composition and glycosaminoglycan metabolism of articular cartilage from habitually loaded and...

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COMPOSITION AND GLYCOSAMINOGLYCAN METABOLISM OF ARTICULAR CARTILAGE

FROM HABITUALLY LOADED AND HABITUALLY UNLOADED SITES

SALLY D. SLOWMAN and KENNETH D. BRANDT

The uronic acid (proteoglycan, PG) content of cartilage from habitually unloaded sites of normal canine femoral condyles has been shown to be lower than that from habitually loaded regions, even though the glycosaminoglycan (GAG) synthesis is similar. We in- vestigated whether the GAG degradation in unloaded cartilage would be greater than that in loaded cartilage, and we obtained comparative biochemical data concern- ing the PGs and organization of the extracellular matrix of noi-ma1 loaded and unloaded cartilage. PG extractability (qetermined by sequential guanidinium chloride extracts of cartilage), percentage of PGs form- ing large aggregates, and hydrodynamic size of the PG monomers (determined by Sepharose 2B chromatography) were essentially the same in loaded and unloaded cartilage. As expected, the uronic acid content of unloaded cartilage was 20% lower than that of loaded cartilage (P < 0.02), while the water and DNA contents of the 2 tissues were not statistically different. There was no difference in the rate of net "S04-GAG synthesis in organ cultures of loaded and unloaded cartilage. More- over, there was no appreciable difference in the rates of "S04-GAG degradation of loaded and unloaded cartilage, ais determined by "SO4 pulse-chase studies. We

From the Rheumatology Division, Indiana University School of Medicine, Indianapolis.

Supported in part by grants from the NIAbDK (AM-20582 and AN[-7448), an Arthritis Foundation Clinical Research Center grant, aind an award from the Grace M. Showalter Trust.

Sally D. Slowman, MD: Fellow, Rheumatology Division; Kenneth D. Brandt, MD: Professor of Medicine and Head, Rheumatology Division.

Address reprint requests to Kenneth D. Brandt, MD, Rheumatology Division, Indiana University School of Medicine, 541 Clinical Drive, Indianapolis, IN 46223.

Submitted for publication March 25, 1985; accepted May 29. 1985.

have previously shown that selective cyclic compressive stresses applied in vitro to cartilage from loaded areas of canine femoral condyles may increase "S04-GAG synthesis. The present results suggest that the rates of GAG metabolism in loaded and unloaded cartilage under atmospheric pressure in vitro may not reflect the rates which exist in articular joints under compressive loads in vivo.

A number of reports have suggested that a causal relationship exists between the proteoglycan (PG) content of connective tissue and the mechanical stress to which it is subjected. Thus, the glycosaminoglycan (GAG) content of load-bearing areas of dermis (l), articular cartilage (2-5), and tendon (6,7) is greater than that of habitually unloaded areas of the same tissues. Furthermore, it is clear that such differences can be modulated in vivo (8,9). An increase in compressive loading of the normal canine shoulder has been shown to produce an in vivo increase in the chondroitin sulfate content of the articular cartilage of the humeral head (9). Conversely, when loading of articular cartilage is reduced, e.g., by immobilization of the ipsilateral limb, net PG synthesis (8,lO) is decreased and the glycosaminoglycan content (1 1) is reduced.

This relationship between load-bearing and PG metabolism of connective tissue is also reflected by the results of several in vitro studies. Thus, net GAG synthesis in cultures of articular cartilage (12-19, tendon (16), and embryonic chick chondrocytes (17) has been shown to increase after intermittent loading in compression or tension. Recent studies in our laboratory have shown that cyclic application of a compressive load to normal canine femoral cartilage

Arthritis and Rheumatism, Vol. 29, No. 1 (January 1986)

GAG IN LOADED AND UNLOADED CARTILAGE 89

either increased or decreased net GAG biosynthesis, depending on the duration of loading (18).

In preliminary studies, we found that the level of net GAG synthesis in normal canine femoral condylar cartilage from habitually loaded areas was comparable with that in cartilage from habitually unloaded areas of the same joint (4). Since the uronic acid content of cartilage from unloaded sites is lower than that of cartilage from loaded regions, we hypo- thesized that the rate of GAG degradation in unloaded cartilage would be greater than that in loaded cartilage. The present study was designed to test this hypothesis and to obtain comparative biochemical data concern- ing the PGs and organization of the extracellular matrix in normal loaded and unloaded cartilage. The results failed to confirm our hypothesis, but showed that the rate of GAG breakdown in unloaded cartilage was no different from that in loaded cartilage. In light of the above findings, the differences in PG content of cartilage specimens obtained from various regions of articular cartilage must reflect the degree of loading to which these regions were subjected in vivo.

MATERIALS AND METHODS Tissue and culture conditions. The distal femurs of 7

adult mongrel dogs (weight 25-30 kg) were aseptically removed immediately after the animals were killed with T-61 euthanasia solution (Taylor Pharmacal, Decatur, IL). Slices of cartilage measuring <1.0 inm thick were shaved with a scalpel from loaded articular surfaces of the medial and lateral femoral condyles and from the adjacent medial, lateral, and superior unloaded surfaces. Appf-oximately 300 mg of cartilage (wet weight) was obtained from the loaded regions and 250 mg from the unloaded regions of the femurs of each dog.

The shavings from loaded and unloaded cartilage of the medial and lateral condyles of both knees from each animal were pooled separately and were placed in Corning 60-nim tissue culture dishes containing 10 ml of Ham’s F-12 nutrient mixture, pH 7.4, supplemented with 10% fetal calf serum, streptomycin (100 pg/ml), Fungizone (0.25 pg/ml; Squibb, Princeton, NJ), and penicillin (100 units/ml). NaZ3’SO4 (20 pCi/ml; New England Nuclear, Boston, MA) was added to the medihm, and after 20 hours of incubation at 37°C under 5% COz, 95% air, the medium was decanted, and the cartilage was washed with fresh medium. The spent medium and wash were combined and dialyzed against distilled water for 48 hours at 4°C in Spectrapor No. 3 dialysis tubing (Spectrum Medical Industries, Los Angeles, CA), which has a molecular weight cutoff of 3,500 daltons. Triplicate samples of washed cartilage (14-20 mg/sample) were removed and utilized for analysis of the 35S04-GAG content, as described below.

Determination of net GAG synthesis. After incubation with NaZ3%04, the washed cartilage slices were weighed,

and digested for 24 hours at 56°C in Tris buffer, pH 8.0 (1 ml/lS mg of cartilage), containing pronase (1 mg/ml; Calbiochem-Behring, La Jolla, CA). The digests were then dialyzed against distilled water for 48 hours at 4°C. Net GAG synthesis was determined from the sum of the nondialyzable 35S04 counts per minute in 0.1-ml portions of the spent medium and 0.5-ml portions of the pronase digest, which were added separately to 10 ml of scintillation fluid (Scint-A; United Technologies Packard Instrument Co., Downers Grove, IL) and counted in a model LS 7000 Beckman liquid scintillation spectrometer. Results were adjusted for differences in wet weight of the cartilage.

Pulse-chase studies. The remainder of the washed tissue (approximately 200-250 mg each of loaded and unloaded cartilagej was suspended in Ham’s F-12 nutrient mixture containing 1 mM SO4, as Na2S04, without radioac- tive label. Incubation was continued at 37°C under 5% COz, 95% air, with replacement of the spent medium every 48 hours. Samples of loaded and unloaded cartilage (approximately 15 mg each) were removed in triplicate at various intervals (3-144 hours), weighed, and digested with pronase. The digests were dialyzed against distilled water for 48 hours, after which 1.0-ml portions of the retentate were added to 10 ml of scintillation fluid, as described above. The nondialyzable 35S04 content of the triplicate samples at each interval was determined and adjusted for differences in cartilage wet weight. The results were expressed as a percentage of the nondialyzable cpm at time 0.

Sequential extraction of PGs from the cartilage. Car- tilage from the loaded and unloaded regions of the medial and lateral condyles of 4 dogs was obtained as described above, weighed, and pooled separately. Approximately 250 mg (wet weight) of cartilage was immediately suspended in 25 ml of cold 0.4M guanidinium chloride (GuHCI) in O.05M sodium acetate, pH 5.9 (19), containing the protease inhibitors EDTA (0.01M), 6-aminohexanoic acid (0. lM), benzamidine hydrochloride (O.OOSM), and phenylmethyl- sulfonyl fluoride (0.025 mM) for metalloproteases, cathepsin D activity, trypsin-like activity, and serine proteinases, respectively (20,21). After stirring for 24 hours at 4°C in Corer: tubes, the sllspension was centrifuged at 15,000 revolutions per minute for 20 minutes at 4°C in a Beckman model J-21B centrifuge, and the supernatant was dialyzed against distilled water for 48 hours.

The cartilage residue remaining after extraction with 0.4M GuHCl was resuspended in cold 4.OM GuHCl in O.05M sodium acetate, pH 5.9, containing the above protease inhibitors plus soybean trypsin inhibitor (1 mg/100 ml of buffer; Sigma, St. Louis, MO) (21). The suspension was stirred for 24 hours at 4“C, after which it was centrifuged at 15,000 rpm for 20 minutes at 4°C. The supernatant was dialyzed against distilled water, and the pellet containing the cartilage residue was digested with pronase, as described above. The uronic acid contents of triplicate 0.5-ml aliquots of the 0.4M and the 4.OM GuHCl extracts, and of the GAGS isolated from the tissue after digestion of the residue with pronase, were determined. Results were adjusted for differences in wet weight of the tissues.

Gel chromatography. The GuHCl extracts were dialyzed against distilled water for 48 hours at 4°C and lyophilized Uni-Trap; Virtis, Gardiner, NY). The dried powdered

90 SLOWMAN AND BRANDT

Table 1. habitually loaded and habitually unloaded sites

Composition of normal canine articular cartilage from

Source of cartilage* Composition and No. of analysis dogs Loaded Unloaded Pt

Uronic acid content,

DNA content,

H 2 0 content,

% of dry weight 9 5.04 2 0.33 3.96 ? 0.28 10.02

F g h g wet weight 6 0.30 ? 0.14 0.43 ? 0.21 NS

'3% of wet weight 9 14.2 f 0.8 13.2 f 0.9 NS

* Results are mean 2 SE. t Student's t-test for paired observations. NS = not significant.

product (4-6 mg) was subsequently dissolved overnight in 1 ml of 0.05 sodium acetate, pH 6.5, and applied to a Sepharose 2B column (100 X 1.0 cm) (Pharmacia, Uppsala, Sweden), which was eluted with the acetate buffer at a rate of 4 ml/hour. The uronic acid content of 1-ml effluent fractions was determined as described below. Partition coefficients(K,,) were calculated from the formula:

K,, = (V, - VO) (V, - VO)

where V, represents the peak fraction in the elution diagram, Vo the void volume, and V, the total column volume.

Incubation of PG aggregates with hyaluronic acid Pl+3 hydrolase. Samples (4-5 mg) of the dialyzed and lyophilized 4.0M GuHCl cartilage extract were dissolved overnight in 1 ml of 0.05 sodium acetate, pH 6.5, and were made 0.1M with respect to citric acid and 0.2M with respect to Na2HP04. The pH was adjusted to 5.6, and 5 turbidity- reducing units (TRU) of Streptomyces hyalurolyticus hyaluronidase (Miles Laboratories, Elkhart, IN) was added to the solution, which was then incubated for 5 hours at 37°C. Sepharose 2B chromatographs of the PG samples before and after hyaluronidase treatment were compared. To ensure that the hyaluronidase preparation was free of proteolytic activity and that the hyaluronic acid (HA)-binding region of the proteoglycan subunit (PGS) retained its ability to interact with HA following treatment with the enzyme, purified PGS (22) was incubated overnight with hyaluronidase (2 mg of PGS/5 TRU of enzyme) at 37°C under the abtove conditions, after which the enzyme was inacti- vated by boiling for 8 minutes. HA (Sigma) was then added to provide an HA:PGS ratio of 1:38, and the solution was incubated for 42 hours at 20"C, after which the sample was chromatographed on the Sepharose 2B column.

Analytic methods. For determination of the dry weight of the tissue, slices (approximately 10-15 mg) of loaded and unloaded cartilage were placed separately in 2 changes of acetdne for 48 hours, after which they were dried to constant weight at 80°C. After the dried cartilage was weighed and digested with pronase, as described above, the GAGS were isolated by precipitation with 9-aminoacridine hydrochloride and converted to their sodium salts with Bio-Rad AG 50W-X8 (Na+) cation exchange resin. After the resin was removed by filtration, the uronic acid content of the filtrate was determined by a modified carbazole reaction

(23). The results were expressed as percent of cartilage dry weight.

The DNA content of slices (approximately 15-20 mg) of loaded and unloaded cartilage was measured by the diphenylamine method (24) and expressed as pg of DNA/mg of cartilage wet weight.

Histology and histochemistry. Full-thickness samples of cartilage, with a portion of the underlying subchondral bone attached, were obtained with a Craig biopsy needle (3 mm in diameter) from loaded and unloaded surfaces of the medial and lateral femoral condyles of 5 dogs. The cartilage was fixed in 10% buffered formalin and embedded in paraffin. Histologic sections ( 6 ~ ) , perpendicular to the surface, were cut on a microtome and stained with Safranin 0-fast green. Cartilage depth from surface to tidemark was measured with a reticule eyepiece attachment. Loaded and unloaded cartilage samples from the same dog were processed and stained concurrently.

Statistical analysis. All statistical analyses utilized Student's t-test for comparison of paired observations.

RESULTS Gross appearance, histology, and histochemis-

try. The femoral articular cartilage from each animal appeared normal and was smooth, bluish-white, and glistening, with no surface irregularity. In all histologic samples the cartilage surface and tidemark were in- tact. The unloaded cartilage was approximately 40% thinner than the loaded cartilage. Safranin 0 staining was decreased in unloaded cartilage, with a prominent pericellular and interterritorial uptake around the deeper chondrocytes, in contrast with the more uni- form, intense staining of loaded cartilage. However, the cell distribution and cell morphology in loaded and unloaded cartilage were indistinguishable. No cell clusters were seen.

Analytic data. The uronic acid content of loaded cartilage averaged 5.0 k 0.3% of tissue dry weight; that of unloaded cartilage was 20% lower, i.e., 4.0 ? 0.3% (P < 0.02) (Table 1). However, the water and DNA contents of loaded and unloaded cartilage were not statistically significantly different (Table 1).

Extractability of PGs from the cartilage. Based upon the proportion of the total tissue uronic acid content found in the 2 GuHCl extracts and the pronase digest, there was no difference between loaded and unloaded cartilage with respect to PG extractability (Table 2). Thus, 0.4M GuHCl extracted only approximately 18% of the total tissue PGs, while the largest proportion (44.2-59.8%) of the total PGs was extracted with 4.0M GuHCI. In every case, the cartilage residue, which was digested with pronase, contained approximately 30% of the total tissue PGs.


Table 2. Percentage of total tissue nondialyzable uronic acid in sequential extracts of canine knee cartilage from habitually loaded and habitually unloaded sites*

Source of 0.4M GuHCl 4.0M GuHCl Pronase Dog cartilage extract extract digest

#lo20 Loaded

#lo27 Loaded

#I031 Loaded

#lo35 Loaded

Unloaded

Unloaded

Unloaded

Unloaded

Mean -t SE Loaded Unloaded

18.4 15.8 11.7 13.4 13.8 12.0 28.8 26.1

50.8 47.8 53.8 44.2 58.4 59.8 50.6 50.3

30.8 36.4 34.5 42.4 27.8 28.2 20.6 23.6

18.2 ? 3.8 53.4 ? 1.8 28.4 ? 2.9 16.8 ? 3.2 50.5 ? 3.3 32.6 ? 4.2

* GuHCl = guanidinium chloride.

According to the sum of the uronic acid contents of the GuHCl extracts and the pronase digest of the residue, the total uronic acid content of unloaded cartilage was approximately 18% lower than that of loaded cartilage from the same knee (17.1 ? 1.3 pglmg wet weight and 20.9 2 1.5 pg/mg wet weight, respectively) ( P < 0.02). This difference was consistent with the 20% difference in the uronic acid content of loaded and unloaded cartilage, which was obtained by direct analysis of the GAGs isolated after pronase digestion of the whole tissue.

Gel chromatography of the PGs. The Sepharose 2B chromatographs indicated that 1636% of the PGs in the 4.0M GuHCl extract of loaded cartilage and 19-32% of those in the extract of unloaded cartilage were sufficiently large so as to elute in the void volume (Table 3). The K,, of those PGs which were retarded by Sepharose 2B ranged from 0.22-0.31 for samples of loaded cartilage and from 0.22-0.26 for samples of unloaded cartilage. In all cases, incubation with HA

p1+3 hydrolase eliminated the void volume peak on Sepharose 2B chromatography, indicating that the extracts contained PGs that were interacting with HA. The hyaluronidase was free of significant pro- teoglycanase activity since it did not affect the ability of PGs to interact with HA to form complexes large enough to elute with the void volume of the Sepharose 2B column.

The Sepharose 2B elution profiles of the 0.4M GuHCl extracts were similar to those of the 4.OM GuHCl extracts, i.e., some 2&30% of the total sample was eluted in the void volume. Due to the relatively small amount of PG in the 0.4M GuHCl extracts, no attempt was made to dissociate the PG-HA complexes in these samples with HA p1+3 hydrolase.

PG metabolism. Based upon the sum of nondialyzable 3sS04 radioactivities in the medium and the pronase digests, net GAG synthesis in loaded cartilage was similar to that in unloaded cartilage. In both cases, approximately 3.7 x lo4 cpm was incor- porated into GAG per mg wet weight of cartilage. The tissue remained metabolically active in culture throughout the 7-day incubation period, as indicated by a linear increase in 35S04 incorporation into GAGs after incubation with NaZ3?304.

The data from the pulse-chase experiments indicated that the rates of PG degradation in loaded and unloaded cartilage were similar. In both cases, approximately 50% of the total nondialyzable cpm was lost over the 144-hour chase period (Figure 1).

DISCUSSION

The results of the present study are consistent with previous observations that the uronic acid content (and thus, the proteoglycan content) of unloaded

Table 3. cartilage*

Results of Sepharose 2B chromatography of proteoglycans extracted from canine articular

4.OM GuHCl extract after incubation with

0.4M GuHCl extract 4.0M GuHCl extract HA PI-3 hydrolase

Dog cartilage % i n Vo K,,, V, % in Vo K .,", V, % in Vo K,, , V, Source of

#I027 Loaded 18 0.40 34 0.22 6 0.30 Unloaded 23 0.36 24 0.22 6 0.26

#I031 Loaded 33 0.36 36 0.26 4 0.34 Unloaded 21 0.37 32 0.22 ND ND

#lo35 Loaded 23 0.46 16 0.31 I 0.50 Unloaded 24 0.50 19 0.26 I 0.61

* GuHCl = guanidinium chloride; Vo = void volume; V, = material eluting from the column after the void volume peak; HA = hyaluronic acid; N D = not done because of insufficient material.


o iz 24 48 i 2 & I io I44

TIME OF CHASE (hours)

Figure 1. Results of pulse-chase studies, expressed as percentage of nondialyzable 3sS04 cpm at time 0, of habitually loaded cartilage (.-a) and habitually unloaded cartilage (0-0) obtained from adult dogs. Results are means t SE. Numbers in parentheses are nuinberii of dogs from which cartilage samples were taken.

cartilage is lower than that of loaded cartilage. This was apparent when the uronic acid values were related either to the water content or to the DNA content of the tissues, since these were essentially the same in unloaded cartilage as in loaded cartilage. The similarity of water content values in loaded and unloaded cartilage suggests that normal unloaded cartilage is different from atrophic (10,25) or osteoarthritic cartilage (26,27) in which the uronic acid content is de- creaseld but the water content is increased. Presum- ably, the hyperhydration in damaged cartilage reflects an abnormality in the collagen network of the tissue that permits the hydrophilic proteoglycans to expand their molecular domains (28,29).

In the present study, the proportion of total tissue PGs which could be extracted from unloaded cartilage with the nondissociative solvent, 0.4M GuHCl, was essentially the same as that which could be extracted from loaded cartilage (Table 2). Presum- ably, tlhose PGs which were extracted from the tissue with thds solvent did not exist as aggregates in vivo in association with hyaluronic acid and link gly- coprotcins (30). In addition, the tightness of the collagen meshwork and the degree of hydration of the cartilage may influence the yield of PGS extracted by low-ionic strength solvent (3 1). The comparability of yields in the 0.4M GuHCl extracts of loaded and unloaded cartilage therefore suggests that the 2 tissues are similar with respect to macromolecular organization of matrix PGs.

Further evidence of the similarity between loaded and unloaded tissues was provided by the

yields of PGs in the 4.OM GuHCl extract (Table 2). Dissociative solvents, such as 4.OM GuHCl, presumably extract PGs that exist in vivo as large aggregates in non-covalent association with HA and link glycoproteins (32). Under the experimental conditions used in our studies, extraction with 4.OM GuHCl was not quantitative, since some 30% of the total PGs in both loaded and unloaded cartilage remained in the tissue after extraction with this solvent. However, Bayliss et a1 (33) have shown that the yield of PGs extracted with 4.OM GuHCl is related to the amount of surface area of cartilage available to the solvent. Thus, in their studies, virtually all the PGs were extracted from articular cartilage when the tissue had been sectioned on a microtome into slices only 20p thick.

In the present study, PGs extracted from loaded and unloaded cartilage with 4.OM GuHCl appeared to be comparable with respect to their ability to interact with hyaluronic acid, as judged from the Sepharose 2B elution profiles (Table 3). In both cases, approximately 25% of the material was excluded from the gel and presumably represented PGs which were interacting with extracted hyaluronate to form large aggregates. Elimination of the void volume material by treatment of the samples with hyaluronic acid p1-3 hydrolase supports this view. Furthermore, PG monomers from loaded and unloaded cartilage had essentially the same hydrodynamic size, based on the K,, of uronic acid-containing material which was retarded by Sepharose 2B.

In agreement with results of an earlier study (4), the net rates of 35S04-GAG synthesis in loaded and unloaded cartilage were similar. Given the fact that the uronic acid content of unloaded cartilage is lower than that of loaded cartilage, one might have expected that the rate of catabolism of newly synthesized GAGS would have been greater in unloaded cartilage than in loaded cartilage. However, this was not the case, and the results indicated no appreciable difference in the rates of 35S04-GAG breakdown in loaded and unloaded cartilage (Figure I).

The irregularities noted in the 3SS04-GAG degradation rate during the first 24 hours of chase (Figure 1 ) are difficult to explain and may represent a technical artifact. However, despite the apparent aberration in the initial hours of culture, GAG degradation rates in loaded and unloaded cartilage were similar; in both cases, 50% of the initial label was lost at 144 hours.

Based on the above findings, how can the relatively low PG content of unloaded cartilage be explained? The answer may lie in the load history of


the tissue. DeWitt et a1 (17) have shown that embryonic chick epiphyseal chondrocyte cultures respond to repetitive tensile stresses with an increase in GAG synthesis. Likewise, Slack et a1 (16) have shown that repetitive tensile loads applied to embryonic chick tendon in organ culture produce an increase in sulfated GAG synthesis. Studies conducted in our laboratory, in which the effect of cyclic compressive stresses on GAG metabolism was examined in normal canine articular cartilage in vitro, showed that certain defined cyclic loads produce a rapid increase in net sulfate GAG synthesis (18). Notably, this change was rapidly reversible after elimination of the compressive load and subsequent culture of the cartilage under atmospheric pressure (18). Thus, GAG metabolism in cartilage is affected by loading of cartilage. However, whether this effect is directly due to compressive stress, or to strain, or to other factors is presently unclear.

Nonetheless, the data are consistent with the views that the higher PG content of loaded cartilage, compared with that of unloaded cartilage, is due to in vivo stimulation of GAG biosynthesis at that site under conditions of normal joint use. In view of the prompt reversibility of changes in GAG metabolism when stressed cartilage was placed under atmospheric conditions in vitro, it is not surprising that no differences in the rates of GAG synthesis were found in the present study, since the determinations were per- formed on cartilage slices cultured at atmospheric pressure. Finally, the present data emphasize that in vitro studies of cartilage GAG metabolism which are carried out under atmospheric pressure in the laboratory may not reflect metabolic rates which exist in the tissue during joint loading in vivo.

ACKNOWLEDGMENTS We are grateful to Michael Kinch, James Bean, and

Majorie Albrecht for technical assistance and to Margaret Britner and Roberta Fehrman for secretarial support.

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