Effect of interleukin-1 on hyaluronate synthesis by synovial fibroblastic cells

Summary Interleukin-1 (IL-1) stimulates fibroblast-mediated hyaluronate (HA) synthesis in vitro. In the present study the degree of polymerization of such HA was studied using HPLC (high performance liquid chromatography) with a size exclusion column combined with¹²⁵I-HABP assay used to measure the HA concentration in various HA molecular weight fractions separated using HPLC. IL-1 stimulated HA was more polydisperse than that produced by resting fibroblasts with a molecular weight varying from more than 4×10⁶ daltons to less than 7.1×10³ daltons. This IL-1 effect may contribute to the low molecular weight HA produced by freshly explanted arthritic synovial tissue and to the low viscosity of arthritic synovial fluid in vivo.     

The synthesis of hyaluronic acid by human synovial fibroblasts is influenced by the nature of the hyaluronate in the extracellular environment

Summary Various cell lines of human synovial fibroblasts derived from synovium obtained at the time of biopsy or total joint-replacement surgery have been established. The synthesis of ³H-labelled hyaluronic acid (HA) in these cells has been determined, and the effects of adding HA of varying molecular size to the cultured cells examined. The results obtained clearly show that the in vitro synthesis of HA by these cells is influenced by the concentration and molecular weight (MW) of the HA in their extracellular environment. Synovial fibroblasts derived from an osteoarthritic joint demonstrated the most marked response on exposure to exogenous HA, showing a stimulation of HA synthesis with preparations of weight-average molecular weight (Mw)>5×10⁵ in a concentration dependent manner. HA preparations with Mw<5×10⁵ showed little or no effect except at high concentrations where a suppression of biosynthesis was observed. A model to explain these findings is proposed.     

High-dose corticosteroids during bleomycin-induced alveolitis in the rat do not suppress the accumulation of hyaluronan (hyaluronic acid) in lung tissue.

A single intratracheal injection of bleomycin in rats induced, 4 days later, a considerable accumulation of hyaluronan (hyaluronate, hyaluronic acid) in the lung tissue. This connective tissue reaction was quantified biochemically by analysing hyaluronan (HA) in bronchoalveolar lavage fluid (BAL) and lung tissue extracts. The molecular weight of the HA recovered during lavage was 0.2-0.3 X 10(6) daltons. The HA accumulation was related to an increase in lung water content and associated with an increased influx of eosinophils, neutrophils and lymphocytes into BAL fluid. High-dose corticosteroid treatment (prednisolone 15 mg.kg-1 bw per day) to bleomycin injured rats had no effect on the lung tissue content of HA, the recovery of HA during BAL or the molecular weight of HA accumulated in the alveolar space. Furthermore, steroids did not influence the increased lung water content, or the appearance of inflammatory cells in lavage fluid. These findings indicate that the early connective tissue response to the bleomycin lung injury is mediated by a mechanism which is unaffected by systemic high-dose corticosteroids.     

Quantitative measurement of serum hyaluronic acid molecular weight in rheumatoid arthritis patients and the role of hyaluronidase

Aim: Hyaluronic acid (HA) is a glycosaminoglycan and is essential for protecting the cartilage surface by its physical property. It is known that serum HA concentration in rheumatoid arthritis (RA) patients is higher than in healthy volunteer. However, molecular weight (MW) of serum HA in RA patients is not clear, since it needs a large sample volume to assay serum HA MW. The aim of this study is to establish the method for measuring serum HA MW in small sample sizes and to assess the association between serum HA MW and hyaluronidase (HAase) activity. Methods: MW of serum HA in RA patients was measured using high‐performance liquid chromatography and HA‐binding protein. Additionally, the correlation between serum HA and HAase activity was examined using zymographic measurements. Results: Serum HA MW peaked at 1–2 × 10⁵ Da in all cases. However, in certain cases two peaks were observed, one each at low (1–2 × 10⁵ Da) and high (8–14 × 10⁵ Da) MW. HAase activity was lower in cases exhibiting this two‐peaked serum HA MW pattern than in those cases with only a single peak. Conclusion: The novel method developed for this study permits accurate measurement of serum HA MW. The correlation observed between serum HA MW and HAase activity suggests that serum HA MW may reflect the condition of subjects’ joints.     

Does hyaluronan have a role in endothelial cell proliferation of the synovium?

Hyaluranate (HA) is a major constituent of synovial fluid, but its concentration and molecular size differ in normal and inflamed joints. HA can induce or inhibit angiogenesis depending on both its size and its concentration. Endothelial-cell endocytose-labeled macromolecular HA and HA oligosaccharides and binding studies have identified an HA-specific receptor on the endothelial cell surface (KD, 10(-10) mol/L; approximately 2,000/cell). The molecular weight of HA-binding proteins was found to be 90 to 125, 78, and 46 kd.     

Oxygen derived free radicals and synovial fluid hyaluronate.

High performance liquid chromatography with TSK 5000 PW or TSK 6000 PW size exclusion columns combined with a 125I labelled hyaluronic acid binding protein assay was used to study the effects of oxygen derived free radicals on synovial fluid hyaluronate. A continuous flux of free radicals was generated by the xanthine oxidase/hypoxanthine system. When the free radical flux was generated with xanthine oxidase/hypoxanthine in the presence of the iron chelator desferrioxamine and the hydroxyl radical scavenger mannitol a 30-50% decrease in hyaluronate peak was detected, but the molecular weight of synovial fluid hyaluronate remained almost unchanged as a result of reaction with superoxide radicals and hydrogen peroxide. When trace amounts of iron and EDTA were present in the reaction mixture depolymerisation of synovial fluid hyaluronate occurred, and it reached a final molecular weight of about 13,500 daltons. These results suggest that superoxide and hydroxyl radicals may have a different mode of action on synovial fluid hyaluronate. Superoxide radicals and hydrogen peroxide do not induce depolymerisation but, rather, change the molecular configuration of synovial fluid hyaluronate.     

Concentration and molecular weight of sodium hyaluronate in synovial fluid from patients with rheumatoid arthritis and other arthropathies.

The molecular weight distribution of hyaluronate (HA) in synovial fluid (SF) from 10 patients with rheumatoid arthritis (RA), from six patients with other joint disorders, and from five recently deceased persons without joint affections was investigated by a gel chromatographic procedure. A new and highly specific radioassay was used for determination of the HA concentration in the effluent from the chromatographic column, and this allowed analyses on 0.5 ml or less of untreated synovial fluid. The results confirmed the findings by others that the weight-average molecular weight (Mw) of HA in SF from patients with RA (4.8 X 10(6)) was similar to that in other joint diseases (5.0 X 10(6)) and moderately but significantly (p less than 0.001) lower than that of normal SF (7.0 X 10(6)). Furthermore, the molecular weight distribution of HA in the pathological SF was generally broad and varied considerably between individuals. The HA concentration in the pathological SF varied between 0.17 and 1.32 g/l, which is in accordance with previous reports and considerably lower than that of normal SF. Neither the nature of the arthropathy and the extent of the inflammatory process nor the pharmacological treatment had a tendency to influence the HA concentration in the SF, the mean molecular weight of HA, or its molecular weight distribution. Although the concentration of HA in SF drops in joint disease, the total amount of the polysaccharide is greatly enhanced. Also the amount of high molecular weight polysaccharide (Mw greater than 6 X 10(6)) is in excess in joint disease. The pathological state is therefore characterised not by lack of high molecular weight hyaluronate but by a dilution of it.     

Dynamic changes in cervical glycosaminoglycan composition during normal pregnancy and preterm birth

Glycosaminoglycans (GAG) have diverse functions that regulate macromolecular assembly in the extracellular matrix. During pregnancy, the rigid cervix transforms to a pliable structure to allow birth. Quantitative assessment of cervical GAG is a prerequisite to identify GAG functions in term and preterm birth. In the current study, total GAG levels increased at term, yet the abundance, chain length, and sulfation levels of sulfated GAG remained constant. The increase in total GAG resulted exclusively from an increase in hyaluronan (HA). HA can form large structures that promote increased viscosity, hydration, and matrix disorganization as well as small structures that have roles in inflammation. HA levels increased from 19% of total GAG in early pregnancy to 71% at term. Activity of the HA-metabolizing enzyme, hyaluronidase, increased in labor, resulting in metabolism of large to small HA. Similar to mice, HA transitions from high to low molecular weight in term human cervix. Mouse preterm models were also characterized by an increase in HA resulting from differential expression of the HA synthase (Has) genes, with increased Has1 in preterm in contrast to Has2 induction at term. The Has2 gene but not Has1 is regulated in part by estrogen. These studies identify a shift in sulfated GAG dominance in the early pregnant cervix to HA dominance in term and preterm ripening. Increased HA synthesis along with hyaluronidase-induced changes in HA size in mice and women suggest diverse contributions of HA to macromolecular changes in the extracellular matrix, resulting in loss of tensile strength during parturition.     

Potential mechanism of action of intra-articular hyaluronan therapy in osteoarthritis: are the effects molecular weight dependent?

BACKGROUND: Hyaluronan, or hyaluronic acid (HA), is the major hydrodynamic nonprotein component of joint synovial fluid (SF). Its unique viscoelastic properties confer remarkable shock absorbing and lubricating abilities to SF, while its enormous macromolecular size and hydrophilicity serve to retain fluid in the joint cavity during articulation. HA restricts the entry of large plasma proteins and cells into SF but facilitates solute exchange between the synovial capillaries and cartilage and other joint tissues. In addition, HA can form a pericellular coat around cells, interact with proinflammatory mediators, and bind to cell receptors, such as cluster determinant (CD)44 and receptor for hyaluronate-mediated motility (RHAMM), where it modulates cell proliferation, migration, and gene expression. All these physicochemical and biologic properties of HA have been shown to be molecular weight (MW) dependent. OBJECTIVE: Intra-articular (IA) HA therapy has been used for the treatment of knee osteoarthritis (OA) for more than 30 years. However, the mechanisms responsible for the reported beneficial clinical effects of this form of treatment remain contentious. Furthermore, there are a variety of pharmaceutic HA preparations of different MW available for the treatment of OA, but the significance of their MWs with respect to their pharmacologic activities have not been reviewed previously. The objective of the present review is to redress this deficiency. METHODS: We reviewed in vitro and in vivo reports to identify those pharmacologic activities of HA that were considered relevant to the ability of this agent to relieve symptoms and protect joint tissues in OA. Where possible, reports were selected for inclusion when the pharmacologic effects of HA had been studied in relation to its MW. In many studies, only a single HA preparation had been investigated. In these instances, the experimental outcomes reported were compared with similar studies undertaken with HAs of different MWs. RESULTS: Although in vitro studies have generally indicated that high MW-HA preparations were more biologically active than HAs of lower MW, this finding was not confirmed using animal models of OA. The discrepancy may be partly explained by the enhanced penetration of the lower MW HA preparation through the extracellular matrix of the synovium, thereby maximizing its concentration and facilitating its interaction with target synovial cells. However, there is accumulating experimental evidence to show that the binding of HAs to their cellular receptors is dependent on their molecular size; the smaller HA molecular species often elicits an opposite cellular response to that produced by the higher MW preparations. Studies using large animal models of OA have shown that HAs with MWs within the range of 0.5 x 10(6)-1.0 x 10(6) Da were generally more effective in reducing indices of synovial inflammation and restoring the rheological properties of SF (visco-induction) than HAs with MW > 2.3 x 10(6) Da. These experimental findings were consistent with light and electron microscopic studies of synovial membrane and cartilage biopsy specimens obtained from OA patients administered 5 weekly IA injections of HA of MW = 0.5 x 10(6)-0.73 x 10(6) Da in which evidence of partial restoration of normal joint tissue metabolism was obtained. CONCLUSIONS: By mitigating the activities of proinflammatory mediators and pain producing neuropeptides released by activated synovial cells, HA may improve the symptoms of OA. In addition, HAs within the MW range of 0.5 x 10(6)-1.0 x 10(6) Da partially restore SF rheological properties and synovial fibroblast metabolism in animal models. These pharmacologic activities of HA could account for the reported long-term clinical benefits of this OA therapy. However, clinical evidence has yet to be described to support the animal studies that indicated that HAs with MW > 2.3 x 10(6) Da may be less effective in restoring SF rheology than HAs of half this size.     

Ectopic production of somatostatin-like immuno- and bioactivity by cultured human pulmonary small cell carcinoma

Eleven continuous cultures of human pulmonary small cell carcinoma cells were examined, and eight were shown to secrete quantities of somatostatin-like immunoreactivity (SRIF-LI) ranging from 0.07-27 ng/ml culture medium/4 days, SRIF-LI was also found in a 2-N acetic acid extract of one of three human pulmonary small cell carcinomas obtained at autopsy as well as in the extract of a solid tumor resulting from inoculation of nude, athymic mice with SRIF-LI-producing, cultured small cell carcinoma cells. The SRIF-LI produced by one continuous cell line, DMS 53, was characterized in terms of its immunological, chromatographic, and biological properties. SRIF-LI from DMS 53 culture media and lysed cells was heat stable and exhibited parallel displacement to synthetic SRIF standard in a double antibody RIA. DMS 53 SRIF-LI was quantitatively retained on an immunoaffinity column of sheep anti-SRIF-Sepharose 4B under neutral conditions and could be eluted with 2 N acetic acid. Gel filtration chromatography of immunoaffinity-purified SRIF-LI revealed multiple molecular weight forms, the largest of which had an apparent molecular weight of 10,000-12,000 daltons and may represent a precursor form. This high molecular weight SRIF-LI form was resistant to exposure to denaturing conditions (8 M urea or 4 M urea plus 0.5% mercaptoethanol), suggesting the absence of noncovalent and/or disulfide linkages. A low molecular weight form coeluted with synthetic SRIF. Additional evidence for the identity of this form with the tetradecapeptide was provided by highly specific reverse phase high performance liquid chromatography. The rate of degradation of high molecular weight SRIF-LI by the cultures was markedly reduced in comparison to that of the SRIF monomer, resulting in a preferential accumulation of high molecular weight SRIF-LI in 4-day culture medium. Bioactivity of DMS 53 SRIF-LI was assessed in 4-day primary monolayer cultures of rat adenohypophyseal cells where 10(-10)-10(-9) M synthetic SRIF elicited a linear log-dose suppression of 5 X 10(-4) M synthetic SRIF elicited a linear log-dose suppression of 5 X 10(-4) M dibutyryl cAMP-stimulated rat GH release. Immunoaffinity-purified SRIF-LI from DMS 53 lysed cells and 1-h serum-free incubation medium, which consisted predominantly of monomeric SRIF, was equipotent to synthetic SRIF, SRIF-LI from 4-day culture medium consisted mostly of the high molecular weight form and exhibited a reduced bioassay potency ratio relative to synthetic SRIF of 0.73 (95% confidence limits, 0.99-0.53). Chromatographically purified high molecular weight SRIF-LI had significant bioactivity with a bioassay to immunoassay ratio of 0.19 (95% confidence limits, 0.33-0.09). The demonstration of ectopic SRIF, production by human pulmonary small cell carcinoma is consistent with the proposed derivation of this tumor from a cell type in the amine precursor uptake and decarboxylation cell series.     

Effect of intraarticular hyaluronan injection on synovial fluid hyaluronan in the early stage of canine post-traumatic osteoarthritis.

OBJECTIVE: To determine how the quantity and molecular weight of synovial fluid hyaluronan (HA) within the synovial fluid (SF) of osteoarthritis (OA) joints is affected by intraarticular injection of HA. METHODS: Dogs in which OA was induced by transection of the anterior cruciate ligament received 5 weekly injections of HA (1.5 x 10(6) Da) in saline (10 mg/0.67 ml) or an equal volume of saline into the operated knee, beginning the day after surgery. Immediately before each injection, SF was aspirated and the volume of SF and the concentration of HA was measured (uronic acid), and the molecular weight of the HA in each sample was estimated by electrophoresis in agarose. RESULTS: The volume of SF in the unstable knee increased after surgery, and the molecular weight decreased from approximately 2.5 x 10(6) Da to approximately 2 x 10(6) Da. Injection of HA did not affect the volume of SF or average molecular weight of HA in samples obtained immediately before each injection or at the end of the experiment, 12 weeks after surgery. The SF HA concentration fell from a baseline value of 2.3 +/- 0.1 mg/ml to 1.1 +/- 0.2 mg/ml the day after surgery and remained low throughout the course of injections. The HA concentration 12 weeks after surgery in the HA injected knees was approximately 40% lower than the preoperative value, although it increased slightly relative to saline injected knees (1.4 +/- 0.3 vs 1.1 +/- 0.01 mg/ml, respectively; p = 0.04). CONCLUSION: Intraarticular injection of HA did not alter the volume of SF or molecular weight of HA in SF of OA canine knees, nor did it restore the HA concentration to that of normal canine SF.     

F8 gene dosage defects in atypical patients with severe haemophilia A

Summary. We performed molecular analysis of the factor 8 gene (F8) in 272 unrelated Spanish patients with haemophilia A (HA) and detected a mutation by routine analysis in 267 of them (98.1%). No mutation was detected in the remaining five patients despite clinical and laboratory confirmation of HA. The aim is to describe the molecular alterations in F8 discovered by gene dosage methodologies in three of these patients. For methodology, F8 sequencing, intragenic marker analysis, multiplex ligation-dependent probe amplification and quantitative real time-PCR were followed. One patient had Klinefelter syndrome (47,XXY) and a large deletion spanning exons 1-12 masked by the other F8 allele; the second patient showed a large duplication spanning exons 2-10 and the third patient revealed a non-contiguous double duplication of exons 14 and 23-25. The remaining two patients had mild HA and dosage results were normal. The application of gene dosage methods is useful to define haemophilic patients in whom mutations are not detected using other routine methods. Nevertheless, in a small percentage of patients (<1%), no molecular pathology can be identified after testing several genetic methodologies.     

Experiments with factor 8 separated from fibrinogen by electrophoresis in free buffer film

Approaches to an understanding of the chemistry of Factor VIII (antihaemophilic factor) have been few and necessarily indirect since Factor VIII occurs in plasma in only trace amounts, possibly less than 1 mg./l., is very labile, and the biological assay methods are subject to large errors. There have been repeated suggestions that the actual active Factor-VIII molecule might be rather small and that in plasma it is associated with another protein (Thelin and Wagner, 1961; Surgenor, 1964). Methods of purification of Factor VIII are essentially modifications of methods applicable to fibrinogen. The highest purification of Factor VIII is probably that of Michael and Tunnah (1963) but the protein of even their best preparation is at least 50 per cent fibrinogen. Methods directed towards removing fibrinogen specifically by conversion to fibrin or heating result in a loss of Factor-VIII activity. It seemed possible, therefore, that if there is an association between Factor VIII and another protein, the partner might be fibrinogen. Cross-linked gels provide the possibility of separating molecules according to their size. The degree of cross linking determines the size of molecules which cannot enter the lattice of the gel particles, and so travel fastest down a column of gel. According to the manufacturers (Pharmacia Ltd., Uppsala, Sweden) Sephadex G-200 has a structure which should totally exclude molecules having a molecular weight above 200,000, so that fibrinogen (molecular weight about 340,000) should be separable under very mild conditions from any substances having molecular weights much below 200,000. Thelin and Wagner (1961) produced evidence that in high salt concentration (0.4 M-NaCl) Factor VIII dissociated to a size comparable to that of albumin (molecular weight about 70,000). Molecules of this size should be readily and completely separable from fibrinogen on a Sephadex G-200 column unless the two had some definite bond. In experiments reported below we tried to separate bovine Factor VIII from fibrinogen in commercial bovine antihaemophilic globulin using columns of Sephadex G-200, but found the Factor-VIII activity at the front of the fibrinogen peak even when the gel filtration took place in M-NaCl or M-urea in an effort to minimize protein-protein interaction. We confirmed the observation of Lewis (1964) that Factor VIII in whole human plasma appears to be totally excluded from Sephadex G-200. Although correlation with molecular weight would not be precise if the molecule were markedly asymmetrical this probably indicates that the Factor-VIII molecule or complex has a molecular weight in excess of 200,000. These experiments do not distinguish between the possibilities that Factor VIII is itself a large molecule, or is a small molecule in association with fibrinogen. It seemed that discussion about the possible association between Factor VIII and fibrinogen could only be settled by repeating this type of molecular sieve experiment with Factor VIII prepared free from fibrinogen by some other method. Even the best preparations of Factor VIII made by methods depending on salt fractionation and chromatography still contained a large proportion of fibrinogen (Michael and Tunnah, 1963). Only electrophoretic methods seemed to offer the possibility of a complete separation of these two factors.     

Effects of the molecular weight of hyaluronic acid and its action mechanisms on experimental joint pain in rats.

OBJECTIVES--It has been shown previously that hyaluronic acid (HA) has an analgesic action on bradykinin induced pain in the knee joints of rats. This study further clarifies the effects of the molecular weight of HA and its mechanism of action in the same model using HA of molecular weight 800 to 2.3 x 10(6) daltons and a bradykinin antagonist. METHODS--Bradykinin and the test HA preparations were given to rats by intra-articular injection, and the severity of pain was evaluated by a change in the walking behaviour. RESULTS--HA with a molecular weight greater than 40 kilodaltons produces analgesic effects with a simultaneous or earlier injection. The ID50 values of HA with molecular weight 40, 310, 860, and 2300 kilodaltons were greater than 2.5, 0.6, 0.07, and 0.06 mg/joint respectively. The duration of the analgesic effect of 860 and 2300 kilodalton HA was 72 hours at 10 mg/ml, whereas that of 310 kilodalton HA was short, being undetectable after 24 hours. The analgesic action of HA of 860 kilodaltons was not changed by pretreatment with four saccharide HA and inhibited by pretreatment with HA larger than six to eight saccharides, capable of binding to HA receptors. Further, HA did not interfere with the analgesic action of the bradykinin antagonist, indicating that HA does not directly bind with bradykinin receptors. CONCLUSIONS--HA with a molecular weight of greater than 40 kilodaltons produced an analgesic effect, and HA of 860 and 2300 kilodaltons produced high and long-lasting analgesia. These effects of HA appear to be caused by the interaction between HA and HA receptors.     

Antiviral Activity and Side Effects of Polyriboinosinic-Cytidylic Acid Complexes as Affected by Molecular Size

The decrease of the molecular size of poly(I.C) to less than 10(6) decreases its ability to induce interferon, protect mice against virus, or enhance the immune response. Immune adjuvant activity appeared more sensitive to molecular weight than the other protective activities. The composition of the complex-the molecular size of the individual homopolymers when one was large and the other small-did not affect antiviral activity; the activity of a complex made from large poly(I) and small poly(C) was similar to one made from small poly(I) and large poly(C). Molecular size of the complex did not profoundly alter the side effects of poly(I.C). At 2 mg/kg, none of the complexes markedly altered phagocytic function. Only the largest complex sensitized the mouse to endotoxin. However, all of the complexes studied profoundly inhibited drug metabolism by the liver microsomal enzymes between 24 and 72 hr after their inoculation. Decreasing the molecular weight did not alter this inhibition.     

Dichotomous effect of aerosolized hyaluronan in a hamster model of endotoxin-induced lung injury

Inhalation of aerosolized low-molecular-weight (150-kDa) hyaluronan (HA) was previously shown by this laboratory to prevent experimentally induced pulmonary emphysema without associated toxicity. Nevertheless, other investigators have found that low-molecular-weight HA may be proinflammatory, prompting the authors to determine if aerosolized HA could possibly enhance pulmonary inflammation in a different model of lung injury involving intratracheal instillation of endotoxin to hamsters. Results indicate that exposure to HA following endotoxin administration significantly increased lung inflammation, whereas pretreatment with HA had the opposite effect.     

Depolymerization of synovial fluid hyaluronic acid (HA) by the complete myeloperoxidase (MPO) system may involve the formation of a HA-MPO ionic complex

Gel filtration analysis (Sephacryl S-1000) indicated that the Mr of purified equine synovial cell culture 3H-hyaluronic acid (HA) (Mr greater than 1.67 x 10(7) Da) decreased in a concentration dependent manner after exposure to hypochlorite (OCl-). Both high (equine) and medium (human, Mr = 5.5 x 10(5) Da) molecular weight HA were cleaved by the complete myeloperoxidase system (MPO/H2O2/Cl-). Purified human neutrophil myeloperoxidase (MPO) bound tightly to HA-Sepharose and we suggest that this is due to a strong ionic interaction between HA and MPO. The formation of such a complex did not disturb MPO activity. The significance of these results in relation to our previous studies concerning the reduction in viscosity and potential cleavage of HA by the product of the MPO/H2O2/Cl- system is discussed.     

Mechanical effects of the intraarticular administration of high molecular weight hyaluronic acid plus phospholipid on synovial joint lubrication and prevention of articular cartilage degeneration in experimental osteoarthritis

OBJECTIVE: To examine in vivo the effects of a mixture of high molecular weight hyaluronic acid (HA) plus phospholipids on joint lubrication and articular cartilage degeneration. METHODS: Experimental osteoarthritis (OA) of the right knee was induced by anterior cruciate and medial collateral ligament transection in 40 rabbits. The animals were subjected to 8 consecutive weekly intraarticular administrations of high molecular weight HA (the HA200 group), conventional molecular weight HA (the HA80 group), or high molecular weight HA plus L-delta dipalmitoyl phosphatidylcholine liposomes (the PHA group) and were killed 1 week after the final injection. The remaining transected right knees (the OA group) and randomly selected nontransected contralateral left knees (the control group) were collected simultaneously. Each group (n = 10) was divided into 2 equal subgroups, one of which was evaluated histologically while the other was subjected to a lubricating ability test using a pendulum friction tester. RESULTS: The injected knees had a tendency to demonstrate less damage to the articular cartilage compared with the OA group, and the histologic findings in all groups except for the PHA group differed significantly from the control group. There was a significant difference in the mean +/- SD friction coefficient between the control group (0.0100 +/- 0.00300) and the OA (0.0206 +/- 0.00649), HA200 (0.0190 +/- 0.00427), and HA80 (0.0177 +/- 0.00712) groups (P < 0.05 for each comparison), but not between the control group and the PHA group (0.0150 +/- 0.00330) (P = 0.15). CONCLUSION: To our knowledge, this is the first in vivo study to examine whether intraarticular injections of phospholipids influence joint lubrication by acting as a boundary lubricant, thus protecting articular cartilage from degenerative changes.     

Factor VIII Recombination After Dissociation by CaCl2

Factor VIII is a large protein molecule of molecular weight 2,000,000 or larger that elutes in the void volume on agarose gel chromatography. It has been shown previously that high concentrations of alkali halides and, more specifically, 0.25 M Ca(2+) dissociate the molecule into a large carrier protein and a small fragment that retains the factor VIII activity. Factor VIII was prepared from normal canine plasma collected in sodium oxalate and heparin and adsorbed with BaSO(4). Results with Ca(2+) dissociation were the same as those obtained with fraction prepared from canine plasma collected in sodium citrate. The addition of 0.1 M epsilon-aminocaproic acid in the dissociation step had no effect. Fractionation of canine hemophilic plasma produced preparations without activity, and no activity was found when these inert preparations were dissociated with Ca(2+). These results indicate that the Ca(2+) dissociation is a true dissociation and not caused by enzymatic degradation by plasmin, thrombin, or activated factors VII, IX, or X. The apparent molecular weight of the small active fragment of factor VIII determined by gel chromatography was about 100,000. Finally, when the large carrier protein and the small active fragment of factor VIII were separated by gel chromatography, mixed, and dialyzed free of Ca(2+), they recombined to form a large active molecule that appeared in the void volume on agarose gel chromatography.     

Comparison of two hyaluronan drugs in patients with advanced osteoarthritis of the knee. A prospective, randomized, double-blind study with long term follow-up

OBJECTIVES: To compare the long-term effects of high and low molecular weight hyaluronic acid (HA) applications in severe (Kellgren Lawrence stage III) osteoarthritis (OA) of the knee. METHODS: In a prospective clinical trial 184 knees (92 patients) with radiographic Kellgren Lawrence stage III OA were randomized to receive either 3 intra-articular high molecular weight HA (Hylan G-F 20) injections or 3 low molecular weight HA (Orthovisc) injections at one-week intervals. Patients were evaluated by the Hospital for Special Surgery (HSS) Knee Score and were followed-up for 12 months. RESULTS: The total HSS score in high molecular weight HA patients improved from 71.8+/-11.6 to 86.7+/-11.6 and in low molecular weight HA patients from 66.7+/-11.0 to 86.6+/-9.1 at the end of the trial (p < 0.01). There were no statistically significant differences between the groups and both had improved in all parameters at the latest follow-up (p = 0.000). CONCLUSIONS: Three intra-articular injections at intervals of 1 week of both HA preparations resulted in a pronounced reduction in pain and improved function as measured by the HSS score during a period of 52 weeks, without complications.