Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1.

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA.

Chondroprotective effects of high-molecular-weight cross-linked hyaluronic acid in a rabbit knee osteoarthritis model.

OBJECTIVES: We hypothesized that high-molecular-weight (MW) cross-linked (CL) hyaluronic acid (HA) improves joint lubrication and has an enhanced chondroprotective effect. We examined the histopathological changes and friction coefficients in osteoarthritic knee joints after injecting high-MW CL HA. DESIGN: A bilateral anterior cruciate ligament transection (ACLT) model in 20 Japanese white rabbits was used. From week 5 after transection, low-MW HA (0.8 × 10(6) Da; HA80) or high-MW CL HA (6 × 10(6) Da; HA600) was injected weekly into 10 right knee for 3 weeks; normal saline (NS) was injected into the 10 left knee. A sham operation was undertaken to exclude spontaneous osteoarthritis (OA) in five knees. Results were evaluated with macroscopy, histopathology (Kikuchi's score), biomechanical testing, and rheological assessment of the joint fluid viscoelasticity. Statistical analysis was performed using one-way analysis of variance with a 95% confidence interval (CI) (P < 0.05). RESULTS: The macroscopic findings showed severely damaged cartilage in 30% of the NS group and 20% of the HA80 and HA600 groups and intact cartilage in 100% of the sham group. The histological scores and friction coefficients of the HA600 group were significantly lower than those of the NS group (P = 0.007 and P = 0.002, respectively). Viscoelasticity measurements of the joint fluid showed no significant differences between the three treatment groups. CONCLUSION: High-MW CL HA exerts potential chondroprotective effects and produces superior friction coefficients. Our results suggest that HA600 delays the progression of OA effectively and improves joint lubrication significantly.

Mathematical analysis of the flow of hyaluronic acid around fascia during manual therapy motions.

CONTEXT: More research is needed to understand the flow characteristics of hyaluronic acid (HA) during motions used in osteopathic manipulative treatment and other manual therapies. OBJECTIVE: To apply a 3-dimensional mathematical model to explore the relationship between the 3 manual therapy motions (constant sliding, perpendicular vibration, and tangential oscillation) and the flow characteristics of HA below the fascial layer. METHODS: The Squeeze Film Lubrication theory of fluid mechanics for flow between 2 plates was used, as well as the Navier-Stokes equations. RESULTS: The fluid pressure of HA increased substantially as fascia was deformed during manual therapies. There was a higher rate of pressure during tangential oscillation and perpendicular vibration than during constant sliding. This variation of pressure caused HA to flow near the edges of the fascial area under manipulation, and this flow resulted in greater lubrication. The pressure generated in the fluid between the muscle and the fascia during osteopathic manipulative treatment causes the fluid gap to increase. Consequently, the thickness between 2 fascial layers increases as well. Thus, the presence of a thicker fluid gap can improve the sliding system and permit the muscles to work more efficiently. CONCLUSION: The mathematical model employed by the authors suggests that inclusion of perpendicular vibration and tangential oscillation may increase the action of the treatment in the extracellular matrix, providing additional benefits in manual therapies that currently use only constant sliding motions.

Squeeze film lubrication for non-Newtonian fluids with application to manual medicine.

In this paper, we computed fluid pressure and force on fascia sheets during manual therapy treatments using Squeeze Film Lubrication theory for non-Newtonian fluids. For this purpose, we developed a model valid for three dimensional fluid flow of a non-Newtonian liquid. Previous models considered only one-dimensional flows in two dimensions. We applied this model to compare the one-dimensional flow of HA, considered as a lubricating fluid, around or within the fascia during sliding, vibration, and back-and-forth sliding manipulation treatment techniques. The fluid pressure of HA increases dramatically as fascia is deformed during manual therapies. The fluid force increases more during vertical vibratory manipulation treatment than in constant sliding, and back and forth motion. The variation of fluid pressure/force causes HA to flow near the edges of the fascial area under manipulation in sliding and back and forth motion which may result in greater lubrication. The fluid pressure generated in manual therapy techniques may improve sliding and permit muscles to work more efficiently.

Inhibition of PGE2 production by nimesulide compared with diclofenac in the acutely inflamed joint of patients with arthritis.

OBJECTIVE: Cyclo-oxygenase (COX) exists in two isoforms, COX-1 and COX-2. COX-1 is responsible for homeostatic functions, whereas COX-2 is inducible and responsible for the inflammatory effects of prostaglandins. Nimesulide, a selective inhibitor of COX-2, has been shown to relieve pain rapidly in arthritis. We examined the effect of nimesulide on prostaglandin formation in arthritis, to evaluate if this compound gains access to the site of inflammation and whether this is required for analgesia. STUDY DESIGN: This was a single-dose, double-blind, double-dummy, parallel group study of nimesulide 100mg compared with diclofenac 50mg. METHODS: Serial sampling of synovial fluid, whole blood and plasma was performed at baseline and 0.5, 1, 2, 3 and 4 hours after drug administration. Synovial tissue was obtained by needle biopsy at completion of the study period. Synovial fluid prostaglandin E2 (PGE2) was measured by enzyme immunoassay. COX-1 and COX-2 activities in whole blood were estimated by serum thromboxane B2 (TxB2) and endotoxin-induced PGE2 concentrations respectively. Synovial tissue COX-1 and COX-2 mRNA and protein expression were studied by reverse transcriptase polymerase chain reaction and immunohistochemistry respectively. Twenty patients with acute knee inflammation on a background of arthritis of all types completed the study. RESULTS: Patients were allocated randomly to groups to receive nimesulide (n = 10) or diclofenac (n = 10). The mean (+/- SEM) plasma concentration of PGE2 in the nimesulide group decreased from 24.45 +/- 2.71 ng/mL at baseline to 1.74 +/- 2.71 ng/ mL at 2 hours. Diclofenac also inhibited PGE2, but at a later time point (28.15 +/- 2.86 ng/mL at baseline and 0.85 +/- 2.86 ng/mL at 4 hours). The mean (+/- SEM) synovial fluid concentration of PGE2 was 319 +/- 89 pg/mL before treatment; it remained unaltered over 4 hours after the administration of nimesulide or diclofenac (235 +/- 72 pg/mL). In contrast, in six patients receiving long-term treatment with nimesulide or a non-selective NSAID, synovial PGE2 was 61 +/- 24 pg/ mL, suggesting that inhibition of synovial prostaglandin formation is delayed compared with that in plasma. Nimesulide caused partial inhibition of serum TxB2 (a decrease from a mean of 268 +/- 24 ng/mL to one of 164 +/- 27 ng/mL at 2 hours), whereas diclofenac had a greater effect (a decrease from 224 +/- 33 ng/mL, to 76 +/- 27 ng/mL at 3 hours). CONCLUSIONS: Nimesulide, a COX-2 selective inhibitor, has a rapid onset of action in the blood compartment, with early inhibition of PGE2 generation, an index of COX-2 activity. In contrast, it exhibits a delay in achieving therapeutic concentrations in the synovial fluid. Thus factors other than local inhibition of prostaglandins may explain the rapid onset of analgesia that is associated with nimesulide, including a possible central mechanism of pain relief.

Temporomandibular disorders: relationship between joint pain and effusion and nitric oxide concentration in the joint fluid.

OBJECTIVES: To evaluate the relationship between joint effusion, contrast enhancement of effusion, nitric oxide concentration in TMJ fluid and TM joint pain. METHODS: Nonenhanced T1- and T2-weighted and gadolinium-enhanced T1-weighted spin-echo sequences were performed in 77 patients with TMD. The nitric oxide concentration in TMJ fluid was analysed spectrophotometrically by the Griess reaction. RESULTS: Some or marked effusion was seen in five (9%) of the 56 asymptomatic joints and in 55 (56%) of the 98 symptomatic joints. The prevalence of contrast enhancement of joint effusion was significantly higher in the joint pain group than in the joint sound or asymptomatic joint groups (chi2 test, P<0.001). On postcontrast T1-weighted images, there was no evidence of synovial proliferation in patients with TMD. Anterior disk displacement without reduction was detected in 93% of the TMJs with marked effusion. The degree of joint pain correlated with raised nitric oxide concentration (Spearman's rank correlation, P<0.05). CONCLUSIONS: Painful joints are more likely to demonstrate contrast enhancement of joint effusion. Nitric oxide concentration in TMJ fluid is closely associated with inflammatory changes and painful TM joints.

Interrelationship of the kinin system, nitric oxide and eicosanoids in the antigen-induced arthritis in rabbits.

The aim of the present study was to investigate the interrelationship of the kinin system, nitric oxide and eicosanoids in the acute phase of antigen-induced arthritis (AIA) in rabbits. The arthritis was induced in immunized rabbits and the following parameters were evaluated 24 hours later: leukocyte influx (total and differential white cell count), vascular permeability (Evans's blue method), and synovial PMN cell infiltrate. PGE2 and LTB4 (radioimmunoassay) levels were quantified in the synovial fluid. The animals were pre-treated with 20mg/kg/day during 14 days with L-NAME or D-NAME and/or Enalapril (0.12 mg/kg/day-14 days), and/or the B2 antagonist of Bradykinin HOE 140 (0.9 mg/kg). Our results showed that L-NAME was effective in the prevention of AIA with reduction of all Inflammatory parameters analyzed. Enalapril partially reverted the L-NAME anti-inflammatory effects. The simultaneous treatment with HOE 140 abolished this reversion and returned the inflammatory parameters to the levels observed in L-NAME treated animals. Our results suggest that pressoric alterations induced by L-NAME could not account for all its anti-inflammatory action in this model of experimental arthritis. Additionally the contribution of the kinin system in AIA was characterized as well as its interaction with eicosanoids and nitric oxide.

Tryptophan catabolism in synovial fluid of various arthropathies and its relationship with inflammatory cytokines.

Synovial fluids (SF) from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), gout, and osteoarthritis (OA) were investigated for the levels of interleukin (IL)-1 beta, IL-6 and IL-8, tryptophan (Trp) and indoleamine 2,3-dioxygenase (IDO) activity. Significant differences exist in the levels of IL-1 beta between inflammatory arthritides RA, PsA and gout and non inflammatory arthritis, such as OA. The highest concentration of IL-1 beta was found in RA, that showed high levels also of IL-6 and IL-8. In the same disease we also found the highest IDO activity and the lowest Trp concentration. In addition, IDO activity seems to be related with the decrease in Trp, as demonstrated by the inverse correlation found between these two substances in the SF of all patients.

Activated gelatinase-B (MMP-9) and urokinase-type plasminogen activator in synovial fluids of patients with arthritis. Correlation with clinical and experimental variables of inflammation.

OBJECTIVE: To evaluate the occurrence of gelatinase-B (matrix metalloproteinase 9, MMP-9) in synovial fluids (SF) of patients with arthritis to investigate the possible role of this neutral MMP in joint destruction. METHODS: In paired (series I) and unpaired SF (series II) we examined the occurrence of gelatinase-B, prostromelysin-1, and urokinase-type plasminogen activator (u-PA). RESULTS: In the paired SF a parallelism between the presence of activated gelatinase-B and the local arthritis activity scores of the knees was observed. Activated gelatinase-B correlated well with the presence of stromelysin-1 and u-PA, 2 enzymes probably involved in the activation process of gelatinase-B. In the 2nd series, activated gelatinase-B was found in 56 SF samples, whereas 82 samples did not exhibit activated gelatinase-B. The SF samples with the activated form of gelatinase-B showed a significantly higher ability to induce permeability changes in cultured monolayers of human endothelial cells, had more myeloperoxidase activity--secreted by infiltrated leukocytes--and had higher u-PA antigen concentrations, compared to SF samples without the activated form of gelatinase-B. CONCLUSION: Our data suggest that the presence of gelatinase-B is a reflection of the inflammatory condition of the joints of patients with arthritis, and that the activation of gelatinase-B in the joints, which may occur in a u-PA/plasmin dependent and/or a stromelysin dependent way, contributes to the progression of arthritis.

Responses of normal and rheumatic human articular chondrocytes cultured under various experimental conditions in agarose.

The purpose of the present study was to test if agarose could support the maintenance of normal and arthritic human chondrocytes in culture, and under which experimental conditions they could be successfully grown. Cultures of chondrocytes isolated from articular cartilage from patients with rheumatoid arthritis (RA), juvenile rheumatoid arthritis (JRA), and healthy controls were assessed by light microscopy, alcian blue staining, formazan uptake and incorporation of radiosulfate into the extracellular matrix. The results showed that both normal and arthritic chondrocytes proliferated, and synthesized proteoglycan (PG) in agarose in short term and long term culture. Proliferation and PG synthesis occurred at a slower rate in chondrocytes from adult rheumatic patients than from healthy controls. Supplements to the medium influenced chondrocyte proliferation, PG synthesis and release into the medium. Serum from RA patients stimulated chondrocyte responses more than normal human serum (NHS), and NHS promoted PG synthesis more than fetal calf serum (FCS). Exposure to inflammatory synovial fluid (SF) enhanced PG synthesis of healthy chondrocytes, but suppressed it in arthritic chondrocytes. We conclude that species-specific serum is optimal for chondrocyte cultures, and that disease related culture conditions change the chondrocyte response. As metabolic responses of human chondrocytes are maintained in agarose, this culture system appears as a suitable in vitro tool for further studies of human joint disease.

The effect of growth factors and synovial fluid on chondrogenesis in perichondrium.

Reconstruction of cartilage with perichondrium depends on the chondrogenic property of the perichondrial fibrocytes. The present investigation concerns the conditions for the differentiation of fibrocytes into chondrocytes both in vivo and in vitro. For the in vivo studies specimens of rib and auricular perichondrium from adult rabbits were wrapped round silicon rods which were enclosed in dialysis bags. One was placed in the suprapatellar pouch of the knee joint and one was placed intraperitoneally in each rabbit. After two months the bags were extracted, the perichondrium prepared for microscopic examination, and the chondrogenesis evaluated. In vitro the perichondrium was divided into small pieces and incubated with tissue culture medium. The medium was supplemented with fetal calf serum, together with epidermal growth factor, platelet derived growth factor, synovial fluid, or with human serum albumin (control group). After three weeks the explants were prepared for microscopy. Chondrogenesis was judged by the degree of cellular enlargement, capsule formation, deposition of matrix, and activation of the outer fibrocytic layer. In vivo, good cartilage development was found in all specimens placed in the knee joint but, in those placed intraperitoneally, little if any chondrogenesis was seen. In vitro profound differentiation occurred in all cultures supplemented with epidermal growth factor and platelet derived growth factor. An equivalent differentiation was found in perichondrium that had been incubated with synovial fluid. We conclude that the differentiation of perichondrial fibrocytes is initiated in vitro by growth factors. In addition, we have shown that synovial fluid contains factors that promote and enhance the development of cartilage from perichondrium.

[Artificial synovial fluid for the joints]. / Iskusstvennaia sinovia'lnaia zhidkos't dlia sustavov.

The author presents the results of his biomechanical, experimental and clinical studies to produce an artificial synovial fluid for therapeutic correction when administered intraarticularly in inflammatory and degenerative diseases, traumas and operations on the joints. According to the results of the rheologic investigations, the 15% aqueous solution of mean molecular polyvinylpyrrolidone (PVP) and its complex with a biopolymer (PVP-hyaluronate) turned out to be most similar to the natural synovial fluid of the joints. The experimental studies (201 rabbits) revealed a suppression of the inflammatory process in the joints and an antiadhesive action of the artificial synovia which improved the metabolism of the acetabular cartilage and the functions of the joints. In the clinical part of the study (514 patients) the preparations of the artificial synovial fluid and its complexes with other drugs were administered intraarticularly to the patients with rheumatoid arthritis and deforming osteoarthrosis. An improvement in many values of the function of the joints and of the laboratory and biochemical data has been revealed. There have been no side effects in the patients under study; an improvement in the working ability has been noted, including the evaluation of the long-term results (1 to 10 years).

Cytokines in chronic inflammatory arthritis. II. Granulocyte-macrophage colony-stimulating factor in rheumatoid synovial effusions.

A liquid culture technique was used to study 23 synovial fluids (SF) (21 from inflammatory joint diseases and 2 noninflammatory SF) and supernatants of two cultured rheumatoid arthritis (RA) synovial tissues for colony-stimulating factor (CSF). The proliferative responses of human peripheral blood macrophage-depleted non-T cells treated with synovial fluids, supernatants of synovial tissue explants, and recombinant granulocyte-macrophage (rGM)-CSF were compared. Aggregates of cells that formed in long-term cultures (15 d) were similar for each applied agent and consisted of macrophages, eosinophils, and large blasts. Tritiated thymidine incorporation was proportional to the concentration of rGM-CSF and was accompanied by an increase in number and size of cellular aggregates formed in the cultures. CSF activity was observed in inflammatory SF, with tritiated thymidine uptake of 3,501 +/- 1,140 cpm in the presence of RA samples (n = 15) compared to 1,985 +/- 628 for non-RA inflammatory SF (n = 7) (P less than 0.05) and 583 +/- 525 for medium (n = 6) (P less than 0.01). The proliferative response to RA SF was often more apparent when the samples were diluted, because at higher concentrations the RA SF was inhibitory. Two RA SF were fractionated by Sephadex G100 column chromatography; low levels of CSF activity were detected in fractions corresponding to Mr of 70-100 kD, but the major CSF activity was found in the 20-24-kD fractions. A polyclonal rabbit anti-GM-CSF antibody eliminated the stimulating activity from both rGM-CSF and RA SF. Finally, a specific RIA identified significant levels of GM-CSF (40-140 U/ml) in the culture supernatants of 3 additional RA synovial tissues. These data document the local production of GM-CSF in rheumatoid synovitis and are the first description of this cytokine at a site of disease activity.

Transcutaneous electrical nerve stimulation in experimental acute arthritis.

Transcutaneous electrical nerve stimulation (TENS) is applied for the relief of various types of pain, including rheumatoid arthritis and osteoarthritis. This study evaluated the influence of TENS on intraarticular (IA) temperature and pressure and synovial tissue of inflamed rabbit joints. Four hours after induction of acute bilateral hind joint arthritis by single intraarticular injection of urate crystals, the knees of 14 anesthetized rabbits were fixed at 90 degrees between femur and tibia. TENS was supplied from an electrical stimulator to the left knee joint while the other joint was unstimulated as a control. A microprobe needle for temperature measurement was inserted into the knee joint. Intraarticular pressure was recorded by polygraph connected to an IA needle. Readings were carried out at 10-min intervals three times before and four times after 5 min of TENS. A significant increase in IA temperature from mean 36.2C to 36.6C and significant reduction of IA pressure (mean decrease of 0.8 mmHg, from 2.5 mmHg to 1.7 mmHg) were found in the stimulated joints, which also showed a reduction of synovial fluid volume and total leukocyte count in comparison to the controls. While the nonstimulated synovial membrane showed massive leukocytic infiltration with neutrophilic exudate and fibrin clot in the intraarticular cavity, the stimulated synovial membrane demonstrated moderate homogeneous leukocytic infiltration through all layers, with absence of inflammatory exudate in the intraarticular cavity. These results suggested that the analgesic effect of TENS on arthritis may be partially attributed to decrease in IA synovial fluid pressure, volume, and leukocyte count. Therefore, TENS may be useful for reducing pain from inflammatory arthritis.