The application of an indenter system to measure structural properties of articular cartilage in the horse. Suitability of the instrument and correlation with biochemical data.

A recently developed indenter system that aims at determination of local structural properties of the cartilage surface was evaluated for suitability in the horse. To this aim, maximum indenter force was measured of the articular surface and related to biochemical characteristics of the cartilage at different sites of the distal metacarpal bone (MC). Significant topographical variation exists in structural properties of the articular surface of the MC. The dorsal margin showed a significantly lower maximum indenter force than more centrally located areas, indicating an increased compliance under load. A high maximum indenter force correlated positively with high glycosaminoglycan levels. No correlation was found between measured indenter force and collagen content of the cartilage. It was concluded that the instrument is able to quantify differences in structural properties of the cartilage surface which reflect glycosaminoglycan content and hence is capable of giving some indication of cartilage quality. The instrument is a potentially useful tool, but use is limited due to geometrical constraints.

Use of recombinant human osteogenic protein-1 for the repair of subchondral defects in articular cartilage in goats.

The objective of this pilot study was to examine in vivo the potential of recombinant human osteogenic protein-1 (rhOP-1, also called bone morphogenetic protein-7, BMP-7) for treatment of subchondral lesions by induction of new hyaline cartilage formation. Subchondral left knee defects in 17 mature goats were treated with fresh coagulated blood mixed with (1) rhOP-1 combined with collagen (OP-1 device, 400 microgram/mL); (2) rhOP-1 alone (OP-1 peptide, 200 microgram/mL); (3) OP-1 device with small particles of autologous ear perichondrium; (4) OP-1 peptide with small particles of autologous ear perichondrium; or (5) autologous ear perichondrium alone (controls). rhOP-1 was combined with either collagen (OP-1 device) or not (OP-1 peptide). The defects were closed with a periosteal flap. The formation of cartilage tissue was studied by histologic and biochemical evaluation at 1, 2, and 4 months after implantation. One and 2 months after implantation there were no obvious differences between control and rhOP-1-treated defects. Four months after implantation, only one out of three controls (without rhOP-1) showed beginning signs of cartilage formation while all four rhOP-1-treated defects were completely or partly filled with cartilage. A significant linear relationship was found between rhOP-1 concentration and the total amount of aggrecan in the defects. These results suggest that implantation of rhOP-1 promotes cartilage formation in subchondral defects in goats at 4 months after implantation. Therefore, rhOP-1 could be a novel factor for regeneration of cartilage in articular cartilage defects.

Increase of decorin content in articular cartilage following running.

The effect of long distance running exercise (40 km/day for 15 weeks, five days a week) on the decorin content of articular cartilage from the knee joint was studied in female beagle dogs. Samples from load bearing sites on the lateral plateau of the tibia (TL), and pooled material from two minimum load bearing sites on the posterior section of lateral (FLP) and medial (FMP) femoral condyles were analyzed. The running exercise protocol did not lead to significant changes in the overall glycosaminoglycan content of the cartilage. However, the amount of decorin significantly increased in the TL samples, and also in the FMP pool. These results support earlier in vitro observations that decorin synthesis is stimulated by loading, independent of the synthesis of aggrecan.

Cyclic loading is harmful to articular cartilage from which proteoglycans have been partially depleted by retinoic acid.

We studied whether cyclic loading is harmful to degraded cartilage. Sets of four cartilage-bearing sesamoid bones were dissected from 5-year old cows. One bone from each set was cultured for 17 h in control medium to serve as an ex vivo control. The three others were cultured for 1 week in control medium to which 0, 10 or 300 ng/mL retinoic acid (RAc), which depletes the cartilage matrix of proteoglycans, had been added. Two were then cultured for another week in control medium. During the last week, one of the two was subjected to a cyclic load (1 MPa, 0.2 Hz). Following treatment with RAc, glycosaminoglycan content and synthesis were significantly decreased, as confirmed by safranin O staining and autoradiography. They were further diminished by loading during the second week of culture. Increased amounts of 3-B-3(-)epitope were found in cartilage that had been treated with 300 ng/mL RAc and then loaded. While loading cartilage matrix that was only slightly degraded proved to be damaging, loading severely degraded cartilage matrix apparently induced osteoarthritic-like changes.

Irreversible changes in glycosaminoglycan composition of anatomically intact bovine articular cartilage induced by intermittent loading.

The changes in matrix composition induced by I MPa intermittent (0.2 Hz) loading of anatomically intact bovine articular cartilage in vitro are studied. The kinetics of chondrocyte response was determined in experiments where sesamoid bones of adult cows were loaded for 0, 3, 5 and 7 days. The reversibility of the induced changes were studied in sesamoid bones that were loaded for 5 days and subsequently cultured without loading for another 1, 2 and 3 weeks. Water content was not affected by loading, nor by subsequent unloaded culture. Glycosaminoglycan content was not affected by loading, nor by subsequent unloaded culture for another 2 weeks. However, after 3 weeks of culture following 5 days of loading, a significant decrease in glycosaminoglycan content was found. Glycosaminoglycan synthesis was already increased after 3 days of loading. Loading for longer periods (5 and 7 days) further increased the glycosaminoglycan synthesis rate. Glycosaminoglycan synthesis decreased during the first week of subsequent culture without loading. In the third week it dropped to a lower level than observed for the control. The amount of 3-B-3(-) epitope was increased and the length of newly synthesized glycosaminoglycans was decreased by intermittent loading. Subsequent unloaded culture did not further increase the amount of 3-B-3(-) epitope. However, after 3 weeks of unloaded culture the newly synthesized glycosaminoglycans were as long as those synthesized in the control. The results suggest that the changes in glycosaminoglycan chain length were reversible. However, the overall chondrocyte response to our loading regime seems to be detrimental to the tissue: expression of 3-B-3(-) epitope induced by loading together with the drop in glycosaminoglycan content and synthesis observed at the end of the total culture period indicate that the cartilage is irreversibly damaged.

Transforming growth factor-beta stimulates retinoic acid-induced proteoglycan depletion in intact articular cartilage.

Cartilage-bearing sesamoid bones from the metacarpophalangeal joints of adult cows were cultured with retinoic acid for 1 week and allowed to recover in control medium for another 2 weeks. Retinoic acid decreased the proteoglycan synthesis of the cartilage to 33% of control values, and induced 26% loss of proteoglycans from the matrix. During recovery, the synthesis of proteoglycans returned to the control level but their content remained reduced. Transforming growth factor-beta (TGF-beta 1, 5 ng/ml) was added to the culture medium to stimulate the recovery. However, TGF-beta depressed the synthesis of proteoglycans and increased their loss to 61%. Only the large aggregating species, aggrecan, was lost from the matrix. The half-life of proteoglycans synthesized during recovery in control medium was 12.7 days, which was reduced to 8.7 days by TGF-beta. The proteoglycan half-life in control cartilage cultured without retinoic acid or TGF-beta was 33.8 days. Neither retinoic acid nor TGF-beta-induced changes in the hyaluronate content of the tissue. Aggrecans and small proteoglycans synthesized in the presence of TGF-beta were larger than those in controls. The synthesis of the small proteoglycans was stimulated 4.5-fold by TGF-beta, and their content was increased. The results show that TGF-beta can stimulate depletion of aggrecan in retinoic acid-treated cartilage. This indicates a catabolic function of TGF-beta in cartilage remodeling.

Intermittent loading induces the expression of 3-B-3(-) epitope in cultured bovine articular cartilage.

OBJECTIVE: To study the effects of intermittent loading on the proteoglycans synthesized in intact cultured articular cartilage. METHODS: Sesamoid bones carrying articular cartilage were subjected to cyclic loading in vitro for one week. A new procedure to fix and decalcify the tissue was developed and an immunohistochemical analysis of the expression of 3-B-3(-) epitope in the articular cartilage was carried out. The proteoglycans synthesized were quantified and studied using CL-2B chromatography. RESULTS: Loading induced an increase in the synthesis of aggrecan molecules, which were larger and less polydisperse than those from control cartilage. Loading also induced the expression of 3-B-3(-) epitopes on newly synthesized proteoglycans. CONCLUSION: These changes are similar to those found in early experimental and human osteoarthritis (OA). More variables must be studied, but our results suggest that our model might be suitable to study early events in the onset of OA.

Modulation of proteoglycan composition in cultured anatomically intact joint cartilage by cyclic loads of various magnitudes.

The anatomically intact articular cartilage (area approximately 2.5 cm2) of 6-month-old bovine sesamoid bones was cyclically (0.3 Hz) loaded with 0, 5, 25 and 50 kg in vitro for 7 days. The amount and composition of the proteoglycans that were synthesized during the last 17 hours of incubation under each regime were studied. Loads of all magnitudes produced a 45% overall increase in synthesis, which included a disproportionate increase in the synthesis of small proteoglycans. Loading with either 25 or 50 kg induced the synthesis of much larger glycosaminoglycan side-chains and a species of small proteoglycan rich in keratan sulfate. Under these greater loads, synthesis in the deep layer of the cartilage was reduced and there was focal damage to the cartilage surface.

Proteoglycan depletion of intact articular cartilage by retinoic acid is irreversible and involves loss of hyaluronate.

Intact sesamoid bones from bovine metacarpophalangeal joints were cultured with retinoic acid for 9 days and allowed to recover in control medium for up to 17 days. Retinoic acid (300 ng/ml) induced 91.8% inhibition of glycosaminoglycan (GAG) synthesis and 50.6% loss of sulfated GAGs from the cartilage. Retinoic acid also induced 38.2% loss of hyaluronate from the matrix. The synthesis and content of the large aggregating proteoglycan (aggrecan) were preferentially decreased compared with that of the small nonaggregating species. The aggrecan synthesized was similar to control aggrecan in size, aggregation capacity, and composition of its GAGs. GAG synthesis was almost completely restored in control medium within the next 6 days. The GAGs synthesized during recovery were slightly shorter than control GAGs and showed a higher ratio of chondroitin-6-sulfate over chondroitin-4-sulfate. Neither the proteoglycan content nor the hyaluronate content recovered within 17 days. The aggregation capacity of newly synthesized aggrecan was normal. However, the retention of proteoglycans synthesized in the recovery period was much lower in treated cartilage than in control cartilage (T1/2 of 17 and 38 days, respectively). In conclusion, the retinoic-acid-induced proteoglycan depletion was irreversible in spite of the restored synthesis of aggrecan with a normal aggregation capacity. The reduced retention of newly synthesized aggrecan during recovery might be caused by a lack of hyaluronate. This model seems suitable to study aspects of cartilage destruction and repair.

Kinetics of proteoglycan turnover in bovine articular cartilage explants.

The turnover of proteoglycans was studied in explant cultures of mature bovine articular cartilage. The aim of the study was to compare the in vitro turnover rates of newly synthesized proteoglycans and endogenous proteoglycans. Cartilage was maintained in the presence of various serum concentrations in order to determine the conditions of steady-state proteoglycan metabolism. The steady state was achieved in medium containing 20% fetal calf serum. The proteoglycan synthesis rate and the half-life of labeled proteoglycans in steady-state cultures were used to calculate the size of the metabolic pool of newly synthesized proteoglycans in steady state. This metabolic pool was shown to be equal to the total amount of proteoglycans in the matrix. It is concluded that all of the proteoglycans in the matrix have the same half-life in vitro. Taking another approach, aggrecan was isolated from the cartilage and the medium of steady-state cultures prelabeled with [35S]sulfate. The specific activity of the glycosaminoglycans from cartilage aggrecan were compared with that of glycosaminoglycans from medium aggrecan. These proved to be the same throughout the culture period. This shows that newly synthesized aggrecan and endogenous aggrecan have the same turnover rate in vitro. The significance of explant culture systems for the study of proteoglycan turnover is discussed.

Effects of loading on the synthesis of proteoglycans in different layers of anatomically intact articular cartilage in vitro.

The anatomically intact articular cartilage (approximately equal to 2.5 cm2) of whole bovine sesamoid bones was cultured on its bone support. A load of 5 kg was applied intermittently at 0.3 Hz by a specially designed loading apparatus for 7 days. [35S]-sulfate was added during the last 17 h of the experiment. Loading induced a 40% increase in [35S]-sulfate incorporation into aggrecans and an almost 3-fold increase in the synthesis of small proteoglycans. The loading effects occurred mainly in the upper half of the articular cartilage. Samples left unloaded for 7 days and then loaded for 7 days likewise showed an increase in [35S]-sulfate incorporation compared with unloaded controls.

Age related changes in the turnover of proteoglycans from explants of bovine articular cartilage.

Articular cartilage in explant culture synthesized 2 proteoglycan types of different size. The proportion of 35S-sulfate incorporated into the small proteoglycan was higher in mature (17%) than in immature cartilage (11%). The chondroitin sulfate chains of both proteoglycans, synthesized by mature cartilage were shorter than those of immature cartilage, with a higher ratio of 6-sulfated over 4-sulfated disaccharides. Radiolabelled macromolecules from tissue of both ages were released in 2 phases: an initial fast release followed by a period of slow release. The half-lives of both proteoglycan populations were shorter in mature cartilage than in immature tissue (-45%). The amount of proteoglycans released during the initial short phase increases with age, from 23-24% in immature cartilage to 28-29% in mature tissue.

Effect of temperature on the metabolism of proteoglycans in explants of bovine articular cartilage.

The turnover of proteoglycans in bovine articular cartilage was determined in explant cultures, maintained at 32 degrees C or 37 degrees C. Both the rate of proteoglycan synthesis and the release of newly synthesized proteoglycans were decreased in cultures incubated at 32 degrees C compared to 37 degrees C. At both temperatures the newly synthesized proteoglycans were similar in hydrodynamic size and chain length of the glycosaminoglycans. However, the ratio of 6-sulfated disaccharides over 4-sulfated disaccharides of the newly synthesized glycosaminoglycans, differed less from the endogenous ratio at 32 degrees C than at 37 degrees C. At both temperatures, the incorporated 35S-sulfate is released from explants in two pools. Twenty-three percent of the 35S-radiolabel was released into the culture medium during an initial short phase (t1/2 = 1.1 day at 32 degrees C, 1.3 day at 37 degrees C), 77% had a much longer half-life. The lowered temperature markedly decreased the release of 35S-sulfate with a slow turnover (t1/2 = 60 days at 32 degrees C, 38 days at 37 degrees C).

Composition of proteoglycans synthesized in different layers of cultured anatomically intact articular cartilage.

Calf articular cartilage was cultured anatomically intact on its natural bone-support. At day 0 and day 7, respectively, the cartilage was radiolabeled, washed and harvested in 3 successive layers parallel to the articular surface. The proteoglycans were studied after extraction by 4 M guanidine hydrochloride. In the deep layer, the endogenous proteoglycan monomers were slightly smaller, showed an increased polydispersity and the relative amount of keratan sulfate was lower. In addition, chondroitin sulfate side chains were slightly larger and the sulfation degree and proportion of 4-sulfated disaccharides was elevated. At day 0, deep layer chondrocytes incorporated about twice as much [35S]-sulfate into glycosaminoglycans as did superficial chondrocytes. The newly synthesized proteoglycan monomers were the same in all layers with respect to size, dispersity, relative amount of keratan sulfate and size of chondroitin sulfate side chains. The sulfation-pattern, however, changed with depth in the same way as noted in the endogenous proteoglycan population. Small endogenous proteoglycan was present in all layers, but its synthesis was only prominent in the upper layer and decreased with depth. After 7 days culture, the [35S]-sulfate incorporation had increased in the upper half of the cartilage. There was a strong increment in the proportion of 6-sulfated disaccharides of newly synthesized glycosaminoglycan in all layers. The synthesis of small proteoglycan was markedly reduced, especially in the upper layer.

Effect of nalidixic acid, pipemidic acid and cinoxacin on chondrocyte metabolism in explants of articular cartilage.

Explants of immature bovine articular cartilage were exposed to nalidixic acid, pipemidic acid and cinoxacin at one and ten times the human therapeutic plasma level for 7 days. Only nalidixic acid had significant effects on the chondrocyte metabolism. 20 micrograms/ml nalidixic acid caused an increase of 35S-sulfate incorporation into glycosaminoglycans at day 7. Two hundred micrograms/ml nalidixic acid inhibited the incorporation of 3H-thymidine into DNA. The incorporation of 35S-sulfate into glycosaminoglycans was decreased at day 0, while at day 7 the incorporation had returned to the control value. Pipemidic acid and cinoxacin had no significant effects on either the 3H-thymidine or the 35S-sulfate incorporation.

Studies on the extraction of different proteoglycan populations in bovine articular cartilage.

Large proteoglycan monomers and small dermatan sulfate proteoglycans were extracted from explants of bovine articular cartilage with increasing (0-4 M) concentrations of guanidinium chloride (GuHCl). The first extractions were followed by a second extraction with 4 M GuHCl. The amount of proteoglycans extracted in the first buffer depended on the GuHCl concentration. At low concentrations of GuHCl, a relatively high amount of small proteoglycans was obtained. Fifty percent of the small proteoglycans was extracted in buffer with 0.85 M GuHCl, while 2.0-2.2 M GuHCl was needed to extract half of the large proteoglycans. Immediately after synthesis, 35S-labeled large proteoglycans were extracted much easier (50% at 1.4 M GuHCl), and those extracted at low concentrations of GuHCl were less capable of aggregation with hyaluronic acid. After 7 days of 'chase' these differences between endogenous and 35S-labeled proteoglycans had disappeared.

A Staphylococcus aureus factor exclusively removes large proteoglycan monomers from explants of articular cartilage.

Exposure of explants of viable, as well as freeze-thawed, bovine articular cartilage to culture filtrates of Staphylococcus aureus increased the release of proteoglycans from the tissue significantly within 24 h. The S. aureus factor exclusively releases the large, 4 M guanidinium extractable proteoglycans from the matrix; the small proteoglycans are not affected by this factor. The large proteoglycans, released by Staphylococcal culture filtrate, had a slightly smaller hydrodynamic volume compared to the proteoglycans remaining in the matrix. Purified large proteoglycans, incubated in S. aureus filtrate, lost their capacity to aggregate with hyaluronic acid. This suggests that some culture filtrates of S. aureus contain a factor that degrades the core protein of the large proteoglycans at or near the hyaluronic acid binding region.

Bovine sesamoid bones: a culture system for anatomically intact articular cartilage.

Medial sesamoid bones from the metacarpophalangeal joints of calves were used for prolonged culture of anatomically intact articular cartilage on its natural bone support. The cartilage remained viable during culture, without signs of degeneration. After 1 wk of culture the cartilage showed an increased proteoglycan synthesis, and some minor changes in the composition of newly synthesized proteoglycans were observed. In the next 7 wk all studied parameters remained constant, except for the rate of proteoglycan synthesis, which declined between 4 and 8 wk to values just below those measured at the start of culture. Despite the fact that newly synthesized proteoglycans showed some altered biochemical properties, the composition of the total pool of proteoglycans did not change during 8 wk of culture. The significance of this phenomenon is discussed. This new in vitro model of intact articular cartilage offers a promising alternative to in vivo studies because in contrast to other in vitro models no surgical injury of the cartilage is introduced.

Effect of sulfate concentration on glycosaminoglycan synthesis in explant cultures of bovine articular cartilage.

The effects of the sulfate- and FCS concentration on the rate of synthesis and the biochemical properties of glycosaminoglycans, synthesized in bovine articular cartilage in vitro, were studied. 20% FCS in the culture medium stimulated the rate of synthesis. In media without FCS, the rate of synthesis decends from day 0 on. The differences in incorporation rates of [35S]-sodium sulfate and 1,6-[3H]-glucosamine-HCl into glycosaminoglycans in serum free media containing 9 microM and 900 microM sulfate were used to discuss the inorganic sulfate concentration in cartilage. In 9 microM sulfate medium, the newly synthesized glycosaminoglycans contain higher levels of unsulfated disaccharides than the endogenous glycosaminoglycans. In each culture medium, the ratio 6-sulfated disaccharides to 4-sulfated disaccharides of the newly synthesized glycosaminoglycans becomes higher after 3 days in culture. The glycosaminoglycan synthesis is underestimated, when chondrocytes are cultured in media containing less than 200 microM sulfate.