Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression.

OBJECTIVE: We investigated the relationship between the molecular weight (MW) distribution of hyaluronan (HA) in synovial fluid (SF) and risk of knee osteoarthritis (OA) progression. METHODS: HA MW was analyzed for 65 baseline knee SFs. At 3-year follow-up, knees were scored for change in joint space narrowing (JSN), osteophyte (OST) progression, or occurrence of total knee arthroplasty (TKA). HA MW distribution was analyzed using agarose gel electrophoresis (AGE), and its relationship to OA progression was evaluated using logistic regression. The association between HA MW and self-reported baseline knee pain was analyzed using Pearson's correlation coefficients. RESULTS: Knee OA was categorized as non-progressing (OST-/JSN-, 26 knees, 40%), or progressing based on OST (OST+/JSN-, 24 knees, 37%), OST and JSN (OST+/JSN+, 7 knees, 11%) or total knee arthroplasty (TKA, 8 knees, 12%). The MW distribution of HA in baseline SFs was significantly associated with the odds of OA progression, particularly for index knees. After adjusting for age, gender, BMI, baseline X-ray grade and pain, each increase of one percentage point in %HA below 1 million significantly increased the odds of JSN (odds ratios (OR) = 1.45, 95% CI 1.02-2.07), TKA or JSN (OR = 1.24, 95%CI 1.01-1.53) and the odds of any progression (OR = 1.16, 95% CI 1.01-1.32). HA MW distribution significantly correlated with pain. CONCLUSION: These data suggest that the odds of knee OA progression increases as HA MW distribution shifts lower and highlight the value of reporting MW distribution rather than just average MW values for HA.

Tapping mode atomic force microscopy of hyaluronan: extended and intramolecularly interacting chains.

The extracellular matrix polysaccharide hyaluronan has been examined by tapping mode atomic force microscopy. High molecular weight hyaluronan was deposited on mica from dilute aqueous solution and imaged in air. Long unbranched chains could be observed and were found to be compatible with the known covalent structure of hyaluronan. In addition, chains with evidence of intramolecular association were observed. In the simplest cases, the association took the form of loops stabilized by antiparallel double-stranded (probably double-helical) segments. In other cases, the polarity of the associated regions could not be determined. Extensive intramolecular association in long hyaluronan chains resulted in a fenestrated structure of the same type as that formed by intermolecular association at higher concentrations.

Reduction of sensory responses to passive movements of inflamed knee joints by hylan, a hyaluronan derivative.

Hyaluronan (sodium hyaluronate) is a glycosaminoglycan that is present in all joint tissues. Painful arthritic joints have been characterized by hyaluronan of reduced elastoviscosity. The purpose of this investigation was to determine whether hyaluronan has an influence on joint nociceptor sensitivity and whether restoration of elastoviscosity would decrease nerve responses from nociceptive afferent fibers in arthritic joints. Nerve impulse activity was recorded from nociceptive afferent fibers of the medial articular nerve in anesthetized cats. An acute experimental arthritis was produced by intra-articular injection of kaolin and carrageenan. This caused, within 3 h, the development of ongoing nerve activity and enhancement of nerve impulse responses to passive movements in the normal range of the joint. Intra-articular injection of an elastoviscous solution of hylan, a hyaluronan derivative, significantly reduced both the ongoing activity and the movement-evoked responses in 1-2 h. This effect was not obtained when a nonelastoviscous solution of hylan was injected into the inflamed joint. The results indicate that intra-articularly injected elastoviscous solutions of hylan reduced nociceptive activity in inflamed joints through an elastoviscous, rheological effect on nociceptive afferent fibers through the intercellular matrix in which these fibers are embedded.

Hylan gel biomaterial: dermal and immunologic compatibility.

Hylan, a hyaluronan derivative, was chemically cross-linked with divinyl sulfone to produce a water-insoluble gel. This gel was fragmented into a gel slurry and evaluated for particle size, biocompatibility, and residence times in selected tissues. Hylan gels used in this study are made up of pseudoplastic, deformable gel particles with greater elasticity (at all frequencies) and greater viscosity (shear rates, 0.01 sec-1) than the water soluble hylan polymer. Hylan gel was injected intradermally and subdermally in mice and was found to produce a minimal reaction at 24 h; thereafter (up to 7 weeks) there was no significant tissue reaction. Intradermal injection of [3H]-hylan gel in guinea pigs revealed a minimal tissue reaction after 1 week, and measurement of radioactivity in the tissue at 1, 2, and 4 weeks revealed only a slight decrease in the total amount of injected radioactivity. The immunogenic activity of hylan gel was evaluated in rabbits; unmodified hylan gel, degraded hylan gel, and hylan gel ovalbumin conjugate were used to immunize rabbits. No antibody production to any hylan gel sample was detected, although control rabbits immunized with ovalbumin developed titers > 400 of antiovalbumin antibodies by day 21, as measured by the passive cutaneous anaphylaxis assay (PCA). Last, serum from owl monkeys (Aotus trivirgatus) in which hylan gel had been placed intravitreally for up to 3 years contained no detectable anti-hylan gel antibodies (PCA assay). Skin tests on these monkeys were also negative.

Viscosupplementation: a new concept in the treatment of osteoarthritis.

Viscosupplementation is a new medical concept that has as its therapeutic goal the restoration of rheological homeostasis in pathological structures such as osteoarthritic joints. When the normal viscoelasticity of a solid tissue compartment or the elastoviscosity of a liquid tissue compartment is decreased under pathological conditions, normal function and regenerative processes are impaired. By introducing viscosupplementary devices, the normal rheological state of such compartments is restored or augmented. These devices stay in the tissue compartment for various periods of time, depending on the nature of the viscosupplement and the pathophysiology of the tissue compartment.

Effect of hylan on cartilage and chondrocyte cultures.

The protective role of hylan, a hyaluronan [hyaluronic acid (HA)] derivative, was studied in explanted bovine cartilage and isolated chondrocytes. Cartilage and chondrocytes were exposed to degradative enzymes (lysate from activated polymorphonuclear leukocytes), oxygen-derived free radicals (ODFR), conditioned media from mononuclear cells (MCCM), and interleukin-1 (IL-1), in the presence and absence of hylan. The effect of HA was also studied. In cartilage explants susceptibility to pertubation was evaluated in terms of 35S release and proteoglycan depletion and was compared to control cultures; high viscosity hylan was found to reduce 35S release in cartilage explants caused by degradative enzymes, ODFR, MCCM, and IL-1. The hylan effect was reversible and viscosity-dependent. In chondrocyte cultures, high viscosity hylan was effective in reducing cell injury caused by degradative enzymes and ODFR. The data suggest that the glycosaminoglycan hylan, as well as native HA, may mediate exposure to and/or response to stimuli associated with initiation of degenerative processes in cartilage tissues.

Hylan gel composition for percutaneous embolization.

Viscoelastic, pseudoplastic, radiopaque injectable hylan gel for percutaneous embolization was developed and evaluated by in vitro and in vivo tests. The embolization gel is composed of cross-linked hylan (hyaluronan, hyaluronate), tantalum, microcrystalline cellulose, hexamethonium chloride, and thrombin. Upon delivery through small-lumen catheters to the appropriate vascular site, the gel induces formation of a solid blood/gel coagulum. Results from animal studies (rat aorta, rabbit auricular artery) demonstrate that formation of complete and long-lasting arterial blockage is readily achievable without complications due to blood flow, partial vessel obstruction, uncontrolled polymerization, or movement of the gel or its components (specifically thrombin and hexamethonium chloride) into the circulation. Microscopic evaluation indicates that arterial occlusion initially occurs as a result of the injected gel and formed fibrin; at 7 weeks and beyond, arteries are occluded by injected gel, inflammatory cells and fibrosis (scar tissue).

Matrix engineering.

Matrix engineering is a technology that utilizes hyaluronan (HA, hyaluronic acid) based matrices to control, direct or augment tissue regenerative processes. Hyaluronan and the concept of matrix engineering have become established tools in ophthalmic and orthopaedic medicine. The clinical indications for HA are limited by the physical properties and short residence time of the natural HA molecule. To expand and improve upon its current medical applications, a family of HA derivatives was prepared by chemical modification and cross-linking. Relative to the non-modified HA molecule, the hylan family of polymers provides more versatile physical forms, improved mechanical properties and an extended residence time. Hylan can also be used as a surface coating to improve blood compatibility. The chemical, physical and biological properties of hylans will be reviewed, focusing on the specific therapeutic indications they enable.

Preliminary studies on the use of a viscoelastic solution in arthroscopic surgery of the temporomandibular joint.

Arthroscopic surgery of the temporomandibular joint includes the potential for iatrogenic damage of intracapsular structures during introduction of instruments and manipulation of the tissues. A modification of an elastoviscous solution of crosslinked sodium hyaluronate, called hylan fluid, was used for irrigation during surgery in 55 temporomandibular joints. Forty-nine of the joints were monitored postoperatively in a study to measure safety and efficacy of the material during the arthroscopic procedure. The hylan fluid was found to be as safe as the standard irrigating fluid. The hylan fluid also significantly protected the joint surfaces and facilitated the surgical procedure.

Morphology and ultrastructure of human vitreous fibers.

Significant alterations in vitreous structure occur with aging and disease. There is controversy as to the nature of the normal structure of the vitreous and no studies have correlated macroscopic structure with ultrastructure in the same eyes. Twenty-eight fresh, untreated human eyes were examined after removal of the sclera, choroid and retina. Dark-field slit illumination of the whole vitreous revealed the presence of macroscopic fibrous structures. The fibers had an antero-posterior orientation with anterior insertions at the vitreous base and posterior insertions in the premacular vitreous cortex. Transmission electron microscopy demonstrated the presence of collagen fibrils and no membraneous structures. Parallel collagen fibrils packed in bundles were also detected. Macroscopic vitreous fibers most likely result from alteration of the hyaluronic acid-collagen complex with aggregation of collagen fibrils into bundles as seen on electron microscopy. Identifying the mechanisms underlying this process of fiber formation could clarify the pathogenesis of vitreous liquefaction and the pathophysiology of posterior vitreous detachment.

Clinical uses of hyaluronan.

The availability of elastoviscous solutions of highly purified hyaluronan has created two new therapeutic methods in human and veterinary medicine: viscosurgery and viscosupplementation. Viscosurgical tools and implants are widely used in ophthalmology and have been suggested for use in otology. Visco-supplementation of joint fluid using elastoviscous hyaluronan solutions is widely used in the treatment of equine traumatic arthritis. It was also suggested for use in idiopathic osteoarthritis in humans, but this application has not received wide acceptance. Cross-linked forms of hyaluronan have been developed and given the generic name of hylans. Water-insoluble soft gels of hylans are ideally suitable as viscosurgical implants to prevent postoperative adhesions and to control scar formation. Hylan solutions are being used in arthroscopic viscosurgery. Hylan devices in various forms (gels, tubes, membranes) have been used in animal studies for matrix engineering, the purpose of which is to control and direct tissue regeneration and augmentation.

The role of the vitreous in the intraocular pressure rise after neodymium-YAG laser capsulotomy.

Autologous liquid vitreous injected into the anterior chamber of the phakic owl monkey eye leads to markedly increased intraocular pressure (IOP) peaking at one to two hours. In contrast, neodymium-YAG laser shock waves focused in the center of the anterior chamber of the same animal led to a mild decrease in IOP. Debris produced by laser pulses focused on the residual cortex of owl monkey eyes that had undergone extracapsular surgery failed to increase the IOP. Similarly, injection of dialyzed vitreous did not have any significant influence on IOP. We conclude that the disruption of the integrity of the anterior cortical gel and the subsequent release of a dialyzable intravitreal substance with a molecular weight of less than 10,000 daltons into the anterior chamber may contribute to the IOP rise after surgical discission, including neodymium-YAG laser posterior capsulotomy.

Human vitreous fibres and vitreoretinal disease.

Adult human vitreous structure was studied by dark-field Slit Microscopy in fresh, unfixed preparations of vitreous dissected off the sclera, choroid and retina. Macroscopic fibres oriented in an antero-posterior direction were detected. These fibres inserted at the vitreous base and oriented towards the macula. Significant alterations were observed in this vitreous structure with advanced age. The role of these structures in vitreo-retinal disease is discussed.

Exogenous Na-hyaluronate in the anterior chamber of the owl monkey and its effect on the intraocular pressure.

Exogenous, ultrapure (sterile, pyrogen-free), non-inflammatory fraction of Na-hyaluronate (NIF-NaHA) was introduced into the anterior chamber of owl monkeys (Aotus trivirgatus), replacing approximately 48% or 77% of the aqueous humor and creating post-injection intraocular pressures (IOPs) below normal (5-10 mmHg) or above normal (40-60 mmHg), respectively. Five different molecular weight samples (MW 1.7, 3.4, 3.7, 4.5 and 4.9 X 10(6)) were used. All solutions contained 1% NIF-NaHA and, because of the varying molecular weights, the viscosities of the solutions ranged between 10 000 and 930 000 cSt. The IOP and the rate of export of the exogenous NIF-NaHA from the anterior chamber were measured. All solutions caused an increase in the IOP, and the maximum level occurred at 4 hr after injection. In all cases, the IOP returned to normal 24 hr after injection. The highest and most persistent increase in IOP was observed after the injection of the solution with the lowest viscosity (10 000 cSt). The smallest increases in IOP over the post-operative value were observed after replacement of the aqueous humor using those samples with viscosities of 10 0000 to 300 000 cSt. The turnover (export rate) of injected NIF-NaHA depends for the most part on the viscosity of the injected solution. With increasing viscosity the rate constant, and therefore the half-life, of the injected NIF-NaHA decreases. The volume fraction of the viscous solution replacing the aqueous humor is also a determining factor in establishing the turnover rate. The molecular weight of the injected NIF-NaHA did not change during that time (48 hr) in which a sufficient amount of sample for analysis could be obtained. No evidence was found for the presence of any kind of hyaluronic acid-degrading agent in the anterior chamber.

Pathogenesis of cystoid macular edema: an anatomic consideration of vitreoretinal adhesions.

Although it had been widely accepted that vitreous traction plays a key role in the pathogenesis of aphakic cystoid macular edema, vitreoretinal traction is not observable in many patients and other hypotheses have also been investigated. The authors review the present knowledge of the anatomy of the vitreoretinal interface and the pathologic findings in cystoid macular edema. A new hypothesis combining the effects of both traction and inflammation is proposed.

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment.

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.

Isolation and characterization of ichthyosan from tuna vitreous.

Ichthyosan has been prepared from tuna vitreous. Glucuronic acid was found to account for the total uronic acid content of the macromolecule, while the hexosamine content was a mixture of N-acetyl-glucosamine and N-acetyl-galactosamine. When ichthyosan was gel filtered on Sepharose 2B or Sephacryl S-300, using sodium or calcium chloride, the elution profile of the column gave only one peak indicating no separation between glucosamine and galactosamine containing fractions. Similar results were obtained when ichthyosan was chromatographed on DEAE-cellulose using a salt gradient both in the presence and absence of 7.0 M urea. When ichthyosan was gel filtered in 4.0 M guanidine-HCL and subsequently chromatographed on DEAE-Sephacel or DEAE-cellulose, three well separated fractions were present. The two major fractions (II and III) were characterized as chondroitin and hyaluronic acid respectively; while fraction I representing about 3-5% of the total polysaccharide content of ichthyosan was identified as a keratan-like molecule. The same pattern was obtained when ichthyosan was digested with proteolytic enzymes and subsequently chromatographed on DEAE-cellulose or DEAE-Sephacel. Based on these findings it is concluded that in ichthyosan chondroitin, hyaluronate and keratan-like molecular chains are bound to proteins in non-covalent linkages.