Bovine Synovial Fluid Research Articles

The molecular structure of lubricating glycoprotein-I, the boundary lubricant for articular cartilage.

Lubricating glycoprotein-I (LGP-I) was prepared from bovine synovial fluid by density gradient sedimentation and gel-permeation chromatography. The LGP-I sample obtained was able to lubricate articular cartilage in a manner equivalent to that of whole synovial fluid. Chemical, physical, and electron microscope measurements were carried out to determine the structure of the LGP-I molecules. The molecular weight calculated from sedimentation equilibrium measurements was 2 X 10(5), and the solute distribution obtained indicated that LGP-I was relatively monodisperse. The s(0)20,w value was 4.84, and the intrinsic viscosity was 92 ml/g. The molecular weight and diffusion coefficient calculated from later light-scattering measurements was 2.06 x 10(5) and 1.10 x 10(-7) cm2/s, respectively. The electron microscope measurements showed that the LGP-I molecules had a number average length of 204 nm, a weight average length of 222 nm (with a standard deviation of 54 nm), and a width of 1-2 nm. These data and the kinked appearance of the molecules indicate that LGP-I is a partially extended flexible rod. The hydrodynamic measurements also indicate that LGP-I has the same structure in solution, although the apparently high s(0)20,w value, compared to other rod-like molecules, suggests that due to its flexibility LGP-I can occupy a more compact domain than would be expected based on its extended dimensions. The name of "lubricin" is suggested for this lubricating glycoprotein.

The lubricating activity of synovial fluid glycoproteins.

Friction measurements were performed on fractions prepared from bovine synovial fluid by using a cartilage on glass apparatus. A fraction containing lubricating glycoprotein-I (LGP-I) as the only detectable component at concentrations of 30-50 microgram/ml was able to lubricate in an identical manner to whole synovial fluid. These data indicate that LGP-I is th molecule responsible for the lubricating ability of synovial fluid. 125Iodine-labeled LGP-I also lubricated in a manner similar to synovial fluid, whereas when this sample was reduced and alkylated or treated with neuraminidase, the lubricating activity was greatly decreased. In tests to measure binding of 125I LGP-I to cartilage, an initial linear increase in binding was observed, followed by a decrease in binding at higher concentrations. In contrast, both the reduced and alkylated and the neuraminidase treated samples did not show the same concentration-dependent binding to the cartilage. It is suggested, therefore, that at least part of the lubricating ability of LGP-I is dependent upon its ability to bind to articular cartilage.

Problems of encapsulation of total joint replacements

Particulate wear debris of artificial joints, metallic and plastic, and solutions of metals in body fluids are known to have adverse effects. Encapsulation of the articulating parts would prevent wear debris from entering the surrounding tissue and check tissue ingrowth into the joint. The capsule could contain an artificial lubricant to reduce friction and wear and increase the joint life. For the capsule material, several bioelastomers are being tested in an environment of body fluids and lubricants. Three fatigue rigs have been developed for these tests. Tests have shown that a bovine synovial fluid environment causes a tenfold increase in the fatigue life of silicone rubber. A special capsule shape for minimizing stresses due to creasing is being investigated. Electrophoresis has been explored as a method of filtration of wear debris. It has been shown that Co-Cr-Mo alloy wear particles are attracted to polymers having a permanent negative surface charge.

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I, a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.

The isolation and properties of a second glycoprotein (LGP-II) from the articular lubricating fraction from bovine synovial fluid.

A high-molecular-weight glycoprotein (LGP-I) was shown [Swann, Sotman, Dixon & Brooks (1977) Biochem. J. 161, 473--485] to be the major constituent in the articular lubricating fraction from bovine synovial fluid. In addition to the LGP-I component, a second glycoprotein (LGP-II) was also present. After fractionation of bovine synovial fluid by sequential sedimentation in CsCl density gradients, the LGP-I and LGP-II components were separated by gel-permeation chromatography. The LGP-II component was then purified by chromatography on DEAE Bio-Gel A and Bio-Gel P-150. The molecular weight of the LGP-II component was 48,800 calculated from sedimentation-equilibrium measurements. Amino acids represented 53% (w/w) and carbohydrate constituents 36% (w/w) of the molecule. Glutamic acid and lysine (144 and 100 residues/1000 residues) were the major amino acids. Glucosamine, mannose, galactose and N-acetylneuraminic acid [representing 8.0, 6.6, 9.5 and 11.9% (w/w) respectively] were the only carbohydrate constituents detected. Immunodiffusion analysis showed that LGP-II component did not form a detectable precipitin line with antiserum to bovine serum. It appears likely, therefore, that this glycoprotein is synthesized by the joint tissues and is not derived from serum.

The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid.

The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.

Proteins retained with hyaluronic acid during ultrafiltration of synovial fluid.

The identity of the proteins associated with hyaluronic acid after ultrafiltration of bovine synovial fluid was examined. With discontinuous filtration, alpha2 macro- and IgM globulins were retained in hyaluronic complex both from synovial fluid and from a similar mixture of hyaluronic acid and serum. With continuous filtration and stirring to prevent formation of gels, protein was retained with hyaluronic acid in similar proportions. This protein showed immunologic identity with serum albumin, but differed in amino acid composition. After comparison with results of gel filtration, it was concluded that the identity of the retained proteins can be determined by the concentration of hyaluronic acid without variation in other ionic conditions.

Measurement of the Viscosity of Synovial Fluid

An account is given of a viscometer which can be used for measuring the viscosity of synovial fluid when only small quantities are avaliable (0.15 ml) and taking into account the non-Newtonian character of the fluid. A technical description of the apparatus is given. A series of experiments are reported in which bovine synovial fluid was used to study whether and what changes took place in connection with storage, alterations in temperature, and post mortem. It was shown that storage at 4 degrees C for up to 2 weeks did not cause any particular changes, that alterations in temperature from room temperature to body temperature resulted in a moderate decrease in the relative viscosity, and that changes found 6 hours post mortem seemed to be insignificant. The conclusion drawn from the experiments is that the apparatus can be regarded as suitable for use in a more comprehensive study of the changes in viscosity which occur in various joint diseases.

Demonstration of boundary lubrication by synovial fluid.

An account is given of an oscillatory rotating friction measurement apparatus which can be used for demonstration of boundary lubrication by synovial fluid when rubber and glass are employed as rubbing surfaces. A technical description of the apparatus is given. The greatest importance is placed on getting the apparatus to demonstrate reproducible results when saline and normal bovine synovial fluid are used as lubricant. The greatest variations are caused by differences in the cleaning of the rubber and in its tension. On the basis of bovine synovial fluid from ten different hock joints investigations were made of individual variations and changes occurring during storage at 4 degrees C. The individual variations were found to be of no importance; a decrease in the lubricating effect was observed only after storage for two months. Addition of trypsin confirmed that boundary lubrication by synovial fluid can be related to the protein component. The conclusion is that the apparatus is considered suitable for clinical investigations of boundary lubrication in connection with different joint diseases.

Lubrication Mechanism of Hip Joint Replacement Prostheses

A laboratory study has been made of the lubrication and friction characteristics of a plastic on metal (Charnley-Muller) and a metal on metal (McKee-Farrar) total hip joint replacement prostheses. The results show the effect of lubricant, speed, and load on the performance of the joints. Under a 560 lb load the coefficient of friction of the plastic to metal joint was lower than that of the metal to metal at speeds up to the equivalent of fast walking. Bovine serum and synovial fluid, as well as human serum albumin were found to be good lubricants of both types of prostheses. The frictional force produced by the metal on metal prosthesis increased linearly with load, in both the dry and lubricated states. This was not the case with the plastic to metal joint. The results show that at low physiological loads the effort required to articulate the prostheses are comparable while at higher loads the friction force of the metal to plastic is significantly lower. Friction readings taken with no lubricant indicate the serious damage which would result in the human body if a metal to metal prosthesis became devoid of liquid. The load and speed behavior of the prostheses require different interpretation of the data than presently postulated.

Model connective tissue systems. A physical study of gelatin gels containing proteoglycans.

AbstractThe swelling behavior of gelatin gels containing proteoglycans (sulphated proteoglycans from bovine intervertebral discs and a hyaluronate proteoglycan from bovine synovial fluid) when immersed in osmotically active solutions of dextran have been measured. The presence of the proteoglycans markedly affects the internal osmotic contribution to the swelling pressure of the gel. These internal osmotic pressures are considerably in excess of the sum of the osmotic activities of the individual components. This behavior is understood in terms of an entropic interaction between the gelatin and the proteoglycan molecules. By use of the “dilute solution” treatment of Flory, the osmotic pressure excesses are related to the volumes and hence dimensions of the interact acting species. A comparison of these values with those calculated by other means shows good agreement. The osmotic behavior of the complex gels can be understood on a mechanistic basis, if we regard the gelatin and sulphated proteoglycans as spheres and the hyaluronate proteoglycan as a rod.

Hyaluronic acid in synovial fluid. 3. Effect of maturation and aging on the chemical properties of bovine synovial fluid of different joints.

Hyaluronic acid in synovial fluid. 3. Effect of maturation and aging on the chemical properties of bovine synovial fluid of different joints.

Lubrication of animal joints: II The mechanism

The effect of viscosity ( Z) of several lubricants, the frequency of oscillation ( N) and loading ( P) on the coefficient of friction (μ) of excised dog ankle joints have been studied in vitro. Bovine synovial fluid reduced μ from 0·0099, obtained when saline solution was the bath fluid, to 0·0044. Viscosity per se was not an important factor inasmuch as treatment of synovia and synovia concentrate with testicular hyaluronidase, reduced Z from between 5 and 107 centipoise to approximately that of saline, but did not affect μ. The synovial lubricating factor remained in the mucin fraction which was retained by a Millipore filter having a pore dia. of 0·22 microns, and was shown to plate out onto the surface of the cartilage. Viscous purified hyaluronic acid lacked lubricating ability. These data and the relation between μ and a variation in ZN P indicate that combinations of self-pressurized hydrostatic, boundary and elastohydrodynamic mechanisms are involved in the lubrication of animal joints.

Lubrication of Animal Joints

The arthrotripsometer is an apparatus for sensing and recording the instantaneous frictional properties and deformations of articular cartilage in vitro under conditions simulating human locomotion. The frictional force measurements would have been straightforward were it not that the center of rotation of the twenty-one canine ankles studied varied throughout each oscillation. Folding of the friction-force curves about the end points of oscillation served to determine the instantaneous center of rotation and coefficient of friction. The center of rotation was determined as the mid-point between the two curves in the central portion of travel and extrapolated to the trailing end of each oscillation. At the start of each half cycle of oscillation, there occurred an abrupt transition and then a slower buildup of the coefficient of friction. These end effects are attributed to both shearing and small compressive deformations (plowing) of the articular cartilage. The plowing component of sliding friction decreased with increasing speed. The coefficient of friction decreased with increasing load. The friction was much lower in joints than in metallic or rubber bearings. When immersed in physiological saline solution, the mean sliding coefficient of friction of the ankles was .0124 (fifty-seven tests); whereas, bovine synovial fluid reduced this value by an additional 54 per cent to µ = .0057 (nineteen tests). The recovery of articular cartilage from compressive deformation was much quicker when the joint was oscillated than when it was stationary.

A rheological measurement of three synovial fluids

Using aWeissenberg Rheogoniometer, three samples of bovine synovial fluid were tested at room temperature (60 °F) at shear rates from 1.0 × 10−2 to 9.0 × 103 sec−1. There is no apparent explanation for the absence (or small value outside the instruments' range) of normal stress in ankle fluid ‘A’. No information was available as to the rearing environment of the animals. It is clear, however, that synoval fluid can exhibit a high elastic component. The shear rate to which the fluid is subjected during the normal action of the joint is probably quite as high as the maximum value used in these tests.

Purification and Properties of Bovine Synovial Fluid Alkaline Phosphatase

Abstract Alkaline phosphatase from bovine synovial fluid was purified 2300-fold. A molecular weight of 72,300 was determined from sucrose density gradient studies. The following monoesters were hydrolyzed by the enzyme: β-glycerophosphate, galactosamine 6-phosphate, glucosamine 6-phosphate, glucose 6-phosphate, o-phospho-l-serine, o-carboxyphenyl phosphate, phenyl phosphate, and p-nitrophenyl phosphate. Kinetic studies of the enzyme were made with p-nitrophenyl phosphate as substrate. Adenosine triphosphate and pyrophosphate were not hydrolyzed by the enzyme. Activation of the enzyme was observed with Sr2+, Ca2+, and Mg2+ ions. Cyanide and fluoride, as well as ethylenediaminetetraacetate, inhibited the enzyme activated by Mg2+ while the chelating agent, 1,2-bis(-2-dicarboxymethylaminoethoxy)ethane (EGTA), inhibited the enzyme activated by Ca2+. Diisopropylfluorophosphate and p-chloromercuribenzoate were virtually without effect on the activity. The pH optimum increased with increasing substrate concentration. The pH profiles were obtained for pKm, log Vmax, and log Vmax/Km. These data show the existence of two groups at the active site having pK values of 8.6 and 9.6.

Electron microscope observations on the free protein and the protein-polysaccharide complex of bovine synovial fluid.

Electron Microscope Observations on the Free Protein and the Protein-Polysaccharide Complex of Bovine Synovial Fluid

The preparation of hyaluronic acid from bovine synovial fluid

The preparation of hyaluronic acid from bovine synovial fluid

Preparation and stability of hyaluronic acid

The Roseman electrodeposition method provided a convenient method for the preparation of hyaluronic acid. Five repetitions of the process were required, and yields of about 20% were obtained. The products obtained from bovine synovial fluid appeared to be free of protein and to be only slightly degraded relative to that in the original synovial fluid. The best materials had an intrinsic viscosity in 0.2 M phosphate buffer of about 50. The preparation was somewhat improved if the solutions contained 5–10% ethanol or were saturated with toluene. Merthiolate produced degradation of purified hyaluronic acid. Degradation of hyaluronic acid on lyophilization or storage could be made very slight by lyophilization of salt-free (dialyzed) gels and storage of gels or dry products at −10°. Solutions containing 20% ethanol could be kept for long times at room temperature without change of viscosity.

Absence of Blood Clotting Substances from Synovial Fluid

SummaryAnalyses were performed on normal bovine synovial fluid for prothrombin, fibrinogen, Ac-globulin (factor V), autoprothrombin I (factor VII), tissue thromboplastin, and antithrombin. None of these active substances in blood clotting was found. The absence of these proteins, especially prothrombin (MW of 62 700), may be on the basis of selective permeability of the capillaries of the synovial membrane to certain plasma proteins.