Hyaluronic Acid In Synovial Fluid Research Articles

Viscosupplementation in the treatment of osteoarthritis

Viscosupplementation in the treatment of osteoarthritis

Synovial fluid chondroitin sulphate indicates abnormal joint metabolism in asymptomatic osteochondritic horses

Alternative methods to evaluate the joint condition in asymptomatic osteochondrosis dissecans (OCD) and other joint diseases may be useful. To investigate possible changes in synovial fluid composition that may lead to joint conditions in asymptomatic OCD, in mature horses. Animals aged >2 years, of different breeds, with OCD in the intermediate ridge of distal tibia, symptomatic or not, were studied. Synovial fluid samples (10 healthy; 11 asymptomatic OCD; 25 symptomatic OCD) were collected by arthroscopy from 29 horses. Glycosaminoglycans (GAGs) were analysed by a combination of agarose gel electrophoresis and enzymatic degradation with specific GAG lyases. The viscosity, white blood cell (WBC) count, protein concentration and hyaluronic acid (HA) molecular weight were also determined. The method used here to analyse synovial fluid GAGs is reliable, reproducible and specific. The main synovial fluid GAGs are HA and chondroitin sulphate (CS), 93% and 7% respectively in normal horses. In symptomatic OCD, the concentrations of both increased (expressed as GAG/urea ratios), but CS increased more. The CS increased also in asymptomatic OCD. An inflammatory reaction was suggested by the increased WBC counts in OCD. The molecular weight of the synovial fluid HA was reduced in OCD, explaining the lower viscosity observed. The increased CS in synovial fluid of OCD joints in mature horses suggests that the synovial fluid CS and the WBC count are good markers of the joint conditions, allowing the identification of pathological phase in joint diseases. The analysis of synovial fluid GAGs shows that cartilage damage occurs even in asymptomatic OCD, implying that arthroscopic removal of osteochondral fragments should be performed even in asymptomatic OCD.

The Role of Different Hyaluronic Acids in the Articular Cartilage of Rabbit

Purpose:To elucidate if the differences found in the physico-chemical and rheological behaviour of Hyaluronic Acids result in different in vivo activity. For this purpose two Hyaluronic Acids (HA), HA-1 and HA-2, with similar molecular weight but different percentage of concentration variation, were compared through an osteoarthritis model.Methods and Materials:Osteoarthritis was induced in white New Zealand rabbits by anterior cruciate ligament section. After the induction period, the animals were allocated to receive HA-1 or HA-2 intra-articularly in one knee whereas the contralateral knee was used as Operated Control. An additional group of non-operated animals was used as Healthy Controls. Samples of cartilage were taken for different measures: apoptosis, nitric oxide (nitrites) and hyaluronic acid in synovial fluid.Results:The administration of HA-1 had a significant inhibitor effect on apoptosis of the chondrocytes compared to operated untreated animals (p = 0.0089), whereas this difference was not observed in the HA-2 knees. Levels of nitrites determined by HPLC in the HA-1 knees were similar to those in the Healthy group (p = 0.6551) whereas they were significantly higher in Operated Control and HA-2 groups (p = 0.0001). The comparison between HA-1 and HA-2 also revealed significantly lower levels of nitrites in the HA-1 knees (p = 0.0001). Values of hyaluronic acid in synovial fluid did not show statistical differences between the different study groups.Conclusions:HA-1 and HA-2 showed different physico-chemical characteristics and these differences have resulted in different in vivo behaviour. As a consequence, not all the HA with similar molecular weight can be considered as equivalent.

Glucosamine increases hyaluronic acid production in human osteoarthritic synovium explants

BackgroundGlucosamine (GlcN) used by patients with osteoarthritis was demonstrated to reduce pain, but the working mechanism is still not clear. Viscosupplementation with hyaluronic acid (HA) is also described to reduce pain in osteoarthritis. The synthesis of HA requires GlcN as one of its main building blocks. We therefore hypothesized that addition of GlcN might increase HA production by synovium tissue.MethodsHuman osteoarthritic synovium explants were obtained at total knee surgery and pre-cultured for 1 day. The experimental conditions consisted of a 2 days continuation of the culture with addition of N-Acetyl-glucosamine (GlcN-Ac; 5 mM), glucosamine-hydrochloride (GlcN-HCl; 0.5 and 5 mM), glucose (Gluc; 0.5 and 5 mM). Hereafter HA production was measured in culture medium supernatant using an enzyme-linked binding protein assay. Real time RT-PCR was performed for hyaluronic acid synthase (HAS) 1, 2 and 3 on RNA isolated from the explants.Results0.5 mM and 5 mM GlcN-HCl significantly increased HA production compared to control (approximately 2 – 4-fold), whereas GlcN-Ac had no significant effect. Addition of 5 mM Gluc also increased HA production (approximately 2-fold), but 0.5 mM Gluc did not. Gene expression of the HA forming enzymes HAS 1, 2 and 3 was not altered by the addition of GlcN or Gluc.ConclusionOur data suggest that exogenous GlcN can increase HA production by synovium tissue and is more effective at lower concentrations than Gluc. This might indicate that GlcN exerts its potential analgesic properties through stimulation of synovial HA production.

Identification and functional characterization of hyaluronic acid in degenerative joint diseases

Hyaluronic acid (HA), is a long polysaccharide chain that is made of repeating disaccharide units of N-acetylglucosamine and glucuronic acid. In osteoarthritis (OA) and rheumatoid arthritis, the molecular weight and concentration of HA in synovial fluid (SF) are reduced. This might result from abnormal biosynthesis by synovial type B cells and free radical depolymerization of the HA chain. The intra-articular treatment with HA is an accepted therapy for OA. However, the ideal candidate for intra-articular HA has yet to be defined and the exact mechanism of action of HA is unclear. Our study showed that the levels of ROS and acute phase protein profiles were reduced in patients under going HA treatment. HA could increase the cell viability of human chondrocytes under the high oxidative stress conditions also been determined. The primary receptor for HA is CD44, mediating both cell-cell and cell-matrix interactions. We postulate that the biological process initiated by HA-CD44 interaction results in changes in chondrocytes as well as in SF protein profiles. We used the proteomic approaches to investigate the biological functions of HA. There were 31 proteins, functioning in cell rescue, cell defense, cell stress, energy, anaerobic glycolysis, protein fate, cellular organization and cell cycle involved in this regulatory signaling. It suggests that HA involves in the antioxidative activity, anti-inflammation, anti-catabolic effect and cell growth regulation. This study will contribute to our understanding of HA functioning in the progressive degeneration of cartilage and to explore the clinical application of HA for degenerative joint diseases.

Mechanisms Involved in Enhancement of Osteoclast Formation and Function by Low Molecular Weight Hyaluronic Acid

Hyaluronic acid (HA) is a component of the extracellular matrix that has been shown to play an important role in bone formation, resorption, and mineralization both in vivo and in vitro. We examined the effects of HA at several molecular weights on osteoclast formation and function induced by RANKL (receptor activator of NF-kappa B ligand) in a mouse monocyte cell line (RAW 264.7). HA at M(r) < 8,000 (low molecular weight HA (LMW-HA)) enhanced tartrate-resistant acid phosphatase-positive multinucleated cell formation and tartrate-resistant acid phosphatase activity induced by RANKL in a dose-dependent manner, whereas HA at M(r) > 900,000 (high molecular weight HA (HMW-HA)) showed no effect on osteoclast differentiation. LMW-HA enhanced pit formation induced by RAW 264.7 cells, whereas HMW-HA did not, and LMW-HA stimulated the expression of RANK (receptor activator of NF-kappa B) protein in RAW 264.7 cells. In addition, we found that LMW-HA enhanced the levels of c-Src protein and phosphorylation of ERKs and p38 MAPK in RAW 264.7 cells stimulated with RANKL, whereas the p38 MAPK inhibitor SB203580 inhibited RANKL-induced osteoclast differentiation. This enhancement of c-Src and RANK proteins induced by LMW-HA was inhibited by CD44 function-blocking monoclonal antibody. These results indicate that LMW-HA plays an important role in osteoclast differentiation and function through the interaction of RANKL and RANK.

顎関節疾患の病態形成における細胞と細胞外マトリックスについての研究

Recently, many lines of evidence indicated that variable degrees of inflammation exist in temporomandibular disorders (TMD). Synovial fluid (SF) analysis of the temporomandibular joint (TMJ) has revealed that a variety of inflammatory mediators and components of the matrices of the articular cartilage are detectable in the TMJ. We performed SF analysis for the inflammatory cytokines and nitric oxide (NO) in the SF of the TMJ. It is demonstrated that cytokines such as interleukin 1 (IL-1) are frequently detectable in the SF from patients with TMD, not from the asymptomatic controls. Nitric oxide was also detectable mainly from the patients. Furthermore, inducible nitric oxide synthase (iNOS) was expressed in the synovium of the patients with TMD. Hyaluronic acid (HA), a high-molecular-weight polysaccharide, plays a major role in joint lubrication. We demonstrated that the molecular weight of HA in SF from patients with TMD, not from healthy volunteers, is decreased. These data suggested that IL-1 and NO seem to be related to the pathophysiology of the certain types of the TMD. These data also suggested that mechanical stress caused by overloading results in the generation of free radicals such as NO. These radicals may cause degradation of hyaluronic acid, which in turn impairs the joint lubrication and function in patients with TMD.

Thin film rheology and lubricity of hyaluronic acid solutions at a normal physiological concentration.

Using a surface forces apparatus to measure forces, and optical (multiple beam) interferometry to measure surface shapes and separations (to +/-1 A), the normal, viscous, and shear (lubrication) forces between smooth mica surfaces in aqueous hyaluronic acid (HA) solutions were measured. The experimental conditions of loading pressures, pH, and HA concentration were set to closely correspond to physiological human knee-joint conditions. From the force and optical (refractive index) measurements, it was concluded that, like other negatively charged polyelectrolytes, HA does not naturally adsorb on the mica surface which is hydrophilic and weakly negatively charged at physiological conditions: the polymer solution exhibits the bulk viscosity (22.5 +/- 1.5 cP) for films thicker than about 0.4 miccrom of the polymer, whereas for thinner films, the viscosity decreases monotonically toward the value of the pure electrolyte solution (1 cP) as HA is extruded from between the surfaces. This is indicative of a repulsive "depletion" interaction of HA with each mica surface and to a weakly attractive polymer-mediated force between the two surfaces. Thus, free HA in synovial fluid is not expected to act as a good "boundary lubricant." Relaxation measurements on approaching and receding surfaces in HA solutions were also performed, and it is shown that the presence of HA in the bulk solution can improve "hydrodynamic" modes of lubrication, for example, by assuaging the compression stroke. The study includes information that is beneficial to researchers working with biomaterials viscosupplementation devices.

Enhanced synovial production of hyaluronic acid may explain rapid clinical response to high-dose glucosamine in osteoarthritis

Anecdotal reports of rapid symptomatic response to high-dose glucosamine in osteoarthritis are not credibly explained by the traditional view that glucosamine promotes synthesis of cartilage proteoglycans. An alternative or additional possibility is that glucosamine stimulates synovial production of hyaluronic acid (HA), which is primarily responsible for the lubricating and shock-absorbing properties of synovial fluid. Many clinical and veterinary studies have shown that intra-articular injections of high-molecular-weight HA produce rapid pain relief and improved mobility in osteoarthritis. HA has anti-inflammatory and analgesic properties, and promotes anabolic behavior in chondrocytes. The concentration and molecular weight of synovial fluid HA are decreased in osteoarthritis; by reversing this abnormality, high-dose glucosamine may provide rapid symptomatic benefit, and in the longer term aid the repair of damaged cartilage.

Immunohistochemical demonstration of fibronectin in the most superficial layer of normal rabbit articular cartilage.

To locate fibronectin ultrastructurally in the most superficial layer of normal articular cartilage of rabbits, in order to clarify its role in joint physiology. Articular cartilage was obtained from the femoral condyle of seven normal adult rabbits and prepared by a method that included tannic acid fixation. Polyclonal antibodies against rabbit fibronectin were used in an immunohistochemical electron microscopic study, without any enzymic digestion but with a pre-embedding method for the transmission electron microscopy. The cartilage surface was successfully preserved by tannic acid fixation. The most superficial layer in electron photomicrographs was approximately 200-300 nm thick, cell free, and appeared to have two parallel components: the more superficial lamina and the deeper lamina. Gold labelled fibronectin lined this layer in immunohistochemical electron photomicrographs. Fibronectin covering the surface of the articular cartilage may have a role in joint lubrication and protection of the cartilage by binding with the collagenous matrix and hyaluronic acid in synovial fluid. Chondroitin sulphates may act as a charge barrier in close relationship with the collagen fibrils in the deeper lamina. Significant alteration in these functions may be one of the first causal steps leading to destruction of the articular cartilage.

In vitro lead cation-hyaluronic acid interaction.

It is the purpose of this investigation to study the in vitro spontaneous interaction lead-hyaluronic acid using the equilibrium dialysis technique. The following working conditions were used: membrane retention capacity starting from 12000 D; solvent was an aqueous buffer solution (pH 7.4); relation of internal-external volume of 0.5 : 25 mL: dialysis time 24 hours; lead concentration in dialyzing solution 4 micrograms/mL (always outside the bag); temperature 4 degrees C; concentrations of hyaluronic acid 0.3, 1.2, 2.1 and 3.0 mg/mL; concentration of plasma proteins 1.5 mg/mL; lead was determined in the internal solution of the bag, and was measured quantitatively by atomic absorption spectrometry. Testing separately hyaluronic acid and plasma proteins both types of marcromolecules turned out to bind lead. As the concentration of hyaluronic acid increases, the amount of lead retained increases as well, although this relation is only approximately linear. At the normal concentrations of plasma proteins and hyaluronic acid in synovial fluid, the proteins solution bound more lead than hyaluronic acid solution 16.2 +/- 2.1 and 5.7 +/- 0.4 micrograms/mL respectively (P < 0.0001). What these data show ist that both, hyaluronic acid and proteins are involved in the in vitro lead retention. The effect of both ligands must be synergistic, because when the two molecules are present, a higher amount of lead is retained compared to the amount bound separately, suggesting that lead can replace calcium by complexation with mucopolysaccharides; this explains the lead accumulation in synovial fluid since this fluid is rich in hyaluronic acid and proteins.

Concentration and degradation of hyaluronic acid in knee synovial fluid from carrageenin-induced rabbit arthritis.

The concentration and degradation of hyaluronic acid in the synovial fluid of carrageenin-induced arthritic joints of rabbits was studied. A 0.5-ml volume of 1% lambda-carrageenin was intra-articularly injected three times into a right knee joint, and saline into a left. After 5 d from the last injection, inflammatory changes were observed in the synovial membrane and synovial fluid, but not in the articular cartilage. In the inflammatory synovial fluid, lipid peroxide content, phosphatase activity and cell counts were significantly increased, but the copper concentration was not changed. Concentration of polymeric hyaluronic acid and total hyaluronic acid were determined by high-performance liquid chromatography using gel-permeation columns. Total hyaluronic acid was appreciably decreased in the inflammatory fluid. The polymeric hyaluronic acid determined was 38% of the total hyaluronic acid in the inflammatory fluid and 74% in the control fluid. This suggests that in the inflammatory fluid, molecular weights of hyaluronic acid are distributed in the broader range. The concentration of chondroitin sulphates was similar in both the inflammatory fluid and the control fluid, but the content ratio of chondroitin sulphates to hyaluronic acid was higher in the inflammatory fluid. In the inflamed synovial membrane, synthesis of hyaluronic acid as measured by incorporation of [14C]glucosamine into glycoconjugates was increased by about twice that in the control membrane.

Analysis of hyaluronic acid in synovial fluid by reversed-phase liquid chromatography

Analysis of hyaluronic acid in synovial fluid by reversed-phase liquid chromatography

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronan (hyaluronic acid) in biological fluids

An enzyme-linked immunosorbent-inhibition assay for quantitation of hyaluronic acid (HA) is described. The principle of the method depends on the specific binding of HA to the hyaluronic acid-binding region (HABR) of proteoglycan (PG) monomers. The remaining uncomplexed PG monomers were determined by incubation with specific monoclonal antibodies to HABR followed by addition of polyclonal antibodies against PG monomers and enzyme-conjugated antibodies. The HA in samples was quantified by comparing their inhibitory capacity in the assay against a standard inhibition curve obtained using highly purified HA. This method was used to quantitate HA at nanogram levels in normal sera and synovial fluids. The level in normal human sera was found to be 28 ± 17 ng/ml which compared favorably with values obtained using a commercial radioassay kit on the same samples. The assay was used to measure HA in synovial fluid from patients with rheumatoid and osteoarthritis and the results obtained were comparable with data published by others.

CHANGES IN THE VISCOSITY OF HYALURONIC ACID AFTER EXPOSURE TO A MYELOPEROXIDASE‐DERIVED OXIDANT

Both purified hyaluronic acid (HA) and bovine synovial fluid react with OCI-, the major oxidant produced by the myeloperoxidase (MPO)/H2O2/CI- system, resulting in a decrease in their specific viscosity. This reaction is inhibited in the presence of excess methionine. H2O2 alone decreases the viscosity of HA, presumably by the Fenton reaction, in the absence (but not in the presence) of the iron chelator, diethyltriaminepentacetic acid (DETAPAC). In the presence of DETAPAC, incubation of HA with the complete MPO/H2O2/CI- system lowered the viscosity of HA. Analysis of 3H-HA exposed to OCI- by gel filtration chromatography indicated that cleavage of HA occurred only at higher OCI- concentrations. We suggest that the reduction in viscosity of HA by the MPO/H2O2/CI- system may be due to a combination of oxidative cleavage and changes in the conformation of the molecule. We speculate that the changes in the molecular size of rheumatoid synovial fluid HA may be due to the action of the neutrophil MPO/H2O2/CI- system.

ラットにおける実験的関節とう痛モデルとヒアルロン酸ナトリウムの鎮痛作用

Using the model of knee pain reaction induced by intra-articular injection of endogenous pain substances, especially bradykinin (BK) in rats, the mechanism of the analgesic effect of sodium hyaluronate (SPH) was investigated. The simultaneous administration of prostaglandin E2 with BK or hyaluronidase digestion of endogenous hyaluronic acid (HA) in our experiments brought remarkable hyperalgesia on BK-induced knee pain. These results suggest that higher sensitivity to the pain reaction is induced in a diseased joint (higher prostaglandin content, lower concentration and molecular size of HA in synovial fluid) than in a normal one. SPH definitely decreased BK-induced pain, and its analgesic effect was observed for a longer period, depending on its dose in pre-treatment and the degree of its distribution in synovial tissues. As the analgesic effect of SPH was observed in the hyaluronidase-treated joint as well, it is suggested that the increasing viscosity of synovial fluid caused by increasing HA concentration can decrease the pain even without normalizing molecular size of HA in the joint. HA oligomer and other compounds with similar viscosity or with similar polyanionic character as SPH showed no analgesic effect. From these results, it seems that the characteristic steric configurations of higher molecular HA are needed for the manifestation of the analgesic effect. SPH seems to show its analgesic effect by covering pain receptors in synovial tissues and holding endogenous pain substances in its molecule.

Suppression of hyaluronic acid synthesis in synovial organ cultures by corticosteroid suspensions.

The effects of sparingly soluble corticosteroid suspensions prepared for intraarticular therapy, of their vehicles, and of hydrocortisone on synovial hyaluronic acid (HA) synthesis were compared in organ cultures of normal canine villous synovium. Both hydrocortisone and the corticosteroid suspensions suppressed HA synthesis, as did 2 vehicle components, polysorbate 80 and myristyl-gamma-picrolinium chloride. Cultures of synovium from joints of dogs which, 1 day previously, had been injected with methylprednisolone acetate suspension synthesized less HA than did control cultures from noninjected joints of the same animals. The results indicate that suppression of HA synthesis is a mechanism by which these drugs can act to reduce joint HA content and promote resolution of synovial effusions.

The synovial fluid hyaluronic acid in rheumatoid arthritis.

The intrinsic viscosity of hyaluronic acid in synovial fluid decreases significatively in mild and severe arthritis (24% and 37% respectively). Variation in hyaluronic acid concentration parallels the above results. Chondroitin-6-sulfate can be detected in about 30% of the arthritic fluids.

Structure of hyaluronic acid in synovial fluid and its influence on the movement of solutes

Structure of hyaluronic acid in synovial fluid and its influence on the movement of solutes

Distribution of plasma proteins and hyaluronic acid in synovial fluid and serum of human subjects in hydarthrosis

Total protein, fourteen individual plasma proteins and hyaluronate have been analyzed in patients with traumatic hydarthrosis. There is a general increase in the absolute amount of plasma proteins in these synovial fluids, while serum values are close to normal levels. The hyaluronate content is reduced, although the total production is far above the situation in normal synovial fluids. The present data support the existence of a mechanism of joint effusion which is common to several etiologies.