Journal of Orthopaedic Research 9 6 5 M 1 3 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society

Proteoglycan Alterations During Developing Experimental Osteoarthritis in a Novel Hip Joint Model Sven Inerot, Dick Heinegiird, *Sten-Erik Olsson, ?Hans Telhag, and /Lars Audell Department of Physiological Chemistry, University of Lund, Lund; *Laboratory for Comparative Pathology, Wentholmens G i r d , Farentuna; ?Department of Orthopedic Surgery, Malmo General Hospital, Malmo; $Department of Clinical Radiology, the Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden

Summary: Degenerative hip joint disease was induced in dogs by extraarticular surgery that created a condition that mimics hip dysplasia. Decreased acetabular coverage of the femoral head gave altered mechanical load, with ensuing cartilage degeneration. For comparison, degenerative knee joint disease was induced in other dogs by transection of the anterior cruciate ligament of the knee. The femoral head articular cartilage showed macroscopic signs of degeneration within a month. No macroscopical changes of synovitis were present. Chemical analysis of cartilage samples showed loss of proteoglycans. Guanidine hydrochloride extracts of the cartilage contained proteoglycan fragments that could be separated by equilibrium density gradient centrifugation in cesium chloride. The data indicate that proteoglycans are fragmented by proteolytic cleavage and lost from the cartilage. The proteoglycans remaining in the tissue are smaller and have lost the ability to aggregate with hyaluronic acid. Similarly, in experimental knee joint osteoarthritis, the proteoglycan content of the cartilage decreased. The structural changes of those proteoglycans remaining were of a different nature, with no changes in proteoglycan size or aggregation properties, possibly indicating that both degradation and repair took place in the knee articular cartilage and/or that fragments were rapidly lost from the tissue. This may follow from different surgical procedures, only the one used for the hip joint being extra-articular, or from the different anatomy and physiology of the hip joint and the knee joint. Key Words: Osteoarthritisdysplasia. Surgical induction-Proteoglycan-Canine-Hip

Articular cartilage is composed mostly of water (60430%) held in an abundant extracellular matrix. The matrix is composed of collagen (70% of the cartilage dry weight), proteoglycans (20-30% of dry weight) and small amounts of other tissue proteins (24). Collagen forms fibers that are responsible for the tensile strength of the cartilage and the proteo-

glycans are the main factor determining the elasticity (compressive stiffness). Articular cartilage proteoglycans are macromolecules with molecular weights exceeding lo6 daltons (19,20). They contain a central protein core, to which a large number of highly negatively charged glycosaminoglycan chains are covalently attached. The proteoglycans have the capacity to bind to hyaluronic acid by noncovalent specific bonds, thereby creating large proteoglycan aggregates with molecular weights on the order of 50-100 X lo6 daltons. This is an important functional characteristic of the proteoglycans,

Received December 6, 1989; accepted December 20, 1990. Address correspondence and reprint requests to Dr. Dick Heinegird, Department of Physiological Chemistry, Lund University, Box 94, S-221 00 Lund, Sweden.

658

EXPERIMENTAL OA which is useful in the study of alterations in their structure. Several regions in the proteoglycans either containing mainly protein, keratan sulfate, or chondroitin sulfate have been identified (19). These are in the N-terminal end, the hyaluronate binding region (globe 1) and globular domain 2, neither containing any glycosaminoglycanchains and in the Cterminal end, the lectinlike globular 3 domain. The major part of the proteoglycan containing the glycosaminoglycan chains is inserted between these terminal globular domains. It contains two rather distinct domains: one carrying keratan sulfate chains and located close to the N-terminal end (18), i.e., the keratan sulfate-rich region, and one in its turn carrying two chondroitin sulfate-rich domains (18,19). Changes in the relative proportions of these structures may be interpreted in terms of removal of specific regions by degradation of the proteoglycans. Osteoarthritis, a degenerative joint disease with early clinical signs being articular cartilage destruction, is common in both man and domestic animals. The disease is insidious in onset with symptoms appearing rather late. By the time the symptoms are severe enough to get the patient to seek medical care, the changes in the cartilage are usually well advanced. Therefore, to disclose the early changes in osteoarthritis, it has been necessary to develop animal models, where the start of the process is known as the time of surgical intervention (2,6,1013,15,23,29,31,38,40-44,49). Most of these models involve the use of intra-articular surgery to change the load on the cartilage. The most widely used procedure is induction of knee joint instability by transection of the anterior cruciate ligament of the knee in dogs (31,40,41). This model appears to produce a nonprogressive cartilage degeneration (9). Chemical studies of osteoarthritic cartilage have shown marked loss of proteoglycans (5,7,8,32,33, 50,53). Furthermore, the structure of remaining proteoglycans is altered (24). McDevitt et al. (37) and McDevitt and Muir (36) reported an early increase in the ratios of galactosamine/glucosamine and uronic acid/protein in the proteoglycans isolated from knee articular cartilage taken from dogs, in which cartilage degeneration has been induced by transection of the anterior cruciate ligament. In a study of osteoarthritis caused by naturally occurring hip dysplasia in dogs (26), the structure of proteoglycans from the degenerated hip articular cartilage was shown to be altered. The isolated proteoglycan monomer had a smaller size and a higher

659

uronic acid/protein ratio, and their aggregation with hyaluronic acid was reduced. It has been suggested that degradation of the proteoglycans is an important factor in the development of osteoarthritis (22,30,55). In support, Ali and Evans (l), Shoji & Granda (50), and Sapolsky et al. (47,48) reported increased activity of cathepsins and neutral proteases and concomitant loss of uronic acid in osteoarthritic cartilage. One problem in studies of the degenerative process in experimentally induced osteoarthritis is the inflammatory process caused by intra-articular surgery. The inflammation may in itself interfere with normal turnover of cartilage macromolecules (1432). The present investigation studies changes in proteoglycans in a novel model that mimics early osteoarthritis, with no inflammatory response. In this model, an extra-articular approach is used for the induction of osteoarthritis. A hip dysplasia-like condition is induced by osteotomy and inward rotation of the osteotomized part of the pelvis. The change in load-bearing of the femoral head triggers cartilage degeneration without concomitant acute synovitis that is common in those models in which an intra-articular approach is used (28). For comparison, a commonly used knee joint model (transection of the anterior cruciate ligament), in which synovitis is a frequent complication, was studied. MATERIALS AND METHODS

Surgical Method The surgical technique for induction of hip osteoarthritis reported in the present report is a modification of the operation used by Hohn and Janes (21) for the treatment of hip dysplasia in the dog. In its turn, the surgical procedure of Hohn and Janes (21) is an adaptation for the dog of a procedure developed by Salter (45) and used by him for the treatment of congenital dislocation of the hip in children. The surgical intervention described in the present paper decreases the acetabular coverage of the femoral head. Thereby, a slight subluxation of the femoral head is induced, with a resulting shift in loadbearing to the lateral side of the acetabulum. The operation must be done with the dog under general anesthesia using a strict aseptic technique. The dog is placed on the operating table in lateral recumbency, and after the necessary preparation for surgery, a skin incision is made from the cranial

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dorsal iliac spine along the lateral border of the gluteus medius muscle and dorsal to the greater trochanter to the ischiatic tubercle. The skin and the S.C. tissues are retracted to expose the gluteus medius, the tensor fasciae latae and the biceps femoris muscles. The tensor fasciae latae are dissected from the gluteus medius and retracted cranioventrally. The gluteus medius, the gluteus profundus, and the iliopsoas muscles are elevated subperiosteally from the iliac body, which is thereby exposed. The ramus of the ischium is exposed subperiosteally by elevating the internal and the external obturator muscles as far as the obturator foramen. Protecting the sciatic nerve, the iliac body is osteotomized in a stepwise fashion using a Stryker saw, and the ramus of the ischium is osteotomized with a Stryker saw or with a wire saw (Fig. 1). The two ends of the body of the iliac bone are fixed together with a bone screw in such a way that the cranial portion of the iliac bone is lateral to the caudal portion (Fig. 1). This fixation and the fact that the ramus of the ischiatic bone is also osteotomized mean that the osteotomized part of the pelvis changes position. Because this part is attached to the rest of the pelvis at the pelvic symphysis, the acetabular coverage of the femoral head decreases (Figs. 2 and 3). Materials

Hip Model Series Thirteen dogs divided into three series (A, B, and C ) were used in the study (Table 1). Eleven of the dogs were greyhounds and two were German wirehaired pointers. The ages of the dogs varied from 12 to 18 months. The difference between the three series was mainly the method by which the hip joints

were examined postmortem. The dogs in series A were littermates. The greyhounds were kept in large outdoor pens (3 x 10 m), each equipped with a well-insulated doghouse. The two German wirehaired pointers were kept indoors in pens measuring 2 X 3 m. The dogs were fed water ad libitum and were given a well-balanced commercial dog food. A few days before and after surgery the dogs were kept indoors in large cages. The left hip joints of the dogs were operated on with pelvic osteotomy. Extensive analyses were performed on cartilage samples (taken 15-59 days after surgery) from four 1-year-old littermate greyhounds of racing lineage (series A). In addition, samples primarily used for histology taken 119 and 153 days after surgery from two German wirehaired pointers were also included (series C ) . From another series of greyhounds the samples removed were too small for complete analysis (series B). Only some analytical data from these dogs are therefore included. At the time of sacrifice, cartilage was excised from areas of the femoral heads that showed focal degeneration, identified by discoloration, loss of normal glossiness, and erosion of cartilage. When no such sign of degencration was seen, cartilage was taken from a semilunar area just superior to ligamentum teres. This area is the main weight-bearing portion of the cartilage, where also most of the changes are seen in more advanced cases. Normal cartilage from the side not operated on was obtained from corresponding sites. Knee Model Series

Three mongrel dogs of medium size 1 year of age, were operated on with transection of the anterior B

A

FIG. 1. Schematic drawing of left side of a canine pelvis to demonstrate the surgical technique. A: Stepwise osteotomy of the iliac bone (large arrow) and straight osteotomy of the ischiatic bone (small arrow). B: Osteosynthesis of the iliac bone with the cranial fragment lateral to the caudal fragment.

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A

FIG. 2. The macerated pelvis of dog A5 (killed 26 days after surgery). A: Ventral view demonstrating how the osteotomized part of the pelvis including the acetabulum has been tilted to provide less good coverage and support for the femoral head. B: Dorsal view demonstrating the inward tilt of the osteotomized part of the pelvis achieved by the pelvic osteotomies and the side-to-side osteosynthesis of the iliac bone.

cruciate ligament of their left knee as described by Pond and Nuki (41). After 27, 107, and 134 days the dogs were killed. Normal and degenerated cartilage was excised from corresponding sites on the area of the tibia1 plateau not covered by the menisci. Histological Methods

Histological examination of the joint cartilage of the hip joints was performed in six of the dogs. The specimens were fixed in 10% neutral formaldehyde, embedded in paraffin, and cut in 6-pm thick sections. They were stained with hematoxylin and eosin, toluidine blue, and alcian-periodic acidSchiff. A

Chemical Methods

The cartilage samples taken for chemical analysis were frozen in liquid nitrogen immediately after dissection and stored at - 80" until pulverized in liquid nitrogen, as has been described previously (26). Extraction was performed as described elsewhere (16) using 4 M guanidine-HC1 solutions containing protease inhibitors (39). The proteoglycan aggregates (A1 fraction) were isolated by CsCl gradient centrifugation in 0.4 M guanidine-HC1, pH 5.8, starting density 1.62 g/ml, as described previously (26). The proportions of aggregate/monomer in the A1 fractions were determined by chromatography on Sepharose 2B. To obB

FIG. 3. Ventrodorsal radiographs of the pelvis of dog C1 taken 1 (A) and 2 months (B) after surgery. The side-to-side osteosynthesis of the iliac bone, increase in callus formation with time at the osteotomized sites, and decreased acetabular support of the left femoral head are well demonstrated.

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S . INEROT ET AL. TABLE 1. Characterization of alterations with time after surgery

Dog

Breed

A1

Greyhound

A2 A3 A4 A5

Days kept after surgery

Complication

Macroscopical alterations in cartilage

Histological finding in cartilage

None

Greyhound Greyhound Greyhound Greyhound

Stolen on day 10 10 15 19 26

A6 B1 B2

Greyhound Greyhound Greyhound

59 10 19

None None None

B3

Greyhound

None

B4

Greyhound

36 (lost but retrieved) 64

None None Loss of glossiness Rough surface, yellow discoloration Erosion None Slight loss of glossiness Erosion

Paresis

Erosion

Necrosis and pannus formation

B5

Greyhound

Cl

German wirehaired pointer German wirehaired pointer

119

Ventricular fibrillation of the heart None

Deep erosion

153

None

Erosion

Changes varying from fraying to erosion Changes varying from fraying to erosion

c2

Died during surgery

Infection None None None

tain the distribution of all the proteoglycan monomers by chromatography on Sepharose 2B, the protein in the hyaluronic acid binding region of the proteoglycan monomer was unfolded by reduction and alkylation to prevent the proteoglycan monomers from forming aggregates with hyaluronic acid. Size distribution of the chondroitin sulfate side chains was obtained by chromatography on Sephadex G 200 of papain-digested A1 fractions. Hexosamines were quantified after hydrolysis of samples in 4 M HCl for 10 hours at 100°C in sealed tubes under argon. A Durrum automatic amino acid analyzer was used as described previously (25). Glucosamine and galactosamine contents in glycosaminoglycans were determined after chromatography of papain digests of proteoglycans on DEAEcellulose (Whatman DE32) to purify the glycosaminoglycans as described elsewhere (3). It was assumed that glucosamine provided a measure of keratan sulfate and hyaluronic acid, galactosamine a measure of chondroitin sulfate, and their sum a measure of total glycosaminoglycans or proteoglycans. Hyaluronic acid was quantified after treatment of A1 fractions (12 pg/ml) in 0.1 M sodium hydroxide for 40 hours at 20°C. A radioligand method analogous to the one described by Tengblad (54) using '251-labeled hyaluronic acid binding region was used (Wieslander and Heinegird, unpublished report). Hydroxyproline contents in extracts

J Orthop Res, Vol. 9, No. 5 , 1991

Fraying of surface None Fraying of surface

and residues were determined according to Stegemann and Stalder (51). Uronic acid was determined according to Bitter and Muir (4)or by an automated version of this method (17). Protein was determined using the microprocedure of Lowry et al. (27). Statistical Methods

Statistically signifcant differences between chemical parameters from the hip joint operated on and from the contralateral hip joint were evaluated by Wilcoxon rank sum test for paired observations.

RESULTS Hip Model Osteoarthritis Thirteen dogs were operated on. In ten dogs, the surgical intervention and the postoperative period were uncomplicated, but two of these dogs were stolen and only one was retrieved. Of the remaining three dogs, one died of ventricular fibrillation during surgery, and in one dog there was hemorrhage and wound infection with apparent involvement of the hip joint. In the third dog, a traumatic injury to the ischiatic nerve caused paresis of the left hind leg. The 10 dogs without postsurgical complications put weight on the left hind leg within a day or two

663

EXPERIMENTAL OA

after surgery. A slight limp remained for 2 4 weeks after surgery. Postoperative radiographs of the pelvis were taken of all the dogs at varying intervals (Fig. 3). One radiograph was always taken immediately before they were killed. Cartilage Morphology

The articular cartilage from the joint of the side operated on showed macroscopical degenerative lesions at postoperative days 19,26, 36, and 59 (greyhounds) and at 119 and 153 days (German wirehaired pointers) after the operation. Degeneration, i.e., erosion of the cartilage surface, was observed in the dogs that were killed at 36 days and later after the operation. The dog killed 15 days after surgery showed no macroscopical signs of degeneration. These findings indicate that the induced degeneration is progressive and gradual in development. The changes are listed in Table 1. In none of the dogs did the control side (not operated on) show any detectable changes. Acute synovitis was not observed in any of the dogs. In the first series of greyhounds (A) cartilage was taken only for biochemical studies. In the four greyhounds of the B series, small samples of the cartilage (1 mm in diameter) of the left and right femoral head were punched out for histology using a special instrument. In dog B1 (Fig. 4) there was fraying of the surface of the cartilage and some loss of stainability, whereas dog B2, although killed after the longer time of 19 days, showed no histological lesion. In dog B4 (the one with paresis, killed at 67 days), the cartilage was necrotic and pannus formation had appeared. The slight discrepancy between macroscopical and histological findings, particularly in dog B2, may be due to the small samples not being fully representative of the lesion. In dogs C1 (killed at 119 days) and C2 (killed at 153 days), several sections of the entire femoral head were taken from each side for histological examination. Sections were also taken from the acetabulum of each side. Changes of different severity were seen in three sections. They varied from fraying of the surface, cluster formation of the chondrocytes, and loss of stainability to deep erosion (Figs. 5-8). It was also observed that the bone underlying the cartilage with the various lesions had undergone some atrophy. The bone trabeculae were thin and slender in comparison with those of the correspond-

FIG. 4. Histological section of a punched out specimen (1 mrn in diameter) of the articular cartilage from the left fernoral head of dog B1 (killed 10 days after surgery). There is fraying and slight loss of stainability of the surface layer of the cartilage (hematoxylin-eosin x 125).

ing area of the right femoral head (Fig. 6). Also, the calcified part of the joint cartilage seemed to have undergone some atrophy (Fig. 7). Cartilage Composition

Decreasing contents of glycosaminoglycans in degenerating articular cartilage have been a frequent finding in osteoarthritis (7,26,30,37). In the present investigation, at 15 days after operation, the articular cartilage from the operated hip already had a lower content of glycosaminoglycans compared with the control side (Fig. 8) as had all corresponding cartilage samples taken later in the postoperative period. This difference was significant (p < 0.05). Therefore, an early sign of degenerative joint disease is loss of glycosaminoglycans, i.e., proteo-

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FIG. 5. Histological section of the articular cartilage and subchondral bone from the weightbearing area of the left femoral head of dog C2 (killed 153 days after surgery). The cartilage is thinner than normal and the surface is frayed (hematoxylineosin x 60).

glycans, from the affected cartilage. In analogy, available data show that loss of metachromasia is an early event in osteoarthritis (24). Proteoglycan Composition

Studies of structural changes of those proteoglycans remaining in the articular cartilage provided some information on the mechanism of loss of the molecules from the tissue. Proteoglycans were extracted with 4 M guanidine hydrochloride in significantly lower yields (p < 0.05) from the hip which was operated on, compared with the control side (Fig. 9b). It is possible that extractable proteoglyA

cans are lost from the tissue to somewhat greater extent than those not extractable, which are recovered in the residue (Fig. 9b and c). Extracted proteoglycans were purified by density gradient centrifugation under associative conditions to yield the aggregate containing A1 fraction. Analysis of these isolated proteoglycans showed increased relative contents of chondroitin sulfate (measured as uronic acid in glycosaminoglycans) compared with protein (Fig. 10). The uronic acid/ protein ratio was increasingly higher with longer time after the operation. These changes in contents of uronic acid truly represent altered contents of chondroitin sulfate, because the amounts of hyal6

FIG. 6. Histological section of the left (A) and right (6)femoral head of dog C1 (killed 19 days after surgery). The cartilage of the lateral part of the weight-bearing area of the left femoral head (A) is eroded and thin. For comparison, the corresponding part of the right femoral head, which is normal, is shown (6)(hematoxylin-eosin x 6).

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FIG. 7. Histological section of the lateral weight-bearing area of the left femoral head (A) and the corresponding area of the right femoral head (B) of dog C1 (killed 119 days after surgery). In the left femoral head there is erosion, fraying, and loss of stainability, as well as thinning of the articular cartilage combined with some atrophy of its calcified part (toluidine blue x 60).

uronic acid in the preparations were the same (discussed later). The keratan sulfate (glucosamine) contents of the purified proteoglycans (Al) were already significantly (p < 0.05) lower a short time after the operation (Fig. 11). McDevitt et al. (35) similarly observed decreased glucosamine contents of A1 fractions isolated from articular cartilage of dogs after the cruciate ligament had been transsectioned. The changes in the glucosamine contents can be attributed to an altered content of keratan sulfate, because separate quantitation of hyaluronic acid using a specific radioligand assay showed essentially equal amounts of hyaluronic acid in the samples (Table 2). The compositional data indicate that the proteoglycans purified from the articular cartilage of the side operated on are enriched in the

chondroitin sulfate-rich region and have decreased contents in the keratan sulfate-rich region. In addition, lower protein contents indicate that the proteoglycans contain less of hyaluronic acid binding region, which constitutes a substantial portion of the protein in the molecule. The data can be taken to indicate a fragmentation of the proteoglycans such that the high buoyant density chondroitin sulfate-rich region was recovered in the bottom, A1 fraction, while protein and perhaps keratan sulfaterich fragments were recovered in the upper portions of the gradient used for purification. Support for this hypothesis was obtained from analysis of the extract (Fig. llb). No significant difference was observed between relative keratan sulfate (glucosamine in glycosaminoglycans) contents of the ex-

FIG. 8. Histological section of the articular cartilage of the acetabulum from the left hip joint of dog C1 (killed 119 days after surgery). There is marked cluster formation of the chondrocytes (hematoxylin-eosin x 60).

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0non-operated

t

I a

operated

1

06 04 02

b

Extract 081

L

tracts from the articular cartilage taken from the two sides. Because the recovery of keratan sulfate in the A1 fraction was lower from the cartilage of the hip operated on, it appears that some of the keratan sulfate was recovered in the top fraction of the gradient. The data, therefore, strongly indicate that the extract from the articular cartilage of the side operated on contained fragmented proteoglycans and that the major proportion of the material in A1 represented structures of the chondroitin sulfate-rich region. The series B greyhounds showed similar changes in the composition of the femoral head cartilage from the hip operated on. The uronic acid/protein ratio was increased, although not until 36 days after operation (Table 3). Furthermore, the relative content of glucosamine was lower in proteoglycans isolated from the side operated on (Table 3).

C

Gel Chromatography of Proteoglycans 0 15

0 10

0 05

15

19

26

59

119

153

DAYS AFTER OPERATION

FIG. 9. Proteoglycan contents of cartilage, extracts, and residue of femoral head cartilage. Proteoglycan content at various times after pelvic osteotomy. Proteoglycans in the cartilage (a), in an extract with 4 M guanidine hydrochloride (b), and in the residue after extraction (c). Proteoglycans quantified as glycosaminoglycan-hexosamine over hydroxyproline (wt/wt).

Chromatography of the A1 fractions on Sepharose 2B showed that 19 days after surgery the proportion of aggregated proteoglycans from the hip operated on was already greatly reduced and remained so for the later times (Fig. 12). In the case of the German wirehaired pointers, additional A1 samples were chromatographed after the addition of hyaluronate (2% wt/wt). The elution profile was identical to that when no hyaluronate had been added. It therefore appears that the low proportion of aggregates is not an effect of loss of hyaluronate from the A1 preparations. The low aggregate proportion and

0 non-operated 1.o

I

operated

FIG. 10. Uronic acid to protein ratio (wVwt) of isolated proteoglycan A1 fractions at various times after pelvic osteotomy.

L

15

19

26

59

DAYS AFTER OPERATION

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153

667

EXPERIMENTAL OA

0non-operated A1

FIG. 11. Keratan sulfate contents of proteoglycans and extracts of femoral head cartilage. Ratios of giycosarninoglycanglucosarnine (keratan sulfate) to total glycosarninoglycans in isolated proteoglycans (A1 fraction) (a) and in guanidine hydrochloride extracts (b) of fernoral head articular cartilage after pelvic osteotorny.

w :

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DAYS AFTER OPERATION

the lower relative protein contents of the A1 fraction (Fig. 10) indicate that the proteoglycans from the hip operated on contain reduced amounts of hyaluronic acid binding region. Chromatography on TABLE 2. Hyaluronic acid contents of AI-fractions from the hip articular cartilage Days after operation 15 19 26 59 119 153

Side Nonoperative Operative Nonoperative Operative Nonoperative Operative Nonoperative Operative Nonoperative Operative Nonoperative Operative

Hyaluronic acid/ uronic acid (wt/wt)

Sepharose 2B of reduced and alkylated A1 fractions (Fig. 13) showed that at 19 days and later in the postoperative period, the proteoglycans from the side operated on eluted more retarded, indicating degradation. This is not an effect of smaller chondroitin sulfate chains, because their size was the same in all samples (Fig. 14). TABLE 3. Composition of A1 fractions of hip articular cartilage from the B series of dogs operated on (hip-operated greyhounds)

0.028 0.026 0.049 0.041 0.020 0.023 0.036 0.040

Days after operation

Not determined

19

0.062 0.122 0.082

36

~

10

Side

Glucosamine/ total hexosamine (wt/wt)

Uronic acid protein (wtlwt)

Nonoperative Operative Nonoperative Operative Nonoperative Operative

0.154 0.140 0.194 0.170 0.198 0.164

0.825 0.815 0.795 0.780 0.745 0.905

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S . INEROT ET AL. non operated

operated non-operated

operated

2

1

19d

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FRACTION NUMBER FIG. 12. Proportions of aggregated proteoglycans in A1 fractions from femoral head cartilage. Chromatography on Sepharose 2B of the A1 fractions from the dogs operated on with pelvic osteotomy. Side operated on and contralateral control side shown to the right and left, respectively. The time intervals after surgery are, from top to bottom, 15, 19, 26, 59, 119, and 153 days. The absorbance (A,) of the carbazole reaction is given as arbitrary units.

Taken together, the data provide evidence for an early scission of the proteoglycan protein core in degenerating articular cartilage. As a result, large fragments of the chondroitin sulfate-rich region are formed. They have high buoyant densities and are recovered in the A1 fraction. Other fragments containing keratan sulfate and possibly rich in protein have low buoyant density. They are therefore lost in the top fraction of the gradient. It is possible that

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1

153 c

153 d l

2

15

t VO

30

45

t

60

Vt

15 30 45

t

t

VO

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60

FRACTION NUMBER FIG. 13. Size distribution of proteoglycan monomers in A1 fractions from femoral head cartilage. Chromatography on Sepharose 2 8 of the reduced and alkylated A1 fractions from the dogs operated on with pelvic osteotomy. Side operated on and contralateral control side shown to the right and left, respectively. The time intervals after surgery are, from top to bottom, 15, 19, 26, 59, 119, and 153 days. The arrows in the chromatograms from the side operated on show the position of the peak of the control side. The absorbance (As3,,) of the carbazole reaction is given as arbitrary units.

669

EXPERIMENTAL O A non operated

operated

I

dogs that had been operated on with transection of the anterior cruciate ligament to induce knee joint cartilage degeneration. This part of the study served as a control for the model of hip osteoarthritis and to provide a direct comparison with similar work on knee joint osteoarthritis by others. Cartilage

The cartilage from the knee of all three dogs showed degenerative lesions from roughness of surface after 27 days to cartilage erosion at 107 and 134 days. The menisci were severely torn and the cartilage lesions were located in the area not covered by the menisci. The cartilage content of glycosaminoglycans decreased with time after the operation (Fig. 15a) in agreement with observations in studies by others (1,30,33,36), as well as with the results obtained with the hip joint model. Proteoglycan Composition

FRACTION NUMBER

FIG. 14. Size distribution of chondroitin sulfate side chains in A1 fractions from femoral head cartilage. Chromatography on Sephadex G-200of papain-digested proteoglycan A1 fractions from femoral head cartilage. The time intervals after surgery are, from top to bottom, 15, 19, 26, 59, 119, and 153 days, respectively. The absorbance (Asa0) of the carbazole reaction are given as arbitrary units.

some such fragments remain bound to the hyaluronic acid as has previously been described for aggregates fragmented in vitro to yield chondroitin sulfate-rich peptides and hyaluronic acid with bound protein and keratan sulfate-rich structures (18). The probable identification of proteoglycan fragments in the cartilage extends previously published results from other joints, where only an increased loss of proteoglycans without identification of fragments have been shown (24). Knee Model Osteoarthritis The same approach in terms of compositional analysis was used in studies of the cartilage of three

Extractable as well as nonextractable proteoglycans were lost from the tissue, similar to the results obtained with the hip joint model (Fig. 15b and c). Further analysis showed that with time the composition of extracted proteoglycans became altered, indicating degradation of the molecules (Figs. 16 and 17), as has previously been shown by McDevitt et al. (34,36,37). The changes in the structure of extractable proteoglycans, however, occurred at a much later time compared with those observed with the hip articular cartilage proteoglycans, although they were of a similar nature.

Gel Chromatography of Proteoglycans There were no major differences in the proportion of aggregated proteoglycans from the joints operated on compared with the control joints (Fig. 18). McDevitt et al. (34) also observed that proteoglycans isolated from degenerating knee articular cartilage did not show impaired ability to form aggregates. The average size of the proteoglycan monomers from the side operated on were similar to those from the control side (Fig. 19). Chromatograms on Sephadex G-200 of papain-digested proteoglycans showed that the average size of the chondroitin sulfate side chains was the same in all knee cartilage samples (data not shown). DISCUSSION Two different types of induced degenerativejoint disease have been studied, with major focus on that

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non-operated Cartilage

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1 2 t

04

02

27

I

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1

l o t w

z

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I

o 2 a

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r 107

O2

~

1.

L

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b

06-

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I 134

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DAYS AFTER OPERATION

FIG. 16. Uronic acid to protein ratio (wVwt) of isolated proteoglycans (A1 fractions) at various times after transection of the anterior cruciate ligament.

extra-articular procedure there is no apparent inflammatory component during the time period studied. At least in its early stages, therefore, this experimental osteoarthritis represents a truly degenerative joint disease, where the degeneration is induced by an altered weight bearing. The changes observed appear not to be limited to one breed of dogs. In a previous study (26) a number of dogs with spontaneous hip dysplasia and osteoarthritis, i.e., German shepherds and afghan

u -

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FIG. 15. Proteoglycan contents of cartilage extracts and residue from knee joint cartilage. Proteoglycan contents at various times after transection of the anterior cruciate ligament. Proteoglycans in the cartilage (a), extract (b) and residue (c) after extraction with 4 M guanidine hydrochloride. Proteoglycans quantified as glycosaminoglycan-hexosamine over hydroxyproline (wtlwt).

induced in the hip joint by osteotomy of the pelvis to mimic hip dysplasia. The molecular changes in this model are similar to those observed in secondary osteoarthritis due to hip dysplasia (26). Early molecular changes include substantial loss of proteoglycans from the tissue. Structural changes of remaining, extractable proteoglycans indicate fragmentation of the molecules into substructures containing different regions of the proteoglycan. It is likely that this cleavage is proteolytic in nature. A schematic interpretation of the observed changes in molecular structure of the proteoglycans during developing hip osteoarthritis is shown in Fig. 20. Interestingly, in degenerative disease induced by this

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10 0 50

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FIG. 17. Keratan sulfate contents of proteoglycans and extracts of knee joint cartilage. Ratios of glycosaminoglycanglucosamine (keratan sulfate) to total glycosaminoglycans in isolated proteoglycans (A1 fraction) (a) and in guanidine hydrochloride extracts (b) of knee articular cartilage after transection of the anterior cruciate ligament.

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non operated

operated

134 d

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FIG. 18. Proportions of aggregated proteoglycans in A1 fractions from knee joint cartilage. Chromatography on Sepharose 28 of the A1 fractions from the dogs operated on with transection of the anterior cruciate ligament. Side operated on and contralateral control side shown to the right and left, respectively. The time intervals after surgery are, from top to bottom, 27,107, and 134 days. The absorbance (As3,,) of the carbazole reaction is given as arbitrary units.

hounds, showed results of a similar nature as those in the present study, where dogs with no history of hip dysplasia have been studied. It is probable that the operation creates a model, where the osteoarthritic process proper can be studied after surgical induction of conditions mimicking hip dysplasia. The degeneration induced in knee articular cartilage by sectioning of the anterior cruciate ligament represents a more complex situation. First, this joint has an entirely different anatomy and the surfaces of the articular cartilages are only in direct contact in a small area. Furthermore, the menisci modify load and impact. Secondly, the intraarticular surgery usually causes synovitis. The ensuing inflammatory response may release factors such as tumor necrosis factor (TNF) and interleukin 1, which can trigger chondrocytes to degrade their surrounding matrix (1432). It appears, however, that the changes in molecular composition of the knee cartilage, although occurring much later than the changes in the hip joint, are similar in nature. In contrast to those proteoglycans remaining in the cartilage with the hip joint model, those from the knee joint did not show reduced proportion of ag-

t VO

t

t

t

VO

Vt

FRACTION NUMBER

FIG. 19. Size distribution of proteoglycan monomer in A1 fractions from knee joint cartilage. Chromatography on Sepharose 28 of reduced and alkylated A1 fraction from the dogs operated on with transection of the anterior cruciate ligament. Side operated on and contralateral control side shown to the right and left, respectively. The time intervals after surgery are, from top to bottom, 27, 107, and 134 days. The arrows in the chromatograms from the side operated on show the position of the peak of the control side. The absorbance (As3*) of the carbazole reaction is given as arbitrary units.

gregates or decreased size. It therefore appears that the degeneration produced by the knee model is not progressive (9) and reparative changes may mask the degradation of proteoglycans. The present study shows similar compositional and structural changes of proteoglycans in knee joint cartilage, as has previously been obtained by others in studies of the knee joint model (34,36), although different breeds of dogs were used. Whereas the compositional data obtained with the knee joint model indicate fragmentation and loss of proteoglycans from the tissue, the data on function of those remaining, i.e., aggregating capacity and size distribution, do not indicate that degraded molecules are retained. One possible explanation is that in the knee articular cartilage the repair process is more active. Those proteoglycans lost, which

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in newly synthesized hip articular cartilage proteoglycans; i.e., on the order of 15-30 dpm per Fg uronic acid in isolated proteoglycans. The induced disease is similar in nature to that of spontaneously occurring secondary osteoarthritis in dogs with hip dysplasia (26). i

Acknowledgment: Grants were obtained from the Swedish Medical Research Council (No. 05668), The John M. Olin Foundation, Jordbrukets forsakringsbolag, Folksams, Stiftelse, Kock’s stiftelser, Osterlunds stiftelse, the Medical Faculty, University of Lund, and Konung Gustav V:s 80-ksfond. We thank Annika Biorne-Persson for skillful technical assistance.

REFERENCES

i

i

FIG. 20. Tentative changes in the structure of cartilage proteoglycans in developing hip osteoarthritis.

contain higher proportions of keratan sulfate and protein, possibly are replaced with those that are rich in chondroitin sulfate. In support, Sandy et al. (46) observed that in a response to loss of proteoglycans, rabbit cartilage cultured in vitro responded with a several-fold increase of the synthesis of chondroitin sulfate-rich proteoglycans. CONCLUSION The presented surgical technique used in the new model causes a degenerative disease similar to early osteoarthritis. Proteoglycans are fragmented and subsequently lost from the tissue. Repair processes seem to be negligible. Only small amounts of 50mCi 35S-sulfateadministered i.v. were incorporated

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