Microinjury to the synovial membrane may cause disaggregation of proteoglycans in rabbit knee joint...

Journal of Orthopaedic Research 2:207-220, Raven Press, New York 0 1984 Orthopaedic Research Society

Microinjury to the Synovial Membrane May Cause Disaggregation of Proteoglycans in Rabbit Knee Joint

Articular Cartilage

Linda Frost and Peter Ghosh

Raymond Purves Research Laboratories, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales, Australia

Summary: Proteoglycans (PGs) isolated from articular cartilage (AC) of mature rabbits subjected to two or more consecutive intraarticular (IA) injections of sterile saline 24 h apart showed an aggregation defect in the presence of excess hyaluronic acid (HA). Although the PG contents of experimental and control cartilages were indistinguishable, a higher proportion of PGs were extractable from the 3 x IA tissues, as assessed by uronic acid analysis. Proteoglycans from experimental and control cartilages when examined by Sepharose CL-2B chromatography showed two subunit populations, the smaller (KAv = 0.70) containing more ketatan sulphate than the larger (KAv = 0.31). Cultures of AC from IA joints released more 35S0,-labelled PGs into the media over 72 h than control tissues and consisted mainly of PG degradation products although 20% could aggregate in the presence of HA. Ex- amination of PG aggregation 2 weeks after 2 x IA or 3 x IA injections showed that the defect initiated was still present; however, cartilage of immature rabbits was not affected by the 2 x IA procedure. Key Words: Synovium- Microinjury- Articular cartilage-Proteoglycan aggregation-Tissue culture.

In recent years there has been increasing interest in the use of animal models to investigate cartilage degeneration in experimentally induced arthropa- thies. Such models include severance of the ante- rior cruciate ligament (5 1,52,74,75); meniscectomy (13,14,23,28,49,56,57); meniscectomy and ligament transection (43,65,75,82); scarification or excision of cartilage surfaces (40,42,54); intraarticular (IA) injection of enzymes (5,20,21,38,39,41), irritants (12,27,30,46), or drugs (45). These models have provided a useful means of identifying the sequence of

This work was presented in part at the 29th Annual Meeting of the Orthopaedic Research Society, Anaheim, California, March 8-10, 1983.

Address correspondence and reprint requests to Dr. Ghosh at Raymond Purves Research Laboratories, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia.

biochemical events that occurs in the early stages of cartilage breakdown and in the long term may contribute to a clearer understanding of the patho- genesis of osteoarthrosis in man. In addition, many new (3 1,32) and currently used antiarthritic agents have been evaluated in various animal systems to ascertain their effects on cartilage integrity (3,4,50,80).

An essential requirement of such investigations is the establishment of suitable control experiments and it is evident from the existing literature that in some instances workers are not aware, or choose to ignore, the fact that microinjury to the synovial membrane caused by arthrotomy or IA administration of drugs may produce changes in joint articular cartilage. Although an early report by Key (46) that multiple IA iajections of sterile saline into rabbit joints produced arthritic-like changes has been re-

207

208 L. FROST AND P . GHOSH

cently substantiated (27,68,81,86), the effects of such treatment on proteoglycan (PG) aggregation in articular cartilage have not been previously described. In this paper we report the results of our studies on PG aggregation and release in culture from articular cartilage of rabbit knee joints following one, two, or three consecutive IA injections of sterile saline.

MATERIALS AND METHODS

Carrier-free sodium (35S)-sulphate in isotonic saline was obtained from the Radiochemical Centre, Amersham, Bucks., U.K. Guanidinium chloride (GuHC1) (Grade l), bovine testicular hyaluronidase (Grade I), phenylmethylsulphonylfluoride, benzam- idine hydrochloride, L-hydroxyproline, L-cysteine free base, D-glucuronolactone, and chondroitin-6- sulphate were purchased from the Sigma Chemical Company, MO, U.S.A. N-Ethylmaleimide was from Calbiochem-Behring, Carlingford, N.S. W., Australia, m-phenylphenol was an Eastman Kodak product purchased from Laboratory Supply, Sydney, Australia, and papain (puriss) was obtained from Fluka A.G., Buchs, Switzerland. Hyaluronic acid (as Heal-on-Vet@), Sepharose CL-2B, and Sephadex G- 10 were supplied by Pharmacia South Seas Division, Sydney, Australia. Dulbecco’s Mod- ified Eagles Medium was purchased from Gibco Laboratories, Grand Island, NY, U. S . A., foetal calf serum was from the Commonwealth Serum Labo- ratories, Melbourne, Australia, gentamycin was from Essex Laboratories, Sydney, Australia and tissue culture plates were from Sterilin, Teddington, Middlesex, U.K. Sterile isotonic saline (injectable form) was purchased from Boots Drug Co. Pty. Ltd., Sydney, Australia. Hexachlorophene was obtained from Essex Laboratories, Sydney, Australia. All other chemicals were analar grade or equivalent unless specified.

Mature (9-18 months) and immature (<3 months) New Zealand white rabbits obtained from the University of Sydney breeding colony were used for this study. The colony has been established for 23 years, and the date of birth of each animal recorded. This ensured that all animals used were age matched to within a few months. Articular cartilage PGs were prelabelled in vivo by intramuscular (gluteal) injection of Na?%O, (1.5 mCi/kg) 4 days prior to IA saline administration. Immediately before IA saline treatment rabbit joints were swabbed liberally with 70% ethanol containing hexachloro-

phene (5%). One milliliter of sterile saline (British Pharmacopoeia injectable quality) was injected into each rabbit joint using a 25 gauge x 3/4 inch needle. The process was repeated 24 and 48 h later to provide the three experimental situations, 1 X IA, 2 x IA, 3 x IA saline injections. In all instances, entry into the flexed joint was made along a line midway between the patella tendon and collateral ligament. This approach allowed introduction of the needle without injury to adjacent articular cartilage. Age-matched control animals received intramuscular (gluteal) Na;?3O4 (1.5 mCi/kg) but no IA injections and were sacrificed 5 , 6, or 7 days later depending on IA experiment used. Experimental animals were sacrificed with an intracardial injection of sodium pentabarbitone (100 mg) 24 h after the last IA injection. To investigate the ability of animals to recover from the IA treatment a group of mature rabbits were subjected to the 2 x IA (number = 4) and 3 x IA (number = 4) procedure but were sacrificed 14 days after the last injection.

After completion of the experiments, articular cartilage was dissected from the femoral condyles, suprapatella grooves, and tibial plateaux of each knee joint within 30 min of death. The tissues from these two joints were pooled and diced in sterile isotonic saline under aseptic conditions. Synovial fluid was also collected using aseptic conditions and was examined by Dr. V. Ackerman of the De- partment of Microbiology, Royal North Shore Hos- pital for microorganisms. All samples afforded neg- ative results. Aliquots of cartilage were extracted with 4.0 M GuHCl and the PG isolated by cesium chloride density gradient ultracentrifugation or set up in organ culture as described below. In animals used for autoradiography and histochemical studies the intramuscular dose of Na,35S04 used was 5 mCi/ kg, but the method of IA injection of saline (1 x IA, 2 x IA and 3 x IA) was the same as already described. After sacrifice, joint tissues were fixed in 10% neutral buffered Formalin containing 5% ce- tylpyridinium chloride, decalcified in 5% formic acid, washed, and imbedded in paraffin wax. Sag- ittal sections (5 pm) were cut through the synovium adjacent to the patella, femoral condyles, and tibial plateaux. Sections were stained with haematoxylin eosin or Alcian Blue or processed for autoradiography as described previously (29).

Organ Culture of Rabbit Articular Cartilage

Cartilage slices (approximately 1 mm2) from joints of controls and rabbits subjected to IA injection

J Orthop Res, Vol. 2, No. 3, 1984

MICROINJUR Y TO S YNOVIAL MEMBRANE 209

were layered on sterile grids in individual Sterilin plate tissue culture wells as described previously (32,53). Tissues were incubated in 2 ml Dulbeccos Modified Eagles Medium containing gentamycin, buffered with 100 mM sodium bicarbonate at pH 7.2, and supplemented with 20% (vol/vol) foetal calf serum. Cultures were incubated at 37°C with an at- mosphere of 95% air/5% CO, controlled with a Kev- atron COz monitor. Media were changed every 24 h over the culture period of 72 h and the radioactivity present per unit volume of media determined for the three time periods by scintillation spectrometry. The washed cartilage remaining after 72 h culture was then extracted for 48 h with 4.0 M GuHCl in tris buffer pH 7.4 containing the enzyme inhibitors described by Sandy et al. (70). The radioactivity per unit volume in these extracts was determined and when added to the amounts released into media over 72 h afforded the amount of 35S04-labelled PGs extractable from the cartilage by these two procedures. The proportion of 35S0,-labelled PGs released into culture media at each time period (24, 48, or 72 h) was calculated as a percentage of the total extractable 35S04-labelled PGs. The culture media remaining after these experiments was pooled, lyophilized, and examined by Sepharose CL-2B gel chromatography in the absence and presence of hyaluronic acid (HA) as described below. Cultures of nonviable cartilage were also examined using the same conditions and methods as above. Freshly dissected and diced articular cartilage was rendered nonviable by six freeze-thawing cycles in sealed tubes.

Extraction of Proteoglycans from Rabbit Articular Cartilage

Weighed aliquots of freshly diced articular cartilage (100-200 mg wet weight) were extracted with a 1.0 ml of 4.0 M GuHCl, buffered with tris-HC1 pH 7.4 containing enzyme inhibitors as described by Sandy et al. (70). After 48 h the extract was separated from the cartilage residue by low speed centrifugation and the radioactivity present in an aliquot of the supernatant determined by scintillation spectrometry. The washed residues (2 x 3 ml of 0.1 M NaAc) were suspended in 1 .O ml activated papain solution and digested for 16 h at 58°C. Ac- tivated papain was prepared by heating to 58°C for 30 min a mixture of papain (O.Smg/ml), 1.0 mM L- cysteine and 5 mM EDTA in buffered O. 1 M NaAc, pH 7.2.

The radioactivity of an aliquot of the clear tissue digest was determined by scintillation counting. The extractability of PGs from the rabbit cartilages was expressed as the percentage of radioactivity or uronic acid in the extract relative to the total amounts present in extract and residue, the latter being determined after papain digestion. Aliquots of the 4.0 M GuHCl extracts were subjected directly to Sepharose CL-2B chromatography or further purified by CsCl density gradient ultracentrifugation as described below.

Isolation of Rabbit Articular Cartilage Proteoglycans

Dissociative Conditions

Solid CsCl was added to 4.0 M GuHCl extracts of cartilage to give a starting density of 1.5 g/ml. Five milliliter samples were then centrifuged for 48 h at 32,000 rpm (105,000 x g) at 6°C in an MSE swing-out titanium rotor. The gradients were fractionated into 0.5 ml aliquots, and the percentage of radioactivity in each tube was plotted against its density. Fractions with p 2 1.56 g/ml were pooled and subjected to a second dissociative density gradient as described by Santer et al. (71). Fractions with p 3 1.58 g/ml were again pooled (80-95% of total radioactivity) and designated DID, (37) and subjected to Sepharose CL-2B chromatography in the presence and absence of excess HA.

Associative Conditions

Four molar GuHCl extracts of control and 2 x IA cartilage were dialysed against 10 volumes of double distilled water for 16 h. Cesium chloride was added to provide density of 1.61 g/ml and the mixture subjected to density gradient ultracentrifugation as described above. Samples were fractionated into 0.5 ml aliquots and the distribution of radioactivity and density determined as above. Fractions with p 5 1.68 g/ml (A,) fractions (37), were examined by Sepharose CL-2B chromatography in the presence and absence of excess HA.

Gel Chromatography and Hyaluronate-Binding Studies

The purified PG (A,), PG subunit (DID1) fractions, or 4.0 M GuHCl cartilage extracts were examined for their ability to aggregate in the presence

J Orthop Res, Vol. 2 , No. 3, 1984

210 L. FROST AND P. GHOSH

of excess HA according to the method of Har- dingham et al. (34). Samples were chromatographed on a Sepharose CL-2B (1.6 x 95cm) column, eluted with 0.5 M sodium acetate pH 6.8. The column was run at 10 ml/h regulated by a peristaltic pump (Model P-1 Pharmacia, South Seas, Sydney, Aus- tralia) in the absence and presence of excess HA (2% of total PG uronic) as described previously (34). The column void volume (V,) was determined using polymerised DNA and column total volume (V,) using phenol red or Na235S04. Chondroitin-6- sulphate prepared by papain digestion of rabbit articular cartilage was also used as a reference marker. Aliquots (0.5 ml) of the eluant fractions were counted by scintillation spectrometry for radioactivity (dpm) and uronic acid levels were determined by the method of Blumenkrantz and Asboe-Hansen (6). Both dpm and uronic acid values were plotted against elution volume.

The V, fractions obtained by Sepharose CL-2B chromatography of the culture media were lyophilized and also examined by Sephadex GI0 chromatography using 0.5 M sodium acetate pH 6.8 as eluant. The column was calibrated with blue dextran, Na,35S04, and tetrasaccharides prepared by papain and testicular hyaluronidase digestion of 4.0 M GuHCl extracted 35S0,-labelled PGs (48).

Hexosamine Analysis of Column Fractions

Pooled peak fractions eluting at KAV = 0.31 and KA, = 0.70 from the Sepharose CL-2B column corresponding to the PG subunit major and minor peaks, respectively, were dialysed (H,O, 100 vols) and lyophilized. The residues were dissolved in 1 .O ml of 4 N HCI and hydrolysed by heating at 105°C for 16 h. The galactosamine and glucosamine content of these hydrolysates was determined on the long column of a Jeol JLC-6AH automatic amino acid analyser.

Statistical Analysis of Results

All results were analysed using analysis of vari- ance and the Student’s t test. Groups were considered to be statistically significantly different when p < 0.05.

RESULTS

Evidence that PGs of mature rabbit knee joint articular cartilage were affected by two or more IA

injections of sterile saline was provided by the biochemical studies but not by the histological or au- toradiographic methods, which by standard histochemical staining techniques appeared normal (data not shown). Although the synovial membrane appeared normal for the 1 x IA animals, the 2 x IA and 3 x IA tissues showed some evidence of low grade synovitis (Fig. 1) as defined by local cell hy- pertrophy and plasma cell infiltration (33). These sites however were localized, presumably around areas damaged by entry of the needle into the joint, whereas other areas appeared histologically normal.

There was no statistically significant difference in the PG content (based on pg uronic/mg tissue dry weight) between 2 x IA, 3 x IA, and control tissues (Table 1). Although PG extractability as assessed by radioactivity measurements indicated no difference between control and experimental groups the uronic acid derived values obtained for the 3 x IA groups were higher than the controls. This difference was statistically significant (p < 0.01) (Table 1). The biochemical response to synovial microinjury became most apparent when the PGs were examined in more detail. When the 4.0 M GuHCl extracts of mature control cartilage were subjected directly to gel filtration chromatography on Sepharose CL-2B under associative conditions, three fractions were identified, one in the excluded volume V, and two in the included volumes with KAv = 0.31 and 0.70, respectively. The elution pro- file remained unchanged after dialysis of the sample prior to application to the column (data not shown).

After incubation with excess HA followed by Sepharose CL-2B chromatography, the size of the major included peak (K0.31) was reduced but the minor peak (K0.70) remained unchanged (Fig. 2). From these results and the work of others (59,70), we concluded that the excluded fraction corre- sponded to PG aggregate, and the and KO.,, fractions consisted of PG subunits. The K0.70 fractions were larger than chondroitin sulphate (KAv = 0.86) and when pooled, lyophilised, and analysed for galactosamine and glucosamine content were found to have ratios quite different from the major subunit fractions. As shown in Table 2, these smaller PGs consistently contained more glucosamine and less galactosamine than the major PG subunit, indicating that the keratan sulphate content was higher. The level of these small PGs was sub- stantially reduced after ultracentrifugation (Figs. 2B and C) presumably because of their low buoyant

J Orthop Res, Vol. 2 , NO. 3, 1984


FIG. 1. Sections of synovium taken from a rabbit receiving two intraarticular saline injections. Note the moderate synovial hyperplasia and scattered infiltration of plasma cells. Haematoxylin & eosin stain. x 365.

densities, which would band them in the D1D2 fractions, which we did not analyse. From the relative areas of the KO and KO,,, plus K0.70 peaks in the presence of excess HA, we estimated the proportion of nonaggregatable PGs in control rabbit car-

TABLE 1. Proteoglycan (PG) content and 4.0 M GuHCl extractability from articular cartilage of control

and experimental animals

Cartilage

PG content (as pg uronic acidlmg 26.2 1.5 26.1 f 1.0 25.4 f 1.7 tissue dry weight) (no. = 12) (no. = 8) (no. = 8)

PG extractability 86.7 -c 1.4 81.6 f 4.0 82.9 f 1.2 (as dpm) (no. = 16) (no. = 5) (no. = 3)

PG extractability 61.4 3- 0.4 61.5 f 3.2 66.7 f 1.0 (as uronic acid) (no. = 4) (no. = 8) (no. = 6)"

IA, intraarticular injection of saline; no. = number of animals used in

a Statistically significant relative to controls (p < 0.01). each group.

tilage to be 25-30% of the total PGs present. The small PGs that eluted at K0.70 represented approximately 15% of the total PGs present in 4.0 M GuHCl extracts (Table 3). The elution profiles of PGs obtained from the Sepharose CL-2B column using uronic acid values were found to be similar to those obtained monitoring radioactivity (Fig. 2). Hyaluronic acid binding studies using the Al fraction obtained after CsCl density gradient ultracentrifugation under associative conditions and the DID, isolated by using dissociative ultracentrifugation (Figs. 2B and 2C) confirmed that approximately 25-30% of the PGs extracted from the control cartilage aggregated in the presence of excess HA (Fig. 2). As discussed by Santer et d. (71), fractionation of the dissociative gradients at 1.5 g/ ml probably included some endogenous HA in the DID, preparation, and like these authors we observed the presence of a small proportion of PG aggregate at the void volume of the Sepharose CL- 2B profiles prior to the addition of exogenous HA.


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Since we were primarily concerned with the population of PG from the experimental rabbit cartilages that were incapable of aggregating in the presence of excess HA, contamination of our preparations by this glycosaminoglycan (GAG) was not expected to influence our conclusions. Articular cartilage from five mature control rabbits was examined by these procedures and, although some animal variability was evident, a mean and standard error of 28.7 & 2.8% nonaggregatable PGs was obtained based on the radioactivity profiles and 25.7 k 3.4% using the uronic acid values. These results are summarized in Table 3.

When the 4.0 M GuHCl extracts of cartilage of mature rabbits subjected to two consecutive 1 .O ml IA injections of sterile saline were examined directly on Sepharose CL-2B in the presence of HA or after density gradient ultracentrifugation, the proportion of nonaggregatable PG present was

greater than the corresponding preparations of control tissues (Fig. 3). The mean nonaggregatable PG value obtained for the treated group of seven animals was 43.0 k 1.5% using the radioactivity profiles and 38.6 & 1.0 using the profiles obtained by uronic acid analysis. The level of nonaggregatable PGs in extracts of the 2 x IA treated group was found to be statistically greater than for the control group (p < 0.05). The sizes of the PG subunits, however, as estimated from the KAV values were indistinguishable. Although the mean galactosamine/glucosamine ratios for the major (KAV = 0.31) and minor (KAv = 0.70) PG subunit populations isolated from the IA-treated cartilages appeared higher than control values (Table 2), the differences were not statistically different.

Although only investigated to a limited extent, it was consistently found by Sepharose CL-2B chromatography in the presence of excess HA that the

J Orthop Res, Vol. 2, N o . 3, 1984


TABLE 2. Hexosamine content of included peak fractions obtained by Sepharose CL-2B chromatography of rabbit articular cartilage proteoglycans (PG)

Major PG subunit population (KAv = 0.31)

Minor PG subunit population (KAv = 0.70)

Gala Glua GaliGlu Gal" Glu" Gal/Glu

Controls (no. = 4)

Mean f SE

2 X IA (no. = 8)

Mean f SE

3 X IA (no. = 6)

Mean f SE

0.073 0.095 0.102 0.139

0.035 0.054 0.062 0.088 0.060 0.017 0.129 0.082

0.122 0.077 0.078 0.129 0.136 0.057

0.049 0.066 0.081 0.100

0.019 0.027 0.094 0.080 0.044 0.01 1 0.079 0.053

0.075 0.051 0.050 0.097 0.098 0.028

1.48 1.45 1.27 1.39 1.40 f 0.05

1.83 1.96 1.62 1.10 1.36 1.45 1.63 1.55 1.56 f 0.09

1.63 1.51 1.57 1.33 1.38 2.00 1.57 2 0.10

0.059 0.085 0.69 0.068 0.105 0.65 0.041 0.063 0.66 0.063 0.097 0.65

0.66 f 0.01

0.024 0.029 0.81 0.030 0.049 0.61 0.036 0.053 0.66 0.057 0.088 0.65 0.062 0.091 0.68

0.65 0.080 0.82 0.71 0.085 0.84

Insufficient material

0.72 f 0.04

0.059 0.077 0.77 0.029 0.037 0.80 0.042 0.068 0.67 0.078 0.144 0.69 0.056 0.088 0.64 0.043 0.053 0.81

0.73 f 0.03

Concentrations of galactosamine (Gal) and glucosamine (Glu) are expressed as pnol/ml of sample. IA, intraarticular injection of saline. a Since the concentrations of PGs in individual samples varied, the absolute amounts of Gal and

Glu are different in each sample; however, their ratios may be compared. No statistically significant differences were found between experimental groups, but the ratios for major and niinor PG populations were highly significantly different (p < 0.005).

TABLE 3. Distribution of proteoglycan (PG) aggregate and subunits in 4.0 M GuHCl extracts of experimental and control rabbit articular cartilage as determined by Seuharose CL-2B chromatoarauhv

Before HA addition (% of total) After HA addition (% of total)

PG aggregate PG subunit PG (small) PG aggregate PG subunit PG (small) (KAV = 0) (KAv = 0.31) (KAv = 0.70) (KAv = 0) (KAv = 0.31) (KAv = 0.70)

Controls (no. = 5) 28.7 f 2.8 15.2 f 2.1 Dpm (35S04) mean 2 SEM 19.0 f 3.1 63.3 f 2.9 16.7 i 1.4

Uronic acid mean 2 SEM 20.7 2 1.3 63.8 f 3.5 15.6 f 2.2 60.1 f 1.0 25.7 f 3.4 14.3 f 2.5

Dprn (35S04) mean * SEM 16.5 +. 2.0 63.5 f 2.4 17.6 * 4.9 42.8 t 3.2 43.5 f 1.5 13.7 f 2.1 Uronic acid mean f SEM 24.1 f 3.5 61.5 f 4.1 14.5 f 3.5 42.7 f 1.9 38.6 2 1.0 18.5 f 0.9

56.1 f 4.0

Treated (2 x IA) (no. = 5)

Treated (2 x IA) followed by 14 days recovery (no. = 4)

Dprn (35S04) mean f SEM ND ND ND 33.1 * 2.2 51.1 f 2.7 15.8 f 1.0 Uronic acid mean f SEM 30.7 f 2.1 49.8 f 1.6 19.5 f 1.9

Dpm (35S04) mean 2 SEM 18.1 2 3.7 65.4 f 6.9 16.5 f 3.8 36.4 f 3.7 49.0 f 2.4 14.4 f 1.8 Uronic acid mean t SEM 19.4 t 2.5 61.5 f 2.4 19.9 f 1.9 35.7 ? 2.7 45.4 t 2.7 18.8 f 1.0

Treated (3 x IA) (no. = 3)

Treated (3 x IA) followed by 14 days recovery (no. = 4)

Dpm ( 3 5 S 0 , ) mean f SEM ND ND ND 30.5 f 1.5 53.2 f 0.8 16.3 f 2.0 53.4 i 1.4 16.4 2 1.3 Uronic acid mean f SEM

HA, hyaluronic acid; IA, intraarticular injection of saline; ND, not determined; SEM, standard error of the mean.

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J Orthop Res, Vol. 2 , No. 3 , 1984


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proportion of nonaggregatable PGs isolated from cartilage of rabbit joints that received 3 x IA saline was greater than the proportion extractable from cartilage of joints subjected to 2 x IA treatment or control cartilage (Fig. 4). When the PGs of cartilages of rabbits that were subjected to 2 x IA or 3 x IA treatment but not sacrificed until 14 days later were examined by the above procedures, it was found that the PG aggregation levels were still de- pressed relative to control values. These results are summarized in Table 3. In contrast, a single IA injection of saline did not alter the level of PG aggregation in joint articular cartilage relative to control values (data not shown). Furthermore the cartilage of joints from immature rabbits (<3 months old) appeared to be refractory to the 2 x IA saline treatment since the proportion of nonaggregatable PG extracted from control and treated joints was equiv-

alent (Fig. 5) . The effects of three consecutive IA injections on immature rabbit joint cartilage were not investigated.

Organ Culture Studies

The release of 35S0,-labelled PGs into media from cultures of articular cartilage of control or 2 x IA-treated rabbits was followed over 72 h. As can be seen from Table 4 the proportion of PGs present in the media after 24, 48, and 72 h was al- way,s greater in cultures of cartilage from the 2 x IA-treated group than in media controls. This difference was statistically significant.

The polydispersity of PGs released into the media of cultured viable and nonviable rabbit cartilage as well as their ability to aggregate was also examined by gel exclusion chromatography on Sepharose CL-



Elut ion Volume ( m l s l FIG. 4. Radioactivity (A) and uronic acid (B) Sepharose CL- 2B chromatography profiles in the presence (---) and absence (-) of hyaluronic acid (HA) of 4 M GuHCl extracts of cartilage from mature rabbits that received three consecutive intraarticular injections 24 h apart (3 x IA). V,, Vpgs, V,,, and Vso4 are elution volumes corresponding to void volume, proteoglycan subunit, chondroitin sulphate, and Naz5S04, respectively.

2B. The results are shown in Fig. 6. In cultures of both control and 2 x IA-treated cartilage most PGs released into the media over 72 h were incapable of aggregating. Approximately 70% of PGs in the control media was included in the Sepharose CL-2B column in the presence of HA whereas 80% of PGs from the 2 x IA cultures were retarded on the column (Fig. 6). In addition the PGs present in the media of cultures of cartilage from the 2 x IA- treated group were more polydispersed than those present in the control cartilage media as was evident from the relative size and broad elution profiles obtained on Sepharose CL-2B (Fig. 6). Media of viable cartilage cultures of both control and 2 x IA-treated animals contained material corresponding in size to chondroitin-6-sulphate (KAV = 0.86) and free 35S04. However, free 35S04 was di- minished in media of the 2 x IA nonviable cartilage cultures (Fig. 6D) and was virtually absent in the nonviable cartilage cultures of the control group (Fig. 6B).

DISCUSSION

The present study clearly shows that two or more IA injections of sterile saline into the knee joints of mature but not immature rabbits may increase the proportion of nonaggregatable PGs extractable from articular cartilage. As would be expected, this early

biochemical defect involving PG aggregation rather than net loss from the tissue was not readily apparent by the traditional histological techniques. The distribution of radiosulphate and intensity of staining was observed to be more variable in sections of cartilage from the saline-treated joints than in control sections but the differences were not suf- ficiently consistent to allow unequivocal interpre- tation.

Our present and previous findings (3 1,32) that 70-75% of PGs in 4.0 M GuHCl extracts of mature New Zealand white rabbit control articular cartilage were capable of aggregating in the presence of HA compare favourably with the studies of Sandy et al. (70) but conflict with the findings of Oegema and Behrens (59), who reported considerably lower (10- 20%) PG aggregation in purified preparations from articular cartilage of the same breed of rabbit. Mos- kowitz et al. (57) also examined PG aggregation in 4.0 M GuHCl extracts of mature New Zealand white rabbits using the analytical centrifuge. This group found that under associative conditions (A, fractions) only 25% of the PGs present sedimented in the form of 59s aggregates. Although the effects of the addition of exogenous HA on sedimentation behaviour were not investigated, Oegema and Beh- rens (59) reported that sufficient HA was present in rabbit cartilage to enable all PGs present to aggregate; moreover, aggregation in their control A, fractions was not greatly affected by the addition of HA.

Although the reasons for this difference are as yet unresolved, it is possible that the “North Amer- ican” New Zealand white rabbit is different from the Australian strain at least with respect to their cartilage PGs (T. R. Oegema, personal communi- cation). Our values however are closer to the PG aggregation levels reported for other hyaline cartilages (2,37,44,47,51,60,64,71). In this context it is noteworthy that the present study also showed that rabbit articular cartilage contains a small nonaggre- gating PG species (KAV = 0.70), which from galactosamine and glucosamine analysis was demonstrated to be richer in keratan than the main subunit population (KAV = 0.31). This species of PG may be analogous to those recently described in human articular cartilage by Bayliss et al. (2), although in our preparations this population did not reaggregate in the presence of excess HA.

The increased proportion of nonaggregatable PGs in cartilage of joints subjected to the 2 x IA and 3 x IA treatment as demonstrated by the HA-binding

J Orthop Res, Vol. 2 , No . 3 , 1984

216

2 6000 7 ? 4.000

E

a-

I - 0

2,000

0 0

L. FROST AND P . GHOSH

i :5, :: i :

FIG. 5. Radioactivity (dpm) and uronic acid (OD,,,) Sepharose CL-PB chromatography profiles in the presence (---) and absence (-) of excess hyaluronic acid (HA) of proteoglycans (PGs) from immature rabbit control cartilage (A, C, and E) and those subjected to two consecutive intraarticular injections (B, D, F) of sterile saline. Diagrams (A- D) show chromatograms of 4.0 M GuHCl extracts whereas (E and F) are D,D,, preparations obtained by dissociative density gradient ultracentrifugation. V,, Vpgs, V,,, and Vso4 correspond to elution volumes of void volume, PG subunit, chondroitin sulphate, and Naq5S0,, respectively.

0

E 0

2000

I I '----. '. 80 120 40 80 120

Elution Volume (mls)

experiments suggests a loss or defect in the HA-binding region of the PG protein core. Similar observations have been reported in experimentally induced cartilage degeneration in the canine (44,61,62) and rabbit (3 1,3237) as well as in osteoarthritic human cartilage (8,9,85). In addition, latent metalloproteo- glycanases (72,73) have been isolated from human articular cartilage, which can degrade both PG aggregate and subunit in vitro. Although enzymatic cleavage at the HA-binding region of the core protein has been suggested to represent an early step

in the degradation of cartilage PGs ( l ) , others consider the chondroitin sulphate rich region as the more likely site (51,52,58,71). Our tissue culture experiments as well as those of Sandy et al. (70) provided direct evidence for the presence within rabbit articular cartilage of enzyme systems capable of degrading PGs. Most PGs released into media over 72 h were incapable of aggregating in the presence of excess HA and were more polydispersed than PGs extracted by 4.0 M GuHCl from noncultured cartilage (cf. Fig. 6 with Fig. 2).

J Orthop Res, Vol. 2 , No . 3, 1984

MICROINJUR Y TO SYNOVIAL MEMBRANE 217

TABLE 4. Percent of total extractable "SO, proteoglycans (PGs) released into media over 72 h from

cultures of rabbit articular cartilage

Culture period (h)

24 48 72

Control (no. = 8) cumulative mean f SEM 25.5 ? 0.7 33.4 ? 1.3 39.5 f 1.5

(no. = 7) cumulative mean f SEM 28.3 f 1.2 38.0 * 1.7 45.7 2 2.1

Control versus treated p < 0.0Y p < 0.05" p < 0.0Y

Treated (2 x IA)

Percent extractable PGs released into media = dpm (media)/

IA, intraarticular injection of saline; SEM, standard error of dpm (media) + dpm 4.0 M GuHCl extract x 100

the mean. Statistically significant.

Significantly, the degradation that occurred in cultures of cartilage from 2 x IA-treated joints was greater than in control cartilage as demonstrated by the higher proportion of nonaggregatable PGs present as well as their polydispersity. Apparently the degradation of PGs in cultures of rabbit articular cartilage was not entirely dependent on the viability of the chondrocytes, as apart from the raised level of free 35S0,, which may have arisen from lyso- soma1 sulphatase activity, media of nonviable cultures contained a spectrum of PG degradation products quite similar to those present in media of viable cultures (Fig. 6). This result suggests that most enzyme activity within the cartilages was established prior to initiation of the cultures and was not a con- sequence of it. Moreover, we deduce from the culture experiments that the level of enzyme activity present in the cartilage of the 2 x IA group was greater than in control cartilage.

The question as to how microinjury to the synovium might enhance enzyme activity within articular cartilage is of fundamental importance since there is still much debate concerning the role of synovitis in the breakdown of articular cartilage in osteoarthritis. Several avenues are worthy of explo- ration, assuming that the transitory dilution of synovial fluid by sterile physiological saline was not a contributory factor. Enzymes released by stimulated macrophage (14,35,36,84) and other cells present in the synovium might diffuse directly into cartilage. Similarly prostanoids or their precursors produced during acute tissue injury (63,81) could

80 120 L C 80 120 Elution Volume ( m l s )

FIG. 6. Sepharose CL-2B chromatography profiles in the presence (- - -) and absence (-) of excess hyaluronic acid (HA) of lyophilized media collected over 72 h from cultures of viable and nonviable mature rabbit cartilage. A: Media from cultures of viable cartilage from control rabbit joints. B: Media from cultures of nonviable control cartilage. C: Media from viable cartilage cultures of rabbit joints subjected to two consecutive intraarticular injections 24 h apart (2 x IA). D: Media from cultures of nonviable cartilage from 2 x IA treated animals. V,, Vpgs, V,,, and Vso4 correspond to elution volumes of void volume, proteoglycan subunit, chondroitin sulphate, and Na;5S0,, respectively.

enter joint cartilage and stimulate the chondrocytes to release degradative proteinases (24,25,81). Con- sideration must also be given to catabolin, the low molecular weight polypeptide isolated from porcine synovium (69), which has been shown in vitro to induce resorption of cartilage by the chondrocyte (18,19,22,79).

Since the histological study showed only moderate synovial hyperplasia, it is unlikely that degradative enzymes were derived directly from this tissue. In addition such enzymes would have to diffuse across the synovial cavity without inactivation by the enzyme inhibitors, which are present in large amounts (77). The cellular origin of catabolin has not been totally resolved but the synovium or in- vading mononuclear cells are considered (14, 16,17,66,79) to be the most likely sources of this



polypeptide. It is possible, therefore, that catabolin was implicated in the initiation of proteolytic activity in our system. In view of the acute nature of the synovial injury we consider the prostaglandins or their precursors to be the most likely mediators of chondrocyte activation. Prostaglandins are known to be secreted from cultures of rheumatoid synovium and synovial cells (15,67,83) and can influence cartilage metabolism by raising intracellular levels of cyclic AMP. Cyclic AMP has been shown in vitro (7,10,87) to increase the synthesis and release of lysosomal enzymes from a variety of cells and can promote degradation of PGs in rabbit ear (76) and canine (78) hyaline cartilages. Moreover, IA injection of E-prostaglandins into mature rabbit or canine joints induced loss of PGs from the cartilage matrix, whereas immature animals were found to be refractory to this treatment (24). In this context it is noteworthy that in our experiments the PGs of immature rabbit joint cartilages were not affected by the 2 x IA treatment. Others (20) have also noted the capacity of immature joint cartilage to recover from synovial injury.

It thus seems clear from the present investigation that damage to the synovial membrane of rabbit joints can influence the turnover of PG in articular cartilage, the intensity of the effect being related to the frequency of the insult and the age of the animal used. Although the effects of arthrotomy on cartilage metabolism have been well documented (1 1,23,26,40,42,43,54-56,65), there has been limited (27,39,46,68,86) recognition that violation of the synovial membrane by IA injections may also be deleterious to the short term (<14 days) integrity of matrix PGs.

Acknowledgment: We acknowledge financial support for this project from the National Health and Medical Research Council and thank Mrs. Beverly Horsburgh, Department of Veterinary Pathology, University of Sydney, for the preparation of histological sections and autoradiographs and Mrs. Joan Sutherland of these laboratories for the hexosamine analysis. We are also grateful to Mrs. Julianne Renwick for her competent as- sistance in the preparation of this manuscript.

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