magnet working at 11 Tesla, and 'Mestre-C 4.9.9.6' software was used to analyze the spectra. The study assessed the increase of lactate, alanine, acetate, N-acetylglucosamine, pyruvate, citrate, creatine/creatinine, glycerol, HDL choline, and a-glucose in OA synovial fluid. The variations observed in samples from horses with OA compared to those in the control group, and similar data found in other studies, confirm that this technique may be useful in the study of joint metabolism. Its practical application may be in the evaluation of the treatment of OA in athletic horses.

Intraarticular Dirofilaria immitis microfilariae in two dogs

Dirofilaria immitis microfilariae were found in the synovial fluid of two dogs. One dog had clinical and cytological evidence of polyarthritis at the time of presentation. The second dog presented with severe effusion in a single joint and was later diagnosed with synovial sarcoma of the affected joint. These patients were not protected with heartworm prophylaxis and lived in heartworm endemic areas. Though there is documentation of D. immitis microfilaria in the synovial fluid of several clinically normal research dogs with cytologically normal synovial fluid, to our knowledge these are the first documented cases of intraarticular microfilaria in a dog with cytologically confirmed polyarthritis. Based on these unique cases, D. immitis infection should be considered a differential diagnosis in patients with polyarthropathies. Interpretive caution must be used when intraarticular microfilaria are present, as concurrent etiologies may also be present.

Synovial fluid recruits human mesenchymal progenitors from subchondral spongious bone marrow

Microfracture is a frequently used reparative technique that induces a healing response in articular cartilage defects. Penetration of the subchondral bone leads to blood clot formation, allows multipotent mesenchymal cells to access the defect and, subsequently, leads to cartilaginous repair tissue. The aim of our study was to analyze the chemotactic recruitment of human subchondral spongious bone marrow-derived cells by synovial fluid (SF) from normal donors (ND), patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Subchondral spongious bone marrow-derived mesenchymal progenitors were isolated from bone cylinders after high tibial osteotomy and analyzed for the presence of stem cell-related cell surface antigens by flow cytometry. Recruitment of subchondral progenitors by normal SF and SF from donors with degenerated joint diseases was documented by using a modified Boyden chamber chemotaxis assay. The chemotaxis assay demonstrated that synovial fluid has the potential to recruit mesenchymal progenitors in vitro. SF from normal donors and patients with OA showed no difference in the potential to stimulate cell migration. SF obtained from RA donors showed significantly reduced cell recruitment compared to SF derived from OA patients (p = 0.0054) and normal donors (p < 0.0001). The chemotactic activity of SF obtained from normal donors and from patients with degenerative joint diseases suggests that SF may be actively involved in the migration of progenitors in cartilage defects after microfracture.

Tribo-corrosion properties of cobalt-based medical implant alloys in simulated biological environments

Tribological problems and corrosion degradation have been recognized as essential risks for total joint replacements, especially for all-metal arthroplasty. Few studies have focused on the interactions between tribology and corrosion (tribo-corrosion) for implant materials. This paper addresses the importance of understanding tribo-corrosion and the evaluation of such materials in simulated biological environments. Due to the complex effect of proteins on tribo-corrosion, which has been demonstrated in previous studies, this study focuses towards understanding the effects of amino acids as aspects of material degradation. Dulbecco's Modified Eagle's Medium (DMEM) is a cell culture solution. It contains comparable amount and types of amino acids to normal synovial fluid in human joints. NaCl (0.36%) solution was employed to isolate the biological species. Three materials were tested: high carbon (HC) CoCrMo (contains 0.19% carbon), low carbon (LC) CoCrMo (widely used materials for total joint replacement) and stainless steel UNS S31603 (316 L). Integrated electrochemical tests supported by measurement of friction and near surface chemical analysis were carried out to enable their tribo-corrosion behaviour to be fully characterized. As a general conclusion, amino acids were found to react with materials under tribological contacts and form complex organometallic/oxides which lubricate the metallic sample surface. Tribo-corrosion plays a very important role in material degradation in the studied environments. HC CoCrMo shows superior wear, corrosion and tribo-corrosion resistance—the material characteristics and their effect on the different tribo-corrosion processes are discussed.

The role of lubricin in the mechanical behavior of synovial fluid

Synovial fluid is a semidilute hyaluronate (HA) polymer solution, the rheology of which depends on HA-protein interactions, and lubricin is a HA-binding protein found in synovial fluid and at cartilage surfaces, where it contributes to boundary lubrication under load. Individuals with genetic deficiency of lubricin develop precocious joint failure. The role of lubricin in synovial fluid rheology is not known. We used a multiple-particle-tracking microrheology technique to study the molecular interactions between lubricin and HA in synovial fluid. Particles (200 nm mean diameter) embedded in normal and lubricin-deficient synovial fluid samples were tracked separately by using multiple-particle-tracking microrheology. The time-dependent ensemble-averaged mean-squared displacements of all of the particles were measured over a range of physiologically relevant frequencies. The mean-squared displacement correlation with time lag had slopes with values of unity for simple HA solutions and for synovial fluid from an individual who genetically lacked lubricin, in contrast to slopes with values less than unity (alpha approximately 0.6) for normal synovial fluid. These data correlated with bulk rheology studies of the same samples. We found that the subdiffusive and elastic behavior of synovial fluid, at physiological shear rates, was absent in fluid from a patient who lacks lubricin. We conclude that lubricin provides synovial fluid with an ability to dissipate strain energy induced by mammalian locomotion, which is a chondroprotective feature that is distinct from boundary lubrication.

ICOS and B7 costimulatory molecule expression identifies activated cellular subsets in rheumatoid arthritis

To better define important cell subsets expressing activation markers in rheumatoid arthritis (RA), we compared selective lymphocyte and monocyte B7H1, B7H2, B7RP.1, B7RP.2, and inducible costimulatory molecule (ICOS) expression from normal peripheral blood (NL PB), RA PB, and RA synovial fluid (SF) by multicolor flow cytometry and immunohistochemistry. RA SF memory lymphocytes expressed B7RP.1 and B7RP.2, suggesting that T-cells may function as antigen presenting cells (APCs) in RA joints. We found similar results for ICOS expression. RA SF CD14+ monocytes also expressed B7RP.1 (an ICOS ligand) and the homologous ligand B7RP.2, identifying monocytes as potential mediators of antigen processing and lymphocyte activation in RA. Furthermore, we found an increased population of RA SF CD14+ monocytes expressing B7H1 and B7H2. [The FACS analysis was supported by immunohistochemistry, showing intense lymphocyte and APC (macrophages with dendritic morphology) ICOS staining in RA synovial tissue (ST). Overall, these results define elevated populations of memoryT-lymphocytes expressing proinflammatory B7 molecules in RA SF that either stimulate T cells through ICOS (via ICOS ligands B7RP.1 and B7RP.2), or down-regulate RA ST T-lymphocytes through B7H1 and B7H2.] Therefore, in the same joint, there may exist positive and negative influences on the inflammatory response, and perhaps, the negative signals dominate as joint inflammation resolves.

Production of lipid peroxidation products in osteoarthritic tissues: New evidence linking 4‐hydroxynonenal to cartilage degradation

The lipid peroxidation product 4-hydroxynonenal (HNE) is prominently produced in osteoarthritic (OA) synovial cells, but its specific contribution to cartilage destruction is not understood. This study was designed to test whether HNE signaling and binding are involved in OA cartilage degradation through type II collagen (CII) and matrix metalloproteinase 13 (MMP-13) modulation. HNE levels in synovial fluid and in isolated OA chondrocytes treated with free radical donors were determined by enzyme-linked immunosorbent assay. The formation of the HNE/CII adducts was measured in cartilage explants by immunoprecipitation. Levels of CII and MMP-13 messenger RNA and protein were determined by reverse transcription-polymerase chain reaction, Western blotting, and by the use of commercial kits. Levels of HNE/protein adducts were higher in OA synovial fluid compared with normal synovial fluid and were higher in OA chondrocytes treated with free radical donors compared with untreated cells. In cartilage explants, HNE induced CII cleavage, as established by the generation of neoepitopes. The level of HNE/CII adducts was increased in OA cartilage explants incubated with free radical donors. Modification of CII by HNE accelerated its degradation by active MMP-13. In isolated OA chondrocytes, HNE inhibited the expression of CII and tissue inhibitor of metalloproteinases 1 and induced MMP-13 mainly through activation of p38 MAPK. In vitro, HNE binding to MMP-13 activated this enzyme at a molar ratio of 1:100 (MMP-13 to HNE). The increased level of HNE in OA cartilage and the ability of HNE to induce transcriptional and posttranslational modifications of CII and MMP-13 suggest that this aldehyde could play a role in OA.

Measurement of S-nitrosothiols in extracellular fluids from healthy human volunteers and rheumatoid arthritis patients, using electron paramagnetic resonance spectrometry

In human tissues, S-nitrosothiols (RSNOs) are generated by the nitric oxide (NO )-dependent S-nitrosation of thiol-containing species. Here, a novel electron paramagnetic resonance spectrometry assay for RSNOs is described, together with its application to studies of human health and disease. The assay involves degrading RSNOs using N-methyl- d-glucamine dithiocarbamate (MGD) at high pH and spin trapping the NO released using (MGD) 2–Fe 2+. Because dietary nitrate might contribute to tissue RSNOs, the assay was used to monitor the effect of Na 15NO 3 ingestion on plasma and gastric juice RSNOs in healthy human volunteers. Na 15NO 3 ingestion (2 mmol) increased gastric RS 15NO concentrations ( p < 0.01), but there was no significant effect on plasma RS 15NO concentrations. Having established that dietary nitrate was not a confounding factor, we applied the RSNO assay to matched plasma and knee-joint synovial fluid (SF) from rheumatoid arthritis (RA) patients, with healthy subjects as controls. Clinical markers of RA inflammatory disease activity were quantified, as were plasma and SF NO 2 − and NO 3 −. Median RSNO concentrations were 0 (interquartile range 68) nM, 109 (282) nM, and 309 (470) nM in normal plasma, RA plasma, and SF, respectively. The median RSNO concentration was significantly elevated in RA SF compared with RA plasma ( p < 0.05) and in RA plasma compared with normal plasma ( p < 0.05). SF RSNO concentrations correlated positively with SF neutrophil counts ( r s = 0.55, p < 0.05) and inversely with blood hemoglobin concentrations ( r s = −0.52, p < 0.05), but not with NO 2 − or NO 3 −. Thus the raised levels of RSNOs in RA SF correlate with some established markers of inflammation, suggesting the described RSNO assay may have applications in rapid clinical monitoring of NO metabolism in human inflammatory conditions.

TGF‐α and ErbB2 production in synovial joint tissue: increased expression in arthritic joints

Objective: Cell types present in synovial joint tissues and during synovitis are known to produce epidermal growth factor receptor (EGFR)/ErbB‐1/HER‐1 and the potent EGFR‐ligand transforming growth factor‐alpha (TGF‐α) in vitro. Concomitant expression of TGF‐α, EGFR, and ErbB2 gives a strong proliferative drive in vitro and in vivo. However, the presence of TGF‐α and members of the EGFR/EGFR‐ligand family has not been thoroughly investigated in joint tissue in vivo. We aimed to determine whether TGF‐α, EGFR, and ErbB2 are present in human synovial joints, especially during rheumatoid arthritis (RA).Methods: TGF‐α protein was immunodetected in knee synovial fluid (SF) collected from 23 RA patients, eight patients with other arthritic conditions, two osteoarthritis (OA) patients, and six post‐traumatic patients (control). TGF‐α mRNA and TGF‐α, ErbB2, EGFR, and CD68 immunoreactivity were detected in knee synovial biopsies (6 RA/2 OA/6 control) using in situ hybridization and immunohistochemistry. TGF‐α mRNA was determined in SF cells by reverse transcription polymerase chain reaction (RT‐PCR) and/or the Northern blot technique.Results: TGF‐α protein was found in the synovial membrane (SM) and in the majority of SF samples. TGF‐α levels were significantly higher (p<0.001) in SF of RA patients than controls, TGF‐α protein and mRNA were increased and more widespread in SM of RA patients. In addition, white blood cells collected from RA SF expressed TGF‐α mRNA. Immunoreactivity for ErbB2 was found in SM and was more widespread in RA patients than in controls.Conclusion: The presence of TGF‐α in normal SF and SM may indicate a physiological maintenance function. The increased expression of TGF‐α and ErbB2 in RA SF and SM may give rise to an abnormal growth pattern, contributing to inflammatory synovial hyperplasia.

In vivo chondrogenesis of adult bone-marrow-derived autologous mesenchymal stem cells

The purpose of this study has been to investigate the possible effects of the normal joint cavity environment on chondrocytic differentiation of bone-marrow-derived mesenchymal stem cells (MSCs). Autologous bone marrow was aspirated from the iliac crest of male sheep. MSCs were purified, expanded, and labeled with the fluorescent dye PKH26. Labeled MSCs were then grown on a three-dimensional porous scaffold of poly (L-lactic-co-glycolic acid) in vitro and implanted into the joint cavity by a surgical procedure. At 4 or 8 weeks after implantation, the implants were removed for histochemical and immunohistochemical analysis. The cells labeled with red fluorescent PKH26 in the implants expressed type II collagen and synthesized sulfated proteoglycans. However, the osteoblast-specific marker, osteocalcin, was not detected by immunohistochemistry indicating that the implanted MSCs had not differentiated into osteoblasts by being directly exposed to the normal joint cavity. To investigate the possible factors involved in chondrocytic differentiation of MSCs further, we co-cultured sheep MSCs with the main components of the normal joint cavity, viz., synovial fluid or synovial cells, in vitro. After 1 or 2 weeks of co-culture, the MSCs in both co-culture systems expressed markers of chondrogenesis. These results suggest that synovial fluid and synovium from normal joint cavity are important for the chondrocytic differentiation of adult bone-marrow-derived MSCs.

Analysis of cartilage oligomeric matrix protein (COMP) degradation and synthesis in equine joint disease

Cartilage oligomeric matrix protein (COMP) is abundant within cartilage; its turnover and/or degradation have been investigated in various equine joint diseases and it has been suggested that COMP fragmentation might be useful for monitoring such conditions. To determine whether COMP metabolism is compromised in equine osteoarthritis (OA) and whether COMP degradation is a useful joint marker representing cartilage destruction. A monoclonal antibody (mAb) with a higher affinity for degraded COMP allows discrimination of diseased joints by quantifying COMP levels and fragmentation. A mAb (clone14G4) was generated against equine cartilage COMP. The NH2-terminal sequence of enzyme-cut COMP fragments recognised by 14G4 was determined, as was the efficiency of binding to COMP (using a generated COMP peptide). COMP concentration and fragmentation were analysed in synovial fluid (SF) from normal horses and those with OA. The mAb 14G4 had a higher affinity for the smaller fragments of equine COMP, compared with a mAb (clone 12C4) generated against human COMP. The 14G4 epitope was identified as between C134 and F147. The COMP values in OA (mean +/- s.d. 205.8 +/- 90.9 microg/ml) were significantly higher than in the normal (133.1 +/- 31.5 microg/ml) SF. On the immunoblots of OA sample, the proportions of intact COMP were significantly lower, while smaller fragments ranging from 75 to 290 kDa were higher compared with the normal SF. The mAb 14G4 reliably detects COMP degradation as well as synthesis, and fragmentation analysis combined with quantification in SF could be useful to study equine OA.

Age-related changes and sex differences in chondroitin sulfate isomers and hyaluronic acid in normal synovial fluid

Age-related changes and sex differences in chondroitin sulfate isomers and hyaluronic acid in normal synovial fluid

Age-related changes and sex differences in chondroitin sulfate isomers and hyaluronic acid in normal synovial fluid

The effects of factors such as age and sex on the metabolism of chondroitin sulfate (CS) and hyaluronic acid (HA) in knee joint tissues are believed to be profoundly important in the onset of joint diseases including osteoarthritis. To test whether age and sex influence CS isomers and HA in normal synovial fluid, we determined concentrations of chondroitin 6-sulfate (C6S), chondroitin 4-sulfate (C4S), and HA in healthy subjects of different ages. Synovial fluids were obtained from 187 healthy volunteers, 14–89 years of age. Chondroitin 6-sulfate, C4S, HA concentrations, and C6S : C4S ratio showed a significant negative correlation with age. There were no sex-related differences in HA concentration, but the concentrations of C6S and C4S and the C6S : C4S ratio were significantly lower in women than in men in most age groups.

Detection of collagen type II and proteoglycans in the synovial fluids of patients diagnosed with non-infectious knee joint synovitis indicates early damage to the articular cartilage matrix

Objective: We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls. Methods: CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate. Results: The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS ( P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower ( P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher ( P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS. Conclusion: The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.

The diagnostic and therapeutic challenge of femoral head osteoid osteoma presenting as thigh pain: a case report

Schaefer MP, Smith J. The diagnostic and therapeutic challenge of femoral head osteoid osteoma presenting as thigh pain: a case report. Osteoid osteoma, an infrequent but important cause of musculoskeletal pain, is often difficult to diagnose. We present a case of a 31-year-old man who, for 2 years, had left groin pain radiating to the thigh. Symptoms began 1 month after a motorcycle crash in which he sustained only shin abrasions. Initial spine and hip radiographs were negative. Treatment with naproxen provided significant relief, but the symptoms gradually worsened over 6 months. An electromyogram and lumbar magnetic resonance imaging (MRI) of the left lower leg were unremarkable. Hip MRI revealed edema without fracture. Prophylactic femoral pinning for impending stress fracture provided no relief. Rheumatologic evaluation revealed normal serologies and synovial fluid. Cyclobenzaprine and sulfasalazine were started and provided mild relief. At presentation to our institution, he was in significant discomfort, but could ride a bicycle for exercise and was completing a home exercise program. He had antalgic gait and globally restricted hip motion with end-range pain. A neurologic examination showed no abnormalities. Hip and pelvis computed tomography scan revealed increased sclerosis of the femoral head, with a central lucency suggestive of osteoid osteoma. This was confirmed by biopsy. Radiofrequency ablation provided significant symptom relief.

Microarray analysis reveals the involvement of beta-2 microglobulin (B2M) in human osteoarthritis

Objective To assess whether beta-2 microglobulin (B2M) has effects on articular chondrocytes that would implicate B2M involvement in osteoarthritis (OA) pathogenesis.MethodsThe mRNA levels of B2M in fetal and osteoarthritic chondrocytes were detected by RT–PCR. B2M levels in synovial fluid and tissue cultured media from cartilage explants were tested using B2M ELISA kit. Primary cultured chondrocytes were used for proliferation and microarray experiments.Results The average B2M level in OA synovial fluid is significantly higher than that found in normal synovial fluid. However, there was no significant difference in B2M synovial fluid levels amongst differing OA stages. The release of B2M by osteoarthritic cartilage was detectable after 24h in culture and continued to increase during the 72h study period. B2M had an inhibitory effect on chondrocyte growth at 1.0μg/ml, and became significantly inhibitory at 10.0μg/ml. Genes regulated by B2M were detected through microarray technology. Twenty genes were found to be up-regulated by B2M, including collagen type III which is known to be up-regulated in OA. Eleven genes were found to be down-regulated at least two-fold by B2M.Conclusion These results indicate that B2M is highly expressed in OA cartilage and synovial fluid compared to normal, and suggest that B2M may have effects on chondrocyte function that could contribute to OA pathogenesis.Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

The effects of age and sex on chondroitin sulfates in normal synovial fluid

To examine how age and sex influence chondroitin sulfates (CS) in normal synovial fluid, we measured the concentrations of chondroitin 6-sulfate (C6S), chondroitin 4-sulfate (C4S), and hyaluronic acid (HA) in healthy subjects of different ages. Synovial fluid samples were obtained from 82 healthy volunteers, ages 20-79 years. The concentrations of CS and HA and the C6S:C4S ratio varied with age. Their values were highest between 20 and 30 years of age, and thereafter they showed a tendency to decrease. Statistically, the C6S concentration and the C6S:C4S ratio at ages 60-70 years were significantly lower than those at 20-30 years of age. There was also a clear between-sex difference, in which the CS concentrations and the C6S:C4S ratio in women were significantly lower than those in men (P = 0.0003 for C6S, P = 0.02 for C4S, P = 0.002 for C6S:C4S ratio). In sharp contrast, little between-sex difference was found in the HA concentration. In multiple regression analysis, age correlated strongly with the C6S concentration and the C6S:C4S ratio (r = -0.521 and r = -0.617, respectively), weakly with the C4S concentration (r = -0.202), and moderately with the HA concentration (r = -0.483). Sex showed a weak correlation with the concentrations of C6S and C4S and the C6S:C4S ratio (r = 0.307, r = 0.225, and r = 0.237, respectively), and little correlation was seen between sex and the HA concentration. The CS concentrations and the sulfation patterns in normal synovial fluid vary with age and sex, and these physiologic variations need to be taken into account when using synovial fluid CS as markers for arthritic conditions.