Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

Maturation-related differences in the structure and composition of proteoglycans synthesized by chondrocytes from bovine articular cartilage

Calf (2-3-month-old) and steer (approximately 18-month-old) bovine articular chondrocytes were isolated and cultured as high density monolayers. The proteoglycans synthesized on day 5 during a 15-h period of labeling with [35S]sulfate or [3H]glucosamine were isolated and characterized. The majority (greater than 70%) of the newly synthesized proteoglycans were found in the medium. When viewed in the electron microscope, medium-derived proteoglycans of high buoyant density were longer in calf than in steer. The medium and extracts of the cell layer were pooled and the radiolabeled proteoglycans were fractionated by isopycnic density gradient centrifugation performed under dissociative conditions. The low buoyant density fraction contained, in both calf and steer, small-sized nonaggregating proteoglycans containing chondroitin sulfate. The high buoyant density fraction contained greater than 90% of the newly synthesized proteoglycans. The majority were able to interact with hyaluronic acid to form aggregates. Calf high buoyant density fraction proteoglycans were larger, had longer chondroitin sulfate chains and lower ratios of keratan sulfate chains/chondroitin sulfate chains than steer high buoyant density fraction proteoglycans. These maturation-related differences are typical of those present in the proteoglycans of the calf and steer cartilage matrix from which the chondrocytes were isolated. Experiments with beta-D-xylosides showed that steer cultures had the capacity to synthesize twice as many chondroitin sulfate chains/cell as calf cultures. At each xyloside concentration used, chondroitin sulfate chains were longer in calf than steer. At both ages, chain size decreased with increase in rate of synthesis; the relationship between chain size and rate of synthesis was, however, quite different at the two ages. The results of these studies suggest that articular chondrocytes have an inherent program that determines the quality of proteoglycans synthesized at different ages.     

Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem. 254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically ³H-labelled by treatment with galactose oxidase followed by reduction with NaB³H₄. The ³H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and ³H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the ³H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.     

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.     

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.     

Frictional behaviour of bovine articular cartilage

The experimentally measured indentation displacement and friction of normal and degraded (treated with chondroitinase AC) bovine articular cartilage plugs against a smooth steel plate were compared with the predictions based on the biphasic theory using the finite element method. It was found that the measured indentation displacement of both cartilage specimens could be predicted from the biphasic theory and the permeability for the degraded cartilage specimen was increased approximately three times. However, the measured friction coefficient was much lower for short period of loading, and the difference in the finite element prediction of friction coefficient between the normal and degraded cartilage specimens was not observed in the experiment. Therefore, it was concluded that both biphasic and other mechanisms were important in controlling the frictional and lubricating characteristics of articular cartilage in mixed and boundary lubrication regimes.     

Studies on cartilage formation. XVIII. Changes of the composition of glycosaminoglycans in the regenerating articular surface.

The cartilaginous articular surface of the distal part of the femur of adult dogs was removed and the composition of GAGs was determined in the granulation tissue adhering to the bone wound and in that adhering to the articular capsule 7, 33, and 70 days after operation. The articular cartilage and the synovial layer of the articular capsule of intact adult dogs were also studies. The materials were digested with papain and the released GAGs were fractionated according to Svejcar and Robertson's method. The articular cartilage of non-operated dogs contained, on the average, 65.3% ChS, 13% KS, 5.8% HA and 15.8% GAG of lower molecular weight. The synovial layer of the capsule contained 41.1% HA, 15.3% Ch4-S and Ch6-S, 13.7% DS, 21.7% KS, 2% H and 6% GAG of lower molecular weight. The granulation tissue of the articular surface and that adhering to the capsule show a different developmental course. The former differentiates into cartilage, whereas the latter is simply added to the tissue of the capsule. The two tissues are different in GAG composition as early as on the 7th postoperative day. With time an increase of Ch4-S, Ch6-S and KS can be observed in the tissue of the articular surface. The tissue adhering to the capsule is characterized by a high HA and an increasing DS content. From the study of the composition of GAG's (proportion of GAG building stones) a deeper insight can be obtained into the details of GAG biosynthesis characteristic of cartilage than from the analysis of quantitative data of ChS. In the development of GAG composition characteristic of the tissue, the epimerase reactions participating in GAG biosynthesis, and the mechanisms regulating their activities seem to play a primary role.     

"Matrigenin" activity from bovine bone--II. Effects on the glycosaminoglycans of bovine articular cartilage in culture

1. Bovine articular cartilage slices were studied in long term culture by periodically pulse-labelling the cultures with radiolabelled precursors of glycosaminoglycans and isolating the glycosaminoglycans from cartilage. 2. Pretreatment of the cartilage slices with bacterial collagenase resulted in stimulation of the incorporation of radioactivity into the glycosaminoglycans. 3. The addition of a fraction from bovine bone, enriched in "matrigenin" activity, to cultures of cartilage pretreated with collagenase resulted in an additional increase in the stimulation of incorporation of radioactivity.     

Biochemical and physiochemical characterization of pepsin-solubilized type-II collagen from bovine articular cartilage.

Solubilization of collagen from bovine articular with pepsin requires the preliminary extraction of proteoglycans from the ground substance. Biochemical and physiochemical properties of this pepsin-solubilized collagen are independent of the pretreatment (extraction with 1.5M-CaCl2, 5M-guanidinium chloride or 0.2M-NaOH) and of the age range (2-4-year-old and 2-month-old animals). Characterization of the de-natured components, of the CNBr peptides and of the amino acid and cross-link composition shows that the collagen of the hyaline cartilage is all type II. Electrical birefringence measurements showed the presence of tropocollagen molecules (length 280nm) and molecules whose length is slightly less than twice that of the tropocollagen molecules. This latter molecule may be a dimer composed of two monomers linked by intermolecular head-to-tail bonds and whose theoretical length (530nm), according to the quarter-stagger theory, is in good agreement with our measured values (510-530nm). We have verified that the beta-components of this collagen are formed of two alpha-chains linked by the stable intermolecular bond, dehydrodihydroxylysinonorleucine. These dimeric molecules are absent from solutions of skin collagen whose beta-components possess only aldol-type intramolecular cross-links. Although reconstituted fibres from solutions of skin and cartilage collagen are similar, the segment-long spacing crystallites formed with pepsin-solubilized cartilage collagen present a symmetrical and dimeric form corresponding to the lateral aggregation of two monomers with an overlap (90nm) of the C-terminal ends.     

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.     

Membrane transport properties of bovine articular cartilage

The thermodynamic parameters which define transport of nonelectrolytes through bovine articular cartilage membranes were evaluated. H³HO, glucose and sucrose were used as permeants. These solutes permeate more readily through the upper layers (near the articular surface) than through the denser deeper layers approaching bone. Cartilage is similar in many respects to a swollen cellulose gel. Viscous-flow contributes importantly to transprot within cartilage thus greatly enhancing the movement of nutrients.     

Microscale frictional response of bovine articular cartilage from atomic force microscopy

The objective of this study was to compare micro- and macroscale friction coefficients of bovine articular cartilage. Microscale measurements were performed using standard atomic force microscopy (AFM) techniques, using a 5 microm spherical probe tip. Twenty-four cylindrical osteochondral plugs were harvested in pairs from adjacent positions in six fresh bovine humeral heads (4-6 months old), and divided into two groups for AFM and macroscopic friction measurements. AFM measurements of friction were observed to be time-independent, whereas macroscale measurements demonstrated the well-documented time-dependent increase from a minimum to an equilibrium value. The microscale AFM friction coefficient (mu(AFM), 0.152+/-0.079) and macroscale equilibrium friction coefficient (mu(eq), 0.138+/-0.036) exhibited no statistical differences (p=0.50), while the macroscale minimum friction coefficient (mu(min), 0.004+/-0.001) was significantly smaller than mu(eq) and mu(AFM) (p<0.0001). Variations in articular surface roughness (Rq= 462+/-216 nm) did not correlate significantly with mu(AFM), mu(eq) or mu(min). The effective compressive modulus determined from AFM indentation tests using a Hertz contact analysis was E*=45.8+/-18.8 kPa. The main finding of this study is that mu(AFM) is more representative of the macroscale equilibrium friction coefficient, which represents the frictional response in the absence of cartilage interstitial fluid pressurization. These results suggest that AFM measurements may be highly suited for exploring the role of boundary lubricants in diarthrodial joint lubrication independently of the confounding effect of fluid pressurization to provide greater insight into articular cartilage lubrication.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

Ultrasound indentation of bovine knee articular cartilage in situ

We have earlier developed a handheld ultrasound indentation instrument for the diagnosis of articular cartilage degeneration. In ultrasound indentation, cartilage is compressed with the ultrasound transducer. Tissue thickness and deformation are calculated from the A-mode ultrasound signal and the stress applied is registered with the strain gauges. In this study, the applicability of the ultrasound indentation instrument to quantify site-dependent variation in the mechano-acoustic properties of bovine knee cartilage was investigated. Osteochondral blocks (n=6 per site) were prepared from the femoral medial condyle (FMC), the lateral facet of the patello-femoral groove (LPG) and the medial tibial plateau (MTP). Cartilage stiffness (dynamic modulus, E(dyn)), as obtained with the ultrasound indentation instrument in situ, correlated highly linearly (r=0.913, p<0.01) with the values obtained using the reference material-testing device in vitro. Reproducibility (standardized coefficient of variation) of the ultrasound indentation measurements was 5.2%, 1.7% and 3.1% for E(dyn), ultrasound reflection coefficient of articular surface (R) and thickness, respectively. E(dyn) and R were site dependent (p<0.05, Kruskall-Wallis H test). E(dyn) was significantly higher (p<0.05, Kruskall-Wallis Post Hoc test) in LPG (mean+/-SD: 10.1+/-3.1MPa) than in MTP (2.9+/-1.4MPa). In FMC, E(dyn) was 4.6+/-1.3MPa. R was significantly (p<0.05) lower at MTP (2.0+/-0.7%) than at other sites (FMC: 4.2+/-0.9%; LPG: 4.4+/-0.8%). Cartilage glycosaminoglycan concentration, as quantified with the digital densitometry, correlated positively with E(dyn) (r=0.678, p<0.01) and especially with the equilibrium Young's modulus (reference device, r=0.874, p<0.01) but it was not associated with R (r=0.294, p=0.24). We conclude that manual measurements are reproducible and the instrument may be used for detection of cartilage quality in situ. Especially, combined measurement of thickness, E(dyn) and R provides valuable diagnostic information on cartilage status.     

Fibronectin fragments cause chondrolysis of bovine articular cartilage slices in culture.

Elevated fibronectin (Fn) and Fn fragment concentrations are found in the synovial fluid of osteoarthritic and rheumatoid arthritic patients. Fn has been shown to affect expression of chondrocytic matrix proteins, and Fn fragments have been shown to elevate gene expression of neutral proteinases in synoviocytes. For these reasons, we tested the effects of Fn fragments on protease release and resultant proteoglycan release from cartilage in serum-free bovine articular cartilage explant cultures. We have found that 1 microM amino-terminal 29- and 50-kDa gelatin-binding Fn fragments caused over a 50-fold enhancement of gelatinolytic and collagenolytic proteinase release with a 23-fold enhancement of proteoglycan (PG) release. Release was significant at fragment concentrations as low as 20 nM. An integrin-binding 140-kDa fragment mixture was the least active fragment, whereas native Fn had little activity. The relative activities of the fragments correlated with their relative abilities to bind to cartilage. The RGDS integrin-recognition peptide also caused release, although sequence mutants did not. PG release was blocked by actinomycin D, cycloheximide, and deoxyglucose. Fn fragment-mediated PG release was decreased in 10% serum by over 10-fold but was still 2-fold greater than in controls. In the presence of insulin-like growth factor-1, PG release was as great as without serum. We suggest that Fn fragments, as found in diseased synovial fluid, may contribute to protease-mediated damage to cartilage.     

Association of the aggrecan keratan sulfate-rich region with collagen in bovine articular cartilage

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent Kd = 1.1 microM) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the D-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.