Passive loss of proteoglycan from articular cartilage explants

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.     

Electron microscopic studies of proteoglycan aggregates from bovine articular cartilage.

Proteoglycan aggregates from bovine articular cartilage have been visualized by electron microscopy of mixed proteoglycan-cytochrome c monolayers. The proteoglycan aggregates consist of proteoglycan subunits arising laterally at fairly regular intervals (20 to 30 nm) from the opposite sides of an elongated filamentous structure. The filamentous backbone in individual aggregates varies in length from 400 to 4000 nm. The individual proteoglycan subunits in the aggregate vary in length from 100 to 400 nm. However, there is no difference in the average size of the proteoglycan subunits associated with the largest or smallest aggregates. The sizes of the individual aggregates are determined mainly by the lengths of their filamentous backbones. The stoichiometry of binding of subunits to filament, calculated from the data reported here, is close to that for the binding of subunits to hyaluronic acid reported by others.     

A role for the interleukin-1 receptor in the pathway linking static mechanical compression to decreased proteoglycan synthesis in surface articular cartilage

Loading of articular cartilage during weight bearing is essential for the maintenance of cartilage function. Although certain cyclic loading protocols stimulate extracellular matrix synthesis, constant or static compression decreases proteoglycan and collagen synthesis in cartilage explants. The goal of this study was to determine whether the compression-induced decrease in proteoglycan synthesis involves an interleukin-1 (IL-1) signaling pathway. Cartilage explants were compressed 50% in the presence of IL-1 receptor antagonist (IL-1ra), and the incorporation of [35S]sulfate into macromolecules was measured. IL-1ra increased sulfate incorporation in compressed cartilage but not in cartilage maintained at the in situ thickness (0% compression). IL-1alpha and IL-1beta mRNAs were detected in cartilage compressed 50% for at least 3h, while nitric oxide synthase II mRNA was only detected in cartilage compressed 50% for 6h. The data support a role for the IL-1 receptor in the pathway linking static compression to reduced proteoglycan synthesis.     

Static compression is associated with decreased diffusivity of dextrans in cartilage explants

The chondrocytes of adult articular cartilage rely upon transport phenomena within their avascular extracellular matrix for many biological activities. Therefore, changes in matrix structure which influence cytokine transport parameters may be an important mechanism involved in the chondrocyte response to tissue compression. With this hypothesis in mind, partitioning and diffusion of 3-, 10-, and 40-kDa dextrans conjugated to tetramethylrhodamine, and 430-Da tetramethylrhodamine itself, were measured within statically compressed bovine articular cartilage explants using a novel experimental apparatus and desorption fluorescence method. Partitioning and diffusion were examined as functions of solute molecular weight and matrix proteoglycan density, and diffusion was measured versus static compression up to 35% volumetric strain. In general, partition coefficients and diffusivities were found to decrease with increasing solute molecular weight. In addition, for a given solute, diffusivities decreased significantly with increasing static compression. Results therefore suggest a possible role for transport limitations of relatively large molecular weight solutes within the extracellular matrix in mediating the biological response of chondrocytes to cartilage compression.     

Direct quantification of the rupture force of single hyaluronan/hyaluronan binding protein bonds

The non-covalent bond between aggrecan and hyaluronan is critical for maintaining the normal structure and function of the extracellular matrix in articular cartilage. The failure of this bond can cause the loss of aggrecan and destruction of the extracellular matrix of articular cartilage. In this study, the rupture force of the single bond between hyaluronan and hyaluronan binding protein - the complex of the hyaluronan binding region of aggrecan and link protein - was directly measured with a nanomechanical testing system as 40+/-11 pN. The results were compared to a theoretical prediction based on a smart version of the Monte Carlo simulation.     

Interleukin-1-induced alterations in proteoglycan metabolism and matrix assembly

In cartilage, the large chondroitin sulfate proteoglycan exists as aggregates by interacting with link protein and hyaluronic acid. In diseases associated with cartilage degeneration, the proteoglycan does not aggregate because of a defect in the hyaluronate-binding activity. Since interleukin-1 (IL-1) is a secretory product of activated macrophages and may influence the cartilage function in joints, we studied the effects of IL-1 on the synthesis and assembly of proteoglycan by rabbit articular chondrocytes in culture. IL-1-treated cells showed a modest increase in the total proteoglycan synthesis, but also showed a more pronounced decrease in the incorporation of extracellular matrix. Affinity chromatography of the conditioned media on hyaluronic acid-Sepharose revealed that all of the proteoglycan of control cells strongly bound to hyaluronate. The IL-1-treated medium contained two fractions: one that was strongly bound to the column and a second that did not bind. The results demonstrate that the IL-1-treated cells cannot incorporate proteoglycan into the matrix partly because of a defect in the proteoglycan molecules and partly due to other mechanisms regulating proteoglycan assembly.     

A method for testing compressive properties of single proteoglycan aggregates

This study presents a method for direct measurement of the compressive properties of single molecules of proteoglycan aggregate using a state-of-the-art laser tweezers/interferometer system previously developed to test the tensile properties of single molecules. A typical molecule of proteoglycan aggregate showed a highly non-linear resistance to compression after being compressed to about 25% of its original molecule length.     

Identification of a hyaluronic acid-binding protein that interferes with the preparation of high-buoyant-density proteoglycan aggregates from adult human articular cartilage.

Adult human articular cartilage contains a hyaluronic acid-binding protein of Mr 60 000-75 000, which contains disulphide bonds essential for this interaction. The molecule can compete with proteoglycan subunits for binding sites on hyaluronic acid, and can also displace proteoglycan subunits from hyaluronic acid if their interaction is not stabilized by the presence of link proteins. The abundance of this protein in the adult accounts for the reported inability to prepare high-buoyant-density proteoglycan aggregates from extracts of adult human cartilage [Roughley, White, Poole & Mort (1984) Biochem. J. 221, 637-644], whereas the deficiency of the protein in newborn human cartilage allows the normal recovery of proteoglycan aggregates from this tissue. The protein shares many common features with a hyaluronic acid-binding region derived by proteolytic treatment of a proteoglycan aggregate preparation, and this may also represent its origin in the cartilage, with its production increasing during tissue maturation.     

The influence of link protein stabilization on the viscometric properties of proteoglycan aggregate solutions

The dynamic, steady-shear and transient shear flow properties of precisely prepared link-stable (s0 136, 66% aggregate) and link-free (s0 93, 59% aggregate) proteoglycan aggregate solutions at concentrations ranging from 10 to 50 mg/ml were determined using a cone-on-plate viscometer in a mechanical spectrometer. All proteoglycan solutions tested possessed: (1) linear viscoelastic properties - as measured by the dynamic complex modulus under small amplitude steady oscillatory conditions (1 less than or equal to omega less than or equal to 100 rad/s) - and (2) nonlinear shear-rate dependent apparent viscosities and primary normal stress difference under steady shearing conditions (0.25 less than or equal to gamma less than or equal to 250 s-1). Our transient flow data show that all proteoglycan aggregate solutions exhibited transient stress overshoot effects in shear stress and normal stress. From these steady and transient flow data, we conclude that link protein stabilized aggregates have significant effects on their dynamic and steady-shear properties as well as transient flow properties. The transient stress overshoot data provide a measure of the energy per unit volume of fluid required to overcome the proteoglycan networks in solution from a resting state. Thus we found that link-stable aggregates form much stronger networks than link-free aggregates. This is corroborated by the fact that link-stable aggregates form more elastic (lower than delta) and stiffer (higher [G*]) networks than link-free aggregates. The complete spectrum of viscometric flow data is entirely compatible with the proposed role of link protein in adding structural stability to the proteoglycan-hyaluronate bond. In cartilage, the enhanced strength of the networks formed by link-stable aggregates may play an important role in determining the material properties of the tissue and thereby contribute to the functional capacity of cartilage in diarthrodial joints.     

How changes in interconnectivity affect the bulk properties of articular cartilage: a fibre network study

The remarkable compressive strength of articular cartilage arises from the mechanical interactions between the tension-resisting collagen fibrils and swelling proteoglycan proteins within the tissue. These interactions are facilitated by a significant level of interconnectivity between neighbouring collagen fibrils within the extracellular matrix. A reduction in interconnectivity is suspected to occur during the early stages of osteoarthritic degeneration. However, the relative contribution of these interconnections towards the bulk mechanical properties of articular cartilage has remained an open question. In this study, we present a simple 2D fibre network model which explicitly represents the microstructure of articular cartilage as collection of discrete nodes and linear springs. The transverse stiffness and swelling properties of this fibre network are studied, and a semi-analytic relationship which relates these two macroscopic properties via microscopic interconnectivity is derived. By comparing this derived expression to previously published experimental data, we show that although a reduction in network interconnectivity accounts for some of the observed changes in the mechanical properties of articular cartilage as degeneration occurs, a decrease in matrix interconnectivity alone do not provide a full account of this process.     

Elastic anisotropy of articular cartilage is associated with the microstructures of collagen fibers and chondrocytes

Chondrocyte shape and volumetric concentration change as a function of depth in articular cartilage. A given chondrocyte shape produces different effects on the global material properties depending on the structure of the collagen fiber network. The shape and volumetric concentration of chondrocytes in articular cartilage appear to be related to the mechanical stability of the matrix. The present study was aimed to investigate, theoretically, the effects of the structural arrangement of the collagen fiber network, and the shape and distribution of chondrocytes, on the global material behavior of articular cartilage. Articular cartilage was assumed to be a four-phasic composite comprised of a matrix (associated with the properties of the proteoglycan structure), vertically and horizontally distributed collagen fibers, and spheroidal inclusions representing chondrocytes. A solution for composite materials was used to estimate the global, effective material properties of cartilage. Only the elasticity of the solid phase was investigated in the present study. Our simulations suggest that a soft, spheroidal cell inclusion in a fiber-reinforced proteoglycan matrix affects the material properties differently depending on the shape of the spheroidal inclusions. If the long axis of the inclusions is parallel to the collagen fibers, as in the deep zone, the soft inclusions increase the stiffness of the composite in the fiber direction, and reduce the stiffness of the composite in the direction normal to the fibers. Furthermore, we found that Young's modulus normal to the contact surface increases from the superficial to the deep zone in articular cartilage by a factor of 10-50, a finding that agrees well with experimental observations. Our analysis suggests that the combination of proteoglycan matrix, fiber orientation, and shape of chondrocytes are intimately related and are likely adapted to optimize the mechanical stability and load carrying capacity of the structure.     

Tensorial electrokinetics in articular cartilage

Electrokinetic phenomena contribute to biomechanical functions of articular cartilage and underlie promising methods for early detection of osteoarthritic lesions. Although some transport properties, such as hydraulic permeability, are known to become anisotropic with compression, the direction-dependence of cartilage electrokinetic properties remains unknown. Electroosmosis experiments were therefore performed on adult bovine articular cartilage samples, whereby fluid flows were driven by electric currents in directions parallel and perpendicular to the articular surface of statically compressed explants. Magnitudes of electrokinetic coefficients decreased slightly with compression (from approximately -7.5 microL/As in the range of 0-20% compression to -6.0 microL/As in the 35-50% range) consistent with predictions of microstructure-based models of cartilage material properties. However, no significant dependence on direction of the electrokinetic coupling coefficient was detected, even for conditions where the hydraulic permeability tensor is known to be anisotropic. This contrast may also be interpreted using microstructure-based models, and provides insights into structure-function relationships in cartilage extracellular matrix and physical mediators of cell responses to tissue compression. Findings support the use of relatively simple isotropic modeling approaches for electrokinetic phenomena in cartilage and related materials, and indicate that measurement of electrokinetic properties may provide particularly robust means for clinical evaluation of cartilage matrix integrity.     

An in situ calibration of an ultrasound transducer: a potential application for an ultrasonic indentation test of articular cartilage

A change in mechanical properties of articular cartilage would be considered one of the most reliable signs of cartilage degeneration. While an indentation method has the potential to measure the cartilage properties in vivo, an accurate measurement of cartilage thickness in situ is technically difficult. An ultrasound transducer has often been used to measure the cartilage thickness. However, its accuracy is limited by the lack of an accurate measurement of the ultrasound speed of cartilage, for the ultrasound speed varies according to the pathological conditions of the tissue. Therefore, the objective of this study is to develop an in situ calibration method of predicting the true ultrasound speed of cartilage and thus allow the ultrasound transducer to measure the thickness of the tissue with great accuracy. By simultaneously implementing an indentation testing protocol using the ultrasound transducer as an indenter, this method can also provide an indentation stiffness measurement of cartilage.The feasibility of the proposed method was examined using normal and proteoglycan-depleted cartilage specimens. It was found that the true ultrasound speed measured by the in situ calibration method was sensitive to the proteoglycan depletion (1735+/-35 m/s for normal, and 1598+/-28 m/s for proteoglycan-depleted cartilage), and that the measured cartilage thickness was consistently accurate regardless of the tissue condition. The measured indentation stiffness of articular cartilage was also sensitive to the tissue condition. Thus, this study demonstrates that the proposed ultrasonic indentation technique can be used to accurately identify the abnormality of articular cartilage in situ.     

Distribution of chondroitin sulfate in cartilage proteoglycans under associative conditions.

Proteoglycan aggregates and proteoglycan subunits were extracted from bovine articular cartilage with guanidine-HC1 folowed by fractionation by equilibrium centrifugation in cesium chloride density gradients. The distribution of chondroitin sulfates (CS) in the cartilage proteoglycans was studied at the disaccharide level by digestion with chondroitinases. In the proteoglycan aggregate fraction, it was observed that the proportion of 4-sulfated disaccharide units to total CS increased from the bottom to the top fractions, whereas that of 6-sulfated disaccharide units was in the reverse order. Thus, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased significantly with decreasing density. The proportion of non-sulfated disaccharide units to total CS tended to increase with increasing density. These data indicate a polydisperse distribution of CS chains, under the conditions used here, in proteoglycan aggregates from bovine articular cartilage.     

Proteoglycans from bovine nasal and articular cartilages. Fractionation of the link proteins by wheat germ agglutinin affinity chromatography

Two forms of link protein, 46 and 51 kDa, are present in proteoglycan aggregates from both bovine nasal and bovine articular cartilages. Studies reported here show that the link proteins bind to concanavalin A, Lens culinaris agglutinin, Ricinus communis agglutinin, soybean agglutinin, and wheat germ agglutinin lectins. When the link proteins are eluted from these lectins with appropriate competing sugars, the 46- and the 51-kDa link proteins elute together and no separation is achieved. However, when the link proteins bound to wheat germ agglutinin are eluted with a 0 to 4 M guanidine hydrochloride linear gradient, a good separation of the 46- and 51-kDa link proteins is achieved. Wheat germ agglutinin affinity chromatography has been used on a preparative scale to isolate the 51-kDa link protein from mature bovine articular cartilage to homogeneity, in amounts sufficient to examine its effect on proteoglycan aggregate size and stability in sedimentation velocity studies. Proteoglycan aggregates were reassembled from proteoglycan monomers and hyaluronate in the absence of link protein, in the presence of both 46- and 51-kDa link proteins, and in the presence of the individual 51-kDa link protein. The sizes of the aggregates were compared in terms of sedimentation coefficients (s(0)20). The stability of the aggregates was compared in terms of the per cent aggregate present at pH 7 and 5. At pH 7, the sedimentation coefficients (s(0)20) of link-free aggregates, aggregates formed with both link proteins, and aggregates formed with 51-kDa link protein were 72, 93, and 112 S, respectively. Thus, the 51-kDa link protein has a pronounced effect on aggregate size. The link-free aggregate was grossly unstable, and only 36% aggregate was present at pH 5. The aggregate formed with both link proteins was effectively stabilized against dissociation and 79% aggregate was present at pH 5. The aggregate formed with 51-kDa link protein was not effectively stabilized against dissociation, and only 60% aggregate was present at pH 5. Thus, despite its pronounced effect on aggregate size, the 51-kDa link protein does not effectively stabilize the proteoglycan aggregate against dissociation. These results suggest that the 51-kDa link protein may selectively increase aggregate size, while the 46-kDa link protein may be required to effectively stabilize the proteoglycan aggregate against dissociation.     

Extraction of mechanical properties of articular cartilage from osmotic swelling behavior monitored using high frequency ultrasound

Articular cartilage is a biological weight-bearing tissue covering the bony ends of articulating joints. Negatively charged proteoglycan (PG) in articular cartilage is one of the main factors that govern its compressive mechanical behavior and swelling phenomenon. PG is nonuniformly distributed throughout the depth direction, and its amount or distribution may change in the degenerated articular cartilage such as osteoarthritis. In this paper, we used a 50 MHz ultrasound system to study the depth-dependent strain of articular cartilage under the osmotic loading induced by the decrease of the bathing saline concentration. The swelling-induced strains under the osmotic loading were used to determine the layered material properties of articular cartilage based on a triphasic model of the free-swelling. Fourteen cylindrical cartilage-bone samples prepared from fresh normal bovine patellae were tested in situ in this study. A layered triphasic model was proposed to describe the depth distribution of the swelling strain for the cartilage and to determine its aggregate modulus H(a) at two different layers, within which H(a) was assumed to be linearly dependent on the depth. The results showed that H(a) was 3.0+/-3.2, 7.0+/-7.4, 24.5+/-11.1 MPa at the cartilage surface, layer interface, and deep region, respectively. They are significantly different (p<0.01). The layer interface located at 70%+/-20% of the overall thickness from the uncalcified-calcified cartilage interface. Parametric analysis demonstrated that the depth-dependent distribution of the water fraction had a significant effect on the modeling results but not the fixed charge density. This study showed that high-frequency ultrasound measurement together with triphasic modeling is practical for quantifying the layered mechanical properties of articular cartilage nondestructively and has the potential for providing useful information for the detection of the early signs of osteoarthritis.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.

A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.