Distinct tribological endotypes of pathological human synovial fluid reveal characteristic biomarkers and variation in efficacy of viscosupplementation at reducing local strains in articular cartilage.

OBJECTIVE: Viscosupplementation has been used for decades to treat mild to moderate osteoarthritis, yet it is unknown if the lubricating function of different pathological synovial fluids (SF) vary, or if they respond differentially to viscosupplementation. The objectives of this study were to (i) evaluate the friction coefficients and induced shear strains in articular cartilage when lubricated with pathological SF, (ii) identify the effect of hyaluronic acid (HA) supplementation on friction coefficients and shear strains, and (iii) identify SF biomarkers that correlate with lubricating function. METHOD: Human pathological SF was grouped by white blood cell count (inflammatory: >2000 cells/mm3, n = 6; non-inflammatory: <2000 cells/mm3, n = 6). Compositional analyses for lubricin and cytokines were performed. Friction coefficients and local tissue shear strain measurements were coupled using new, microscale rheological analyses by lubricating neonatal bovine cartilage explants with SF alone and in a 1:1 ratio with HA (Hymovis®). RESULTS: Friction coefficients were not significantly different between the inflammatory and non-inflammatory pathologies (p = 0.09), and were poorly correlated with peak tissue strains at the cartilage articular surface (R2 = 0.34). A subset of inflammatory SF samples induced higher tissue strains, and HA supplementation was most effective at lowering friction and tissue strains in this inflammatory subset. Across all pathologies there were clear relationships between polymorphonuclear neutrophil (PMN), IL-8, and lubricin concentrations with cartilage tissue strains. CONCLUSION: These results suggest that pathological SF is characterized by distinct tribological endotypes where SF lubricating behaviors are differentially modified by viscosupplementation and are identifiable by biomarkers.

[Age-related traits of cytogenetic abnormalities in synovial cells of knee-joint in residents of the North of Siberia with arthritis of different etiology depending on the GSTM1 gene polymorphism of glutathion-S-transferase.]

It was studied the frequency of cells with cytogenetic abnormalities in the synovial fluid cells of the knee joint in patients of different age groups suffering from chronic arthritis associated with Lyme borreliosis (CAALB) or post-traumatic arthritis (PTA), depending on the polymorphism of the GSTM1 gene of glutathione-S-transferase. The study included 135 residents of the north of the Tomsk and Tyumen regions, 68 of whom suffered from CAALB, and the rest of the 67 patients who made up the control group were diagnosed with PTA. The results of this study have demonstrated that there are significant age-related differences in the frequency of cytogenetic abnormalities of the synovial fluid cells of the knee joint between young and elderly patients of СAALB. The integrative assessment of clinical and cytogenetic parameters in the group of elderly СAALB patients with mutant GSTM1 (0/0) allele, as compared with the other groups, enable to conclude that there are significant positive correlations between the indices of the severity disruption of articular locomotor function and the frequency of synovial fluid cells with trisomy of chromosome 7.

Anti-arthritic actions of ß-cryptoxanthin against the degradation of articular cartilage in vivo and in vitro.

An inverse correlation between the morbidity of rheumatoid arthritis and daily intake of ß-cryptoxanthin has been epidemiologically shown. In this study, we investigated the effects of ß-cryptoxanthin on the metabolism of cartilage extracellular matrix in vivo and in vitro. Oral administration of ß-cryptoxanthin (0.1-1 mg/kg) to antigen-induced arthritic rats suppressed the loss of glycosaminoglycans in articular cartilage, which is accompanied by the interference of aggrecanase-mediated degradation of aggrecan. Inhibition of the interleukin 1α (IL-1α)-induced aggrecan degradation by ß-cryptoxanthin was also observed with porcine articular cartilage explants in culture. ß-Cryptoxanthin (1-10 µM) dose-dependently down-regulated the IL-1α-induced gene expression of aggrecanase 1 (ADAMTS-4) and aggrecanase 2 (ADAMTS-5) in cultured human chondrocytes. Moreover, ß-cryptoxanthin was found to augment the gene expression of aggrecan core protein in chondrocytes. These results provide novel evidence that ß-cryptoxanthin exerts anti-arthritic actions and suggest that ß-cryptoxanthin may be useful in blocking the progression of rheumatoid arthritis and osteoarthritis.

Periploca forrestii Saponin Ameliorates Murine CFA-Induced Arthritis by Suppressing Cytokine Production.

Periploca forrestii Schltr. has been used as a Chinese folk medicine due to its versatile pharmacological effects such as promoting wounds and rheumatoid arthritis. However, the antiarthritic activity of Periploca forrestii saponin (PFS) and its active compound Periplocin has still not been demonstrated. Here, we evaluated the antiarthritic effects of PFS in adjuvant-induced arthritis (AIA) rats by intragastric administration at a dose of 50 mg/kg. The anti-inflammatory activities of Periplocin were also examined in LPS-induced AIA splenocytes and synoviocytes. PFS significantly ameliorated joint swelling; inhibited bone erosion in joints; lowered levels of IL-6 and TGF-ß1 in AIA rat splenocyte; and reduced joint protein expression levels of phospho-STAT3 and IKKα. Using LPS-induced AIA splenocytes, we demonstrate that Periplocin suppressed the key proinflammatory cytokines levels of IL-6, IFN-γ, TGF-ß1, and IL-13 and IL-22 and transcription factor levels of T-bet, GATA3, and C-Jun genes. Periplocin also suppressed LPS-induced cytokine secretion from synoviocytes. Our study highlights the antiarthritic activity of PFS and its derived Periplocin and the underlying mechanisms. These results provide a strong rationale for further testing and validation of the use of Periploca forrestii Schltr. as an alternative modality for the treatment of RA.

Mesenchymal Stem Cells Obtained from Synovial Fluid Mesenchymal Stem Cell-Derived Induced Pluripotent Stem Cells on a Matrigel Coating Exhibited Enhanced Proliferation and Differentiation Potential.

Induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) serve as a promising source for cell-based therapies in regenerative medicine. However, optimal methods for transforming iPSCs into MSCs and the characteristics of iPSC-MSCs obtained from different methods remain poorly understood. In this study, we developed a one-step method for obtaining iPSC-MSCs (CD146+STRO-1+ MSCs) from human synovial fluid MSC-derived induced iPSCs (SFMSC-iPSCs). CD146-STRO-1-SFMSCs were reprogrammed into iPSCs by transduction with lentivirus-mediated Sox2, Oct-3/4, klf4, and c-Myc. SFMSC-iPSCs were maintained with mTeSR1 medium in Matrigel-coated culture plates. Single dissociated cells were obtained by digesting the SFMSC-iPSCs with trypsin. The dissociated cells were then plated into Matrigel-coated culture plate with alpha minimum essential medium supplemented with 10% fetal bovine serum, 1× Glutamax, and the ROCK inhibitor Y-27632. Cells were then passaged in standard cell culture plates with alpha minimum essential medium supplemented with 10% fetal bovine serum and 1× Glutamax. After passaging in vitro, the cells showed a homogenous spindle-shape similar to their ancestor cells (SFMSCs), but with more robust proliferative activity. Flow cytometric analysis revealed typical MSC surface markers, including expression of CD73, CD90, CD105, and CD44 and lack of CD45, CD34, CD11b, CD19, and HLA-DR. However, these cells were positive for CD146 and stro-1, which the ancestor cells were not. Moreover, the cells could also be induced to differentiate in osteogenic, chondrogenic, and adipogenic lineages in vitro. The differentiation potential was improved compared with the ancestor cells in vitro. The cells were not found to exhibit oncogenicity in vivo. Therefore, the method presented herein facilitated the generation of STRO-1+CD146+ MSCs from SFMSC-iPSCs exhibiting enhanced proliferation and differentiation potential.

Analysis of lactate concentrations in canine synovial fluid.

OBJECTIVE: To report synovial fluid lactate concentrations in normal and pathological canine joints. STUDY DESIGN: Controlled, prospective study. METHODS: Lactate was measured in synovial fluid using a hand-held meter and the rest of the fluid was sent to a commercial laboratory for analysis. Samples were divided into four groups; group 1: control, group 2: osteoarthritis, group 3: immune-mediated inflammatory arthritis, and group 4: septic arthritis. Statistical analysis was performed to compare lactate concentrations between the four groups and to examine the predictive value of lactate in the diagnosis of septic arthritis. A correlation was sought between synovial fluid lactate and synovial fluid total nucleated cell count and total protein. RESULTS: Seventy-four samples were investigated from 55 dogs. Statistical analysis found that lactate concentrations were significantly higher in the septic arthritis group than in each of the other three groups. No significant correlation could be found between synovial fluid lactate concentrations and synovial fluid total nucleated cell count or synovial fluid total protein. Lactate concentration was found to be a useful predictor of septic arthritis, with a low concentration pointing towards exclusion rather than a high concentration to the diagnosis of septic arthritis. CLINICAL SIGNIFICANCE: Synovial fluid lactate concentration is not a good marker for osteoarthritis or immune-mediated inflammatory arthritis, but it is significantly increased in septic arthritis and could help the clinician in ruling out this condition in a quick and cost-effective way.

Synovial fluid and plasma n3 long chain polyunsaturated fatty acids in patients with inflammatory arthritis.

Relationships between n-3 long chain polyunsaturated fatty acids (LC-PUFA) in plasma and synovial fluid (SF) were examined in 36 patients with knee effusion within the context of a variety of rheumatic diagnoses and various stated fish oil (FO) intakes (from 0 to 30mL of standard FO daily) of variable duration. In a sub-group of patients, correlations between PUFA in SF mononuclear cells (MNC) and cell-free supernatants of SF and between SF MNC and peripheral blood (PB) MNC were examined. Correlations were also sought between clinical data (stated FO intake, pain score) and n-3 LC-PUFA. Correlations between plasma n-3 LC-PUFA and SF n-3 LC-PUFA were very strong (r(2)>0.9, p<0.001). The LC-PUFA profiles of SF supernatants differed from those of MNC. PUFA profiles in PB MNC and SF MNC were similar, except for a higher proportion of DHA in the latter. Positive correlations were observed between stated intakes of FO and EPA in plasma and SF (for both r=0.37, p=0.02) and DHA in plasma (r=0.37, p=0.02) and SF (r=0.36, p=0.03). n-3 LC-PUFA in plasma and SF correlated inversely with pain score (plasma r(2)=0.16, p<0.02; SF r(2) 0.32, p=0.001). In conclusion, plasma n-3 LC-PUFA is a strong indicator of SF n-3 LC-PUFA status across a broad range of rheumatic diagnoses and FO intakes. Higher n-3 LC-PUFA in plasma and SF were associated with lesser pain experience.

Systemic lentivirus-mediated delivery of short hairpin RNA targeting calcium release-activated calcium channel 3 as gene therapy for collagen-induced arthritis.

Immune cells, including T cells, B cells, and osteoclasts, in conjunction with their associated cytokines, have been studied as primary molecular therapeutic targets for the management of rheumatoid arthritis (RA) patients. The increase in cytosolic Ca(2+) levels through the activation of store-operated Ca(2+) release-activated channels (CRACs) is involved in mediating a disparate array of cellular responses by these immune cells. This study was undertaken to investigate the feasibility and efficiency of the regulation of Ca(2+) entry in the treatment of RA. To moderately suppress Ca(2+) entry via CRACs, we gene silenced CRACM3, which was induced by systemic application of specific short hairpin RNAs (shRNAs) using a lentiviral-delivery system, in a murine model of collagen-induced arthritis (CIA). The inflammatory responses were determined by measuring the levels of a panel of cytokines and chemokines in the joints and serum. Ag-specific responses were evaluated by determining the cytokine profile of T cells stimulated with autoantigen. We also analyzed the ability of specific CRACM3-shRNA to regulate mature osteoclast function in CIA mice. The therapeutic effect of lentiviral-delivered CRACM3-shRNA was associated with gene silencing of CRACM3, along with the successful biodistribution of the virus. Extracellular Ca(2+) influx in the splenocytes, thymocytes, and knee joint synovial cells was moderately suppressed. Inflammatory responses and autoimmune responses were reduced by CRACM3 gene silencing. A decrease in mature osteoclast activity also was observed in CRACM3-shRNA-treated CIA mice. These results indicate that regulation of Ca(2+) entry through lentivirus-mediated CRACM3 gene silencing is beneficial in the treatment of RA.

Salvia miltiorrhiza injection restores apoptosis of fibroblast-like synoviocytes cultured with serum from patients with rheumatoid arthritis.

Salvia miltiorrhiza injection (SMI) is a water­soluble agent, derived from Salvia miltiorrhiza (SM), that is traditionally used to treat cardiovascular and cerebrovascular diseases. Furthermore it has been demonstrated to possess the ability to induce apoptosis of tumor cells. However, it remains unclear whether SMI can induce apoptosis of rheumatoid arthritis (RA) fibroblast­like synoviocytes (FLS), which are hyperplastic in RA due to defective apoptosis. There is also evidence that allogenic serum may be associated with the induction of apoptosis. The aim of the present study was to investigate the involvement of serum during SMI­induced apoptosis in RA FLS. The results demonstrated that SMI could induce apoptosis of RA FLS, cultured with fetal bovine serum (FBS), in a dose­dependent manner. In addition, SMI decreased the expression of nuclear factor­κB in RA FLS nuclear extracts and inhibited the secretion of tumor necrosis factor­α. Fas ligand expression was not detected in RA FLS, in either the presence or absence of SMI. The pro­apoptotic genes B­cell lymphoma 2 (Bcl­2) associated X protein (Bax) and Fas, were shown to be upregulated following SMI stimulation, whereas the expression levels of the anti­apoptotic gene Bcl­2, were downregulated. Upon replacement of FBS with normal human serum, the apoptotic rate and Bax mRNA expression levels following SMI stimulation, were unchanged. However, culturing RA FLS with patient' serum (RPS), restored the apoptotic rate and Bax mRNA expression levels following SMI stimulation. There may be numerous mechanisms by which SMI inhibits RA FLS proliferation. The present study demonstrated that SMI can restore apoptosis of RA FLS cultured with RPS. These results indicate that SMI may have a potential role in the treatment of synovial hyperplasia of RA.

Total saponins from Discorea nipponica ameliorate urate excretion in hyperuricemic mice.

Rhizoma Dioscoreae Nipponicae, from Discorea nipponica, is a widely used traditional Chinese herb. It is used to treat arthroncus, arthrodynia, and arthritis. Hyperuricemia is an important foundation of gouty arthritis. The current study was aimed at investigating whether the effects of total saponins from Rhizoma Dioscoreae Nipponicae on hyperuricemia were due to renal organic ion transporters in potassium oxonate-induced hyperuricemia mice. Hyperplasia of synovial cells prepared from Wistar rats was induced by IL-1ß (1 × 10(4) µg/mL). MTT was used and to screen active components in the inhibition of hyperplasia by total saponins from Rhizoma Dioscoreae Nipponica, individual pure compounds, and different combinations of these compounds. Sixty Kun Ming mice were randomly divided into six groups: normal, model, allopurinol (40 mg/kg), and three total saponins groups receiving dose (600 mg/kg), middle (300 mg/kg), and low doses (60 mg/kg). Hyperuricemic mice were induced with potassium oxonate (300 mg/kg) intragastrically. The total saponins were given six days and the positive drug allopurinol was given one day before inducing hyperuricemia. The serum and urine levels of uric acid and creatinine and the fractional excretion of uric acid were measured in normal and hyperuricemic mice treated with Rhizoma Dioscoreae Nipponicae and allopurinol. The mRNA and protein levels of the mouse urate transporter 1, glucose transporter 9, organic anion transporter 1, and organic anion transporter 3 were analyzed by real-time-PCR and Western blotting methods, respectively. Total saponins from Rhizoma Dioscoreae Nipponicae could effectively reverse potassium oxonate-induced alterations in renal mouse urate transporter 1, glucose transporter 9, organic anion transporter 1, and organic anion transporter 3 mRNA and protein levels, resulting in enhancement of renal urate excretion in mice. These findings suggested that the total saponins from Rhizoma Dioscoreae Nipponicae had a uricosuric effect on the regulation of renal organic ion transporters in hyperuricemic animals.

[Effects of moxibustion intervention on inflammatory reactions and expression of suppressor of cytokine signaling proteins of synovium cells in rheumatoid arthritis rabbits].

OBJECTIVE: To observe the effect of moxibustion intervention on inflammatory reactions and expression of suppressor of cyfokine signaling 1 (SOCS 1) and SOCS 2 [Which are involved in inhibition of the Janus Kinase-signal transducer and activator of transcrip-tion (JAK/STAT signaling pathway and in sffenuation of cytokine signaling)] in synovium cells of the hind-knee joint in rheumatoid arthritis (RA) rabbits, so as to study its mechanism underlying improvement of RA. METHODS: Forty-two Japanese big-ear white rabbits were randomized into control, model and moxibustion groups respectively, with 14 cases in each group. RA model was established by injection of Freund's Complete Adjuvant (0. 5 mL/kg) into the rabbits' bilateral hind-knee joint cavities. Moxibustion was applied to bilateral "Shenshu" (BL 23) areas, 5 cones every time, once daily for 3 weeks except the Sundays. The perimeters of rabbits' hind legs were measured before and after modeling and after the therapy. The synovial tissue of joint was sampled for analyzing the expression levels of SOCS 1 and SOCS 3 by immunohistochemistry. RESULTS: Before the therapy, the perimeters of bilateral knee joints of the control, model and moxibustion groups were of no statistical significance (P>0. 05). In comparison with the control group, the perimeters of bilateral knee joints were significantly increased on day 1, 7, 14 and 21 in the model group (P<0. 01). Compared with the model group, the perimeters of bilateral knee joints in the moxibustion group were significantly decreased (P<0. 05), suggesting an improvement of the inflammatory reaction after moxibustion intervention. Correspondingly, synovial SOCS 1 and SOCS 3 expression levels were remarkabely higher in the model group than in the control group (P<0. 01), and obviously decreased in the moxibustion group compared with the model group (P<0. 01). CONCLUSIONS: Moxibustion intervention has an anti-inflammatory and detumescent effects in RA rabbits, which may be closely associated with its effects in down-regulating expression of SOCS 1 and SOCS 3 proteins by suppressing negative feedback regulatory JAK/STAT pathway in synovial cells. [KEY WORDS] Moxibustion; Rheumatoid arthritis; Inflammatory reactions; Synovial cells; Suppressor of cytokine signaling proteins; Negative-feedback regulatory factors

[Influence of Bushen Huoxue decoction on beta-catenin, MMP-7 of synoviocytes in rats with knee osteoarthritis].

OBJECTIVE: To observe beta-catenin expression of Wnt signaling pathway in rats with knee osteoarthritis, and influence of Bushen Huoxue decoction on beta-catenin and MMP-7 expression of synoviocytes in rats with knee osteoarthritis (OA). METHODS: Rats model with knee osteoarthritis were established by Hulth method. Primary synoviocytes and OA synoviocytes were cultured with collagenase digestion method. The cultured synoviocytes were divided into normal group, OA model group and Bushen Huoxue decoction group. Western blotting method was used to detect beta-catenin, MMP-7 protein expression of synoviocytes after acting by Bushen Huoxue decoction for 48 h; ELISA method was used to detect MMP-7 expression of synovial supernatant. RESULTS: OA synoviocytes were cultured successfully. Western blotting showed that beta-catenin, MMP-7 expression in OA synoviocytes was significantly higher than normal group, Bushen Huoxue decoction could significantly reduce beta-catenin, MMP-7 expression; ELISA results showed that MMP-7 expression of OA synovial supernatant was significantly higher than normal synoviocytes supernatant, Bushen Huoxue decoction significantly regulated the level MMP-7 down. CONCLUSION: (1) High expression of beta-catenin in OA synoviocytes indicates that Wnt classical signal pathway is activated in rat with knee osteoarthritis; (2) High expression of MMP-7 expression in OA synoviocytes confirms the MMP-7 is downstream genes of Wnt/beta-catenin signal pathway; (3) Activation of Wnt signal pathway and increase of MMP-7 may cause degradation of articular cartilage, and promote the formation of osteoarthritis; (4) Bushen Huoxue decoction can reduce expression of MMP-7, and promote cartilage repair, which may be one of mechanisms of osteoarthritis.

Citrullination of fibronectin modulates synovial fibroblast behavior.

INTRODUCTION: Rheumatoid arthritis is an autoimmune arthritis characterized by joint destruction. Anti-citrullinated protein antibodies are pathologic in rheumatoid arthritis, but the role of the citrullinated proteins themselves is much less clear. Citrullination is the conversion of the arginine residues of a protein to citrulline. In the inflamed rheumatoid joint there is increased protein citrullination. Several proteins are citrullinated in rheumatoid arthritis, including collagen type II, fibrinogen, and fibronectin. Fibronectin is thought to mediate the adhesion of joint-invading synovial fibroblasts to the rheumatoid cartilage in addition to regulating other synovial fibroblast functions. However, the effect of citrullinated fibronectin on synovial fibroblasts is unknown. METHODS: To investigate the effect of citrullinated fibronectin on synovial fibroblast behavior, we cultured normal murine, arthritic murine, and human rheumatoid synovial fibroblasts. We then compared several synovial fibroblast functions in the presence of fibronectin versus citrullinated fibronectin. We assessed adhesion with time-lapse microscopy, migration with transwell assays, focal adhesion kinase and paxillin phosphorylation by western blot, and focal matrix degradation by fluorescent gelatin degradation. RESULTS: Normal synovial fibroblasts have impaired adhesion, spreading, migration, and integrin-mediated phosphorylation of focal adhesion kinase and paxillin on citrullinated fibronectin. Murine arthritic and human rheumatoid synovial fibroblasts also have impaired adhesion and spreading on citrullinated fibronectin, but focal matrix degradation is unaffected by citrullinated fibronectin. CONCLUSION: Citrullination of fibronectin alters synovial fibroblast behavior and may affect how these cells adhere to and invade the joint and travel through the bloodstream. This work suggests an important role for the interaction of synovial fibroblasts with citrullinated matrix in the pathophysiology of rheumatoid arthritis.

[Effects of artesunate on tumor necrosis factor alpha and chemotactic factors in the serum and the synoviocyte culture supernate of collagen-induced arthritis rats].

OBJECTIVE: To evaluate the effects of Artesunate on tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein (MCP-1), and on reduced activation normal T cell expressed and secreted (RANTES) in the serum and the synoviocyte culture supernate of collagen-induced arthritis (CIA) rats. METHODS: Eighty male Wistar rats were selected to establish the CIA rat model. On the 6th day after modeling, 60 rats with the sum of arthritis index of right metapedes and two propodium > or = 6 were selected, and randomly divided into 6 groups (n = 10), i.e., the blank control group, the CIA model control group (treated with normal saline, abbreviated as the CIA group), the MTX positive control group (abbreviated as the MTX group), the large dose Artesunate group (at the daily dose of 20 mg/kg), the moderate dose Artesunate group (at the daily dose of 10 mg/ kg), and the small dose of Artesunate group (at the daily dose of 2.5 mg/kg). Mice were sacrificed 7 days of immune injection and their venous blood was collected to obtain the serum. Meanwhile, the synovial tissues of the knee joint were taken by aseptic techniques and primary cultured for 48 h. The supernate was collected by centrifuge. The changes of MCP-1, RANTES, and TNF-alpha in the serum and the synoviocyte culture supernate were observed in each group before and after treatment using ELISA. RESULTS: Artesunate significantly decreased the expressions of TNF-alpha in the serum and the synoviocyte culture supernate, showing significant difference when compared with the model control groups (P < 0.05). There was no statistical difference in the large dose Artesunate group and the moderate dose Artesunate group when compared with the MTX group (P > 0.05). But statistical difference existed in the large dose Artesunate group, the moderate dose Artesunate group, and the MTX group when compared with the small dose Artesunate group (P < 0.05). Artesunate could significantly decrease the expressions of MCP-1 and RANTES in the serum and the synoviocyte culture supernate, showing statistical difference when compared with the model control group (P < 0.05). But no statistical difference existed when compared with the MTX group (P > 0.05). CONCLUSION: The mechanism of anti-inflammatory action and immune regulation of Artesunate might be correlated with the inhibition of inflammatory factor TNF-alpha and chemotactic factors MCP-1 and RANTES.

The fate of acutely inflamed joints with a negative synovial fluid culture.

PURPOSE: The purpose of this study was to evaluate the management and fate of acutely inflamed joints with a negative synovial fluid culture. METHODS: Between January and December 2009, all the patients who presented to our institution with an acutely inflamed joint and were subjected to microbiological assessment of their synovial fluid, were included in the study. Patients with a positive synovial fluid culture, a prosthetic joint replacement in situ and where an aspirate was obtained for a rheumatological diagnosis were excluded. This cohort was then divided into two groups depending on whether a diagnosis could be established through the course of their treatment. Group I included patients in whom a diagnosis could be established and group II included patients in whom a diagnosis could not be established. A thorough review of the patients' medical records and the hospital database was performed. Following this, a database consisting of the patient demographics, clinical features, investigations, treatment and outcome was created. RESULTS: A total of 144 patients met the inclusion criteria (group I: 95, group II: 49). The most commonly affected joint in both the groups was the knee. The average time to presentation was shorter in group II. Clinical findings at presentation were comparable in both groups. However, inflammatory markers were more likely to be raised in group II in comparison with group I. Eighty-two percent of group II required antibiotic treatment compared with 15% of group I. The mean duration of antibiotic treatment in group I was ten days and in group II was 26 days. Mean hospital stay differed significantly between the two groups, with group II being more than twice as long as compared with group I (p=0.001). The rate of mortality was also higher in group II (8.2%, p=0.03). CONCLUSION: Our study shows that patients presenting with an acutely inflamed joint and a negative synovial fluid culture in whom a diagnosis cannot be established during their hospital stay have a longer hospital stay and an increased rate of mortality as compared with patients in whom a diagnosis can be established.

A 3D system for culturing human articular chondrocytes in synovial fluid.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility (1,2). Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo (3-6). Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism (7,8). Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin (9 10). In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in. Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20%) (11-25). While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional (26-28). Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100%) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) (29,30) that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

Anti-inflammatory and anti-nociceptive effect of Betula platyphylla var. japonica in human interleukin-1ß-stimulated fibroblast-like synoviocytes and in experimental animal models.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine has widely been used Betula platyphylla var. japonica to treat various inflammatory diseases including arthritis. AIM OF THE STUDY: To determine the anti-inflammatory, anti-nociceptive, and anti-arthritic effects of Betula platyphylla in interleukin-1ß (IL-1ß)-stimulated fibroblast-like synoviocytes from human rheumatoid arthritis and in nociceptive and inflammatory animal model. MATERIALS AND METHODS: The inflammatory mediators such as IL-6, tumor necrosis factor (TNF)-α matrix metalloproteinase (MMP)-1, MMP-13, inducible nitric oxide synthesis (iNOS), nitrites, prostaglandin E(2) (PGE(2)) and cyclo-oxygenase 2 (COX-2) activity of Betula platyphylla were tested in IL-1ß-stimulated fibroblast-like synoviocytes. Tail withdrawal in response to thermal stimulation in tail flick test or paw flinching and shaking in response to sc hind paw formalin injection was measured 1h after oral administration of Betula platyphylla. The former was evaluated with a paw pressure test, and the latter was measured using the squeaking score, and paw volume in inflammatory arthritis tests. RESULTS: Betula platyphylla significantly inhibited proliferation of IL-1ß-induced synoviocytes. Betula platyphylla reduced the levels of inflammatory mediators, such as IL-6, TNF-α, MMP-1, MMP13, and PGE(2). In particular, Betula platyphylla significantly inhibited the releases of nitrites and iNOS, as well as release of NFκB, into the nucleus of IL-1ß-treated synoviocytes, even at concentrations as low as 1µg/ml. Oral administrant of Betula platyphylla at 400mg/kg significantly decreased about 27.8% of tail flick withdrawal and inhibited about the number of paw flinches in both phases 1 and 2 of the formalin test. In the carrageenan-induced acute pain and arthritis model, Betula platyphylla dose dependently reduced the nociceptive threshold and the arthritic symptoms at day 8, respectively, and Betula platyphylla at 400mg/kg markedly reduced the inflammatory area about 48% in the ankle joints. This capacity of Betula platyphylla at 400mg/kg was similar to that of the celecoxib-2 inhibitor in carrageenan-induced nociceptive and inflammatory arthritis model. CONCLUSIONS: These results suggest that Betula platyphylla has anti-nociceptive and anti-inflammatory effects in IL-1ß-stimulated RA FLS and in an animal model of arthritis. Thus, the use of Betula platyphylla as a pharmaceutical candidate for the treatment of arthritis should be further studied.

Synovial fluid-derived T helper 17 cells correlate with inflammatory activity in arthritis, irrespectively of diagnosis.

We analyzed peripheral blood (PB) and synovial fluid (SF) mononuclear cells from 16 rheumatoid arthritis (RA), 9 spondyloarthritis (SpA), 3 microcrystal arthritis patients, to define the presence of Th17 and Th1 and their relationship with inflammatory activity, and TCR-zeta chain and ZAP-70 levels. Th17 were significantly higher in SF than in PB and more abundant in microcrystal arthritis patients compared to the other groups. Irrespectively of the diagnosis, SF Th17 percentages correlated with joint (SF total leukocyte count, neutrophil percentage) and systemic (C reactive protein [CRP], fibrinogen, erythrocyte sedimentation rate) inflammation markers. SF Th1 percentages directly correlated with inflammation and disease activity (CRP, swollen joint count [SJC]) indices in SpA, but not in RA patients. These observations support the role of Th17 in the pathogenesis of inflammatory arthritides. The TCR-zeta(dim) lymphocytes in SF were found to produce the highest amounts of cytokines including IL-17, whereas no ZAP-70 impairment was associated to Th17.

Effect and mechanism of AR-6 in experimental rheumatoid arthritis.

The root of Clematis chinensis Osbeck has been used widely in rheumatoid arthritis in Chinese traditional medicine and AR-6 is a triterpene saponin isolated from it. In this present study, we investigated in vivo effects of oral AR-6 in chronic rat adjuvant-induced arthritis (AA) and in vitro effect in macrophage and synoviocytes cells. Arthritic scores and serum inflammatory mediators were evaluated 19 days after AA induction by endermic injection of Freund's complete adjuvant in Sprague-Dawley(S-D) rats. Oral administration of AR-6 to arthritic rats resulted in a clear decrease of clinical signs compared to untreated controls. The synoviocyte and macrophage response ex vivo were then analyzed. Anti-arthritic effects of AR-6 correlated with significant decrease of NO and TNF-alpha produced by peritoneal macrophages, ex vivo and in vitro. AR-6 also significant decreased the proliferation of synoviocyte. These data indicate that AR-6 is a potential anti-inflammatory therapeutic and preventive agent.