High-density cultures of chick embryonic chondrocytes were exposed to intermittent compressive force (ICF) of physiologic magnitude for 24 hours. Proteoglycan synthesis was significantly increased in chondrocyte cultures exposed to ICF as compared with control cultures. Similar effects were found in explants of epiphyseal cartilage. Proteoglycans extracted with guanidine-HCI from cultures exposed to ICF aggregated better with hyaluronic acid than did control cultures, as shown by Sepharose 2B gel chromatography. In addition, the amount of non-extractable proteoglycans was increased in ICF cultures. We conclude that ICF not only increases the synthesis of proteoglycans but also improves the aggregating capacity of proteoglycans and the coherence of proteoglycans with other matrix components. High-density cultures of epiphyseal chondrocytes provide a suitable model to study the processes involved in the perception of and the subsequent cellular response to compressive force by cartilage.The ability of cartilage to function as a weightbearing tissue is related to its chemical composition and the macromolecular organization of its constituents (1). The extracellular matrix of cartilage is primarily composed of collagen and proteoglycan, which account for 50-60% and 10-40% of the dry weight, respectively (2). Collagen confers tensile strength (3) and proteoglycans provide the elasticity and the ability to resist compression (43). Proteoglycan monomers which have an average molecular weight of 1-2 x lo6 daltons (6) possess a high fixed negative charge density, which renders them highly hydrophilic (7).In cartilage the proteoglycans form predominantly very large aggregates, in which many individual monomers are noncovalently associated with hyaluronic acid (8,9). A proteoglycan monomer consists of a protein core which contains 3 regions: the hyaluronic acid binding region with a few short keratan sulfate chains, an intermediate keratan sulfate-rich fragment, and a chondroitin sulfate-rich fragment of variable size (10,ll). The proteoglycan-hyaluronic acid interaction is stabilized by 2 glycoproteins (9,(12)(13)(14)(15)(16).The initial in vivo response of articular cartilage to repetitive trauma is enhanced matrix production without cell proliferation (17), whereas a decrease in loading provokes degenerative changes in the articular cartilage (18,19). This study reports the in vitro response of high-density chondrocyte cultures to intermittent compression. We reported earlier that under in vitro conditions, intermittent compressive force (ICF) decreased cell proliferation of chondrocytes cultured in a monolayer (20). Matrix production, however, should be studied using high-density chondrocyte cultures (21-25). We developed a suitable model (24) and reported that high-density chondrocyte cultures exposed to intermittent compressive force showed a decrease in cell proliferation and an increase in matrix

Sesamoid bone cartilage from the metacarpophalangeal joints of 5-year-old cows was cultured intact on its bone support. The incorporation of sulfate increased similarly in experimental cartilage that was subjected to cyclic loading (0.2 MPa, 0.2 Hz) for a week and in control cartilage that was cultured without loading. The synthesis of a population of small macromolecules decreased in the cultured controls, but was maintained at a constant level in the experimental samples. This population was isolated through Sepharose CL-2B chromatography. Subsequent application to Sepharose CL-4B yielded two distinct peaks. One contained protein-free chondroitin sulfate, keratan sulfate, and possibly some dermatan sulfate glycosaminoglycans. The other, more prominent, peak consisted of dermatan sulfate proteoglycans. This peak material was pooled, and applied to a 4-15% SDS-gel. The material was separated into two major bands, which represented biglycan and decorin. They decreased to less than half their day-0 value, in the cultured control. In the loaded cartilage, biglycan synthesis remained constant while decorin synthesis increased. These findings suggest that biglycan and decorin are involved in the adaptation of articular cartilage to variations in loading regime.

Proteoglycans (A1 fractions) were extracted with 4M guanidine hydrochloride (GuHC1) from human articular cartilage samples of a wide age range. Distinctions were made between hip and knee, and upper and lower layers. The residues of these extractions were digested with purified collagenase, and a second extraction with 4M GuHCl was performed, which yielded appreciable amounts of proteoglycans. When proteoglycans from second extractions were compared with those from first extractions, the following changes were observed: an increase in chondroitin sulfate; a relative decrease in keratan sulfate; a decrease in protein content; and a decrease in the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate. The same changes were found when nonaggregating proteoglycans were compared with proteoglycan aggregates, when proteoglycans from young cartilage were compared with those from mature carailage, when proteoglycans from knee cartilage were compared with those from hip cartilage, and when proteoglycans from upper layers of cartilage were compared with those from deeper layers. It is suggested that the differences found between first and second extrac- tions of cartilage, between upper and lower layers of cartilage, and between knee and hip cartilage are caused by variations in the relative amount of nonaggregating proteoglycans and/or variations in proteoglycan size.The most obvious biochemical alterations in aging cartilage are found in the composition and structure of the proteoglycan population (1-5). Proteoglycan size decreases, chondroitin sulfate chain length decreases, and the length of the core protein shortens (2). The content of keratan sulfate increases relatively, as well as absolutely (2,6). These changes were found to be caused by shifts in the relative amounts of the several proteoglycan subclasses (4,7).The generally used proteoglycan extraction procedure liberates, depending on tissue geometry (8), about 5040% of the proteoglycans from mature articular cartilage. In this study, proteoglycans were isolated from human articular cartilage by 2 consecutive extractions with 4M guanidine hydrochloride (GuHCl) intercalated by a collagenase digestion of the residue of the first extraction (5). The chemical properties of the high buoyant density proteoglycan preparations obtained are described in relation to age and to layer of the cartilage (upper or lower), because of reported differences in the composition of directly extracted proteoglycans that occur with depth of the cartilage (8).Proteoglycans isolated from hip and knee cartilage were compared because the knee joint seems to be prone to developing osteoarthritis at an earlier age than does the hip joint (9).

The effects of various proteoglycan samples, isolated from human articular cartilage of different ages, on the rate of the lateral growth phase of the fibril formation of collagen type II were studied by turbidimetry. In general, proteoglycan aggregates accelerate fibrillogenesis, whereas non-aggregating proteoglycans retard this process. The only exception were non-aggregating proteoglycans from very young cartilage, which stimulated the fibril formation strongly. The extent of stimulation by proteoglycans from hip and knee cartilage were compared. The effects of non-aggregating proteoglycans dominate those of aggregated proteoglycans. Chondroitinase ABC digestion of proteoglycan samples did not change the effects on the fibrillogenesis of collagen type II, when these samples were isolated from 18 years-old knee cartilage. The collagen fibril formation was less stimulated in the presence of ABC-ase digested proteoglycan samples from 0-3 month-old knee cartilage, suggesting a primary role for keratan sulphate and a possible influence of chondroitin sulphate when keratan sulphate is not present. Only proteoglycans from very old cartilage were able to reduce the amount of collagen fibrils formed in vitro. Proteoglycans could not be detected bound to the fibril pellet despite the fact that part of the pellet was not dissolvable in acetic acid. It is concluded that proteoglycans may play a regulatory role in collagen type II fibril formation in articular cartilage.

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