Matrix Vol. 1011990, pp. 402 -411

© 1990 by Gustav Fischer Verlag, Stuttgart

Immunohistochemical Localization of Proteoglycans and Non-Collagenous Matrix Proteins in Normal and Osteochondrotic Porcine Articular-Epiphyseal Cartilage Complex

STINA EKMAN1, DICK HEINEGARD2

, OLOF JOHNELL3 and HERIBERTO RODRIGUEZ-MARTINEZ1

1 Department of Anatomy and Histology, Swedish University of Agricultural Sciences, Box 7011, S-75007 Uppsala, 2 Department of Physiological Chemistry, University of Lund, P. O. B. 94, S-221 00 Lund and 3 Department of Orthopaedic Surgery, University of Lund, Malmo General Hospital, S-21401 Malmo, Sweden.

Abstract

Osteochondrosis is an impaired focal endochondral ossification which appears as a cartilage retention in the subchondral bone of growing pigs. The normal differentiation of chondrocytes does not occur and the matrix calcification is restricted.

The present investigation has compared the content of selected macromolecules in the carti­lage matrix of the normal articular and epiphyseal growth cartilage with the osteochondrotic cartilage, using a peroxidase-antiperoxidase (PAP) immunocytochemical method at light micro­scopicallevel.

Some of the non-collagenous macromolecules (fibromodulin, large aggregating proteo­glycans, fibronectin, 100-kDa subunit protein and 148-kDa protein) were conspicuously promi­nent within the osteochondrotic cartilage, compared to the matrix of the "normal" resting, proliferative and hypertrophic regions.

This indicates that the chondrocytes in the osteochondrotic cartilage do not modify their surrounding matrix adequately, thus precluding normal calcification.

Key words: cartilage, growth, immuno-histochemistry, proteins, proteoglycans, swine.

Introduction

Joint cartilages of growing pigs consist of a thin superfi­ciallayer of articular cartilage and a thicker deep layer of epiphyseal growth cartilage. The latter undergoes a similar pattern of endochondral ossification as the growth plate (Carlson et al., 1985). The joint cartilage of an adult pig is composed of the articular cartilage only.

1 With the technical assistance of Ms. Asa Jansson

The normal endochondral ossification that occurs in the growth cartilages of the pig, as in all animals, is charac­terized by morphological and biochemical changes in dif­ferent tissue domains. These are the so called resting, pro­liferative, hypertrophic and calcified regions. Cellular mor­phology and ultrastructure of the matrix in these regions differ (Farnum and Wilsman, 1983).

The precise macromolecular changes in the matrix of the cartilage prior to its calcification are yet unknown. A decrease in proteoglycan aggregation, and an increase in the relative proportion of small proteoglycans have been

described prior to matrix calcification (Franzen et aI., 1982; Buckwalter and Rosenberg, 1988). Up to date no changes have been demonstrated in the structure and mor­phology of the other major macromolecule present in car­tilaginous matrix, i. e. type II collagen (for reference see Miller, 1976; Eyre et aI., 1989), in the early events of endochondral ossification. However, a loss of collagen type II in the territorial matrix of hypertrophic chondro­cytes has been reported (Horton and Machado, 1988). Interestingly, collagen type X has been found only in the matrix of the hypertrophic chondrocytes (Gibson and Flint, 1985).

A number of non-collagenous matrix proteins, also pres­ent in cartilage (for review, see Heinegard and Oldberg, 1989), may playa role in the endochondral ossification. A 148-kDa protein (CMP) is found only in cartilage, although not in weight-bearing articular cartilage. This protein is present in low amounts in epiphyseal cartilage. An 100-kDa subunit protein (COMP) isolated from articular carti­lage appears unique for cartilage. Fibromodulin, isolated from articular cartilage, influences collagen fibrillogenesis in vitro (Hedbom and Heinegard, 1988) and appears to bind to collagen fibrils. There are two structurally related small proteoglycans, one of which (decorin) also appears to bind to the collagen fibrils. Fibronectin, although a minor component, is found in the extracellular matrix of differ­entiating chondrocytes (Weiss and Reddi, 1981; Horton and Machado, 1988).

Osteochondrosis is a generalized skeletal disease mani­festing in the growth cartilages (Olsson, 1973). Osteochon­drosis has been reported in dog (Craig and Riser, 1965; Olsson, 1973), pig (Reiland 1978; Grondalen, 1974), horse (Rejno and Stromberg, 1978), bull (Reiland et aI., 1978), turkey (Poulos, 1978) and rat (Kato and Onodera, 1984). The pathology of osteochondrosis with ensuing osteochon­dritis dissecans in different animals has been reported as being strikingly similar at the microscopic level. Therefore it would be surprising if the pathogenesis of osteochondritis dissecans in man should differ basically from osteochon­dritis dissecans in animals (Olsson and Reiland, 1978).

The osteochondrotic lesions in the pig are usually bilat­eral and the medial femoral condyle is one predilection site. Almost 100% of the Swedish Landrace and Yorkshire breeds with leg pain had osteochondrotic lesions (Reiland, 1978). The lesions are characterized by chondronecrotic areas in the growth cartilage with no chondrocytes matur­ing to hypertrophic cells and no or only minor cartilage calcification. This results in impaired local endochondral ossification, with retained cartilage extending into the sub­chondral bone. Early osteochondrotic lesions in the pig have been described as focal areas of chondronecrosis in the resting region of the growth cartilage. These defects do not affect the endochondral ossification and are not visible macroscopically (Carlson et aI., 1986).

In ultrastructural studies of the normal and osteochon-

Matrix Proteins in Porcine Growth Cartilage 403

drotic growth cartilage from the pig (Farnum and Wilsman, 1984), the matrix (post-fixed with Osmium Ferrocyanide) contained electron dense material of a different morphol­ogy in the osteochondrotic cartilage compared to the hyper­trophic region. This electron dense material may reflect individual processes in the impaired endochondral ossifica­tion.

Osteochondrosis has not been found in the Mini-pig, having wildhog ancestry (Reiland; personal communica­tion). This animal, then, appears to represent a suitable control when studying the pathology of osteochondrosis.

In the present study, molecular alterations in osteochon­drosis have been visualized. The apparent location and distribution of some of the non-collagenous proteins and proteoglycans in the articular-epiphyseal cartilage complex from healthy - including Mini-pigs - and osteochondrotic pigs have been determined, using a peroxidase-antiperoxi­dase (PAP) immunocytochemical method at the light microscopic level.

Material and Methods

Two Mini-pigs (PigLab, Uppsala), 3- and 5-months old, two Landrace-Yorkshire cross-bred pigs (3 months old) and one Swedish Landrace pig (4 months old) were studied. The animals received their feed rations according to Swed­ish standards (Eriksson et aI., 1972). Immediately after stunning and bleeding, the femorotibial joints were dissec­ted. Vertical, 3- to 5-mm thick, sections covering the entire thickness of the articular and epiphyseal cartilage com­plexes as well as subchondral bone were removed by sec­tion from each condyle (Fig. 1).

Pieces of 3 x 4 x 2 mm cartilage from the articular­epiphyseal cartilage complex, were cut out from the slabs under a dissecting microscope, to identify cartilage exten­sions in the subchondral bone. The tissue was fixed by immersion into a 4% aqueous solution of buffered formal­dehyde, trimmed down, dehydrated, embedded in paraffin, cut into approximately 6-~m thick sections and stained with Hematoxylin and Eosin (HE) and van Kossa's stains.

Complementary pieces were immediately plunged into liquid nitrogen (LN2 ) and stored at - 70°C until analysis. These frozen samples were mounted onto pre-cooled chucks, and cut in a cryostat (Ames Lab-tek, USA) into 4- to 6-~m thick sections. These were left at room temperature (20-22°C) for one hour and then fixed in acetone for 5 min. The sections were first briefly washed in 30 mM phosphate-buffered, 0.8% saline solution (PBS), pH 7.3 and thereafter incubated with 0.3% H20 2 for 15 min in order to block the endogenous peroxidase present. Pre­treatments with normal swine serum and 4% bovine serum albumin (BSA) in PBS were done in order to minimize non­specific binding to immunoglobulins. Eight different poly­clonal primary antibodies raised in rabbits against bovine

404 S. Ekman et a1.

proteins were used (see Table I). For review of these pro­teins see Heinegard and Old berg (1989).

Before immuno-staining, one set of sectioned samples were digested with a drop (40 f-ll) of bovine testicular hyaluronidase (Sigma 1-S, 1 mg/ml) in 150 mM NaCl, 5 mM phosphate, pH 7.4, at room temperature for 30 min.

All antibodies were diluted in PBS with 4% BSA. The sections were incubated with antisera for 30 min, then rinsed in PBS and incubated with an excess of the second antibody (pig anti-rabbit IgG) and finally with the PAP­complex (rabbit anti-peroxidase and peroxidase). The developer 3-amino-9-ethylcarbazole was added to visualize the peroxidase. Nuclear staining was done with Mayers Hematoxylin.

Table 1. Cartilage matrix non-collagenous protein.

Proteoglycans Large aggregating proteoglycan

PC-51 (biglycan)

PC-52 (decorin)

Fibromodulin (59-kDA protein)

Proteins 148-kDa protein (CMP) 100-kDa protein subunit protein (COMP) 62-kDa protein Fibronectin

Cartilage specific, provide resilience Ubiquitous, no known function Ubiquitous, modulates collagen fibril formation Modulates collagen fibril formation

Cartilage specific Cartilage specific

Bone specific? Ubiquitous, Interacts with collagen and glycosaminoglycans

Antibody dilutions of 1150 to 11200 were used. The antibodies were raised in rabbit against bovine proteins (for reference, see Heinegard and Oldberg, 1989).

Fig. 1. Transverse section of the medial (med) and lateral (lat) femoral condyles of a 4-month old Swedish Land­race pig. Note the extension of cartilage (arrows) into the subchondral bone (os­teochondrotic lesion) of the medial condyle. X3.

Controls were run as follows: a) without primary anti­body, to test the method itself; b) with pre-immune serum from the same species as the primary-antibody donor, in order to detect any non-specific binding of antibodies; and c) with a mixture of antigen/antibody to block the specific antibody.

The incubated sections were photographed with a Nikon microphot-FXA Photomicroscope equipped with No­marski interference contrast optics.

Results

Light microscopy

The joint cartilages from the Swedish Landrace-York­shire cross-bred pigs had characteristically a superficial zone of articular cartilage, without any blood vessels. The part closest to the surface of the articular cartilage con­tained flattened chondrocytes.

The deeper located growth cartilage occupied about two thirds of the thickness of the joint cartilage. In contrast to the avascular articular cartilage, the growth cartilage con­tained many morphologically viable blood vessels in so called vascular channels. The cells in the basal region were hypertrophic and the inter-territorial matrix in the deeper basal areas presented calcification. Blood vessel penetration and ossification occurred in the chondrocyte lacunae found most closely to the subchondral bone.

Focal areas of chondronecrosis could be seen in the rest­ing zone of the growth cartilage (Figs. 2 a), often together with degenerated blood vessels, i. e. lumina outlined or filled with cellular debris. Groups of chondrocytes called clusters were found in the viable growth cartilage surround­ing the areas of necrosis. These clusters were denser towards the border of the basal growth cartilage. The ossifi­cation appeared not to be affected.

The articular cartilage from the 4-month old pure-bred Swedish Landrace pig presented a morphology similar to that described in the cross-bred pigs. The growth cartilage from both lateral and medial condyles showed areas of chondronecrosis. These were found in all the different regions of the growth cartilage and also protruding, as pale areas, into the subchondral bone (Figs. 1 and 2 b). Towards the border of the morphologically viable growth cartilage, clusters of chondrocytes could be seen. Some of these, located et the cartilage-bone interface, were surrounded by calcified matrix. Vascular channels were seen both in the normal growth cartilage and in the chondronecrotic tissue. All the blood vessels in vascular channels found in the chondronecrotic areas were lined by debris, indicating degenerative changes (Fig. 2 b).

The joint cartilages from the Mini-pigs had a similar general morphology, but the different regions were thinner and no areas of chondronecrosis were present (Fig. 2 c). The number of vascular channels in the growth cartilage varied with the age of the pig, such that, only a few vascular channels were seen in the older pig. No chondronecrotic areas were seen in any of the Mini-pigs.

Vascular channels undergoing chondrification were found in all the examined pigs (Fig.2a).

Immuno-eytoehemistry

The intensity of the immune reaction with the PAP­technique, was graded as mild, moderate or intense.

No staining was seen when the primary specific antibody was omitted in the incubation. Different dilutions of the non-immune serum were tested. With a 11200 dilution no immune reaction was observed. A non-specific cellular staining was in some cases obtained also after blocking of antibody-binding by added antigen.

No immune reactivity with antibodies against PC-S2 (decorin) and 62-kDa bone protein was seen in the cartilage from any of the five pigs. Neither was any immune reactiv­ity apparent after predigestion with hyaluronidase.

Antibodies to PC-LA (Heinegard et aI., 1985) and COMP gave similar matrix staining patterns. The most intense immuno reactivity was observed in the articular cartilage matrix from all three domestic pigs (Figs. 3 a -d). Interestingly, the deeper parts of the articular cartilage showed a more intense staining for COMP than the superfi­cial layer did (Fig. 3 c-d). This staining difference in the articular cartilage could, however, at least in part be artefactual, due to an uneven thickness of the section (Fig. 3 c). A non-specific cellular staining was also present in most of the chondrocytes in the growth cartilage. A mild immune reaction was obtained in the extracellular matrix of the epiphyseal cartilage from all regions. The staining reaction in the hypertrophic zones appeared to be most intense within the pericellular matrix (Fig. 3 b). Hyalu­ronidase digestion did not result in an intensified staining,

Matrix Proteins in Porcine Growth Cartilage 405

except when anti-COMP was used. In this case a mild increase in the staining reaction could be noticed in the extracellular matrix.

The cartilage from the Mini-pigs showed a mild immune reaction in the articular cartilage, while staining in the superficial parts was more intense.

The immune reaction seen in the osteochondrotic carti­lage was intense in the extracellular matrix and in the clustered cells (Figs. 3 e-f).

Only a very mild staining with anti-PC-S1 (biglyean) was found in the matrix of the articular cartilage, restricted to the matrix of the chondronecrosis and in the clustered chondrocytes in the osteochondrotic cartilage of the domestic pigs (Fig. 4). The immune reaction increased slightly in the matrix of the articular cartilage after pre­digestion with hyaluronidase. No staining was found in the cartilage of the Mini-pigs.

Antibodies to fibromodulin showed an intense immune reaction in the matrix of the articular cartilage (Fig. 5 a - b) and a mild non-specific staining in the resting and prolifera­tive cells in the growth cartilage of the domestic pigs. An artefactual staining spot is present in the articular cartilage shown in Fig. 5 a, caused by focal de attachment of the section to the glass slide. Mild to moderate immune reac­tion was demonstrated in the matrix of the chondronecrosis and the clusters (Fig. 5 c). A slight increase in staining was found after hyaluronidase digestion. The staining was intense in the superficial layer of the articular cartilage from the Mini-pigs.

No (Fig. 6 a) or a very mild immune reaction was seen with anti-eMP (148-kDa protein) and then only after pre­treatment with hyaluronidase. This was found both in the matrix of articular and epiphyseal cartilages of all pigs. A moderate immune reaction, however, was present in the matrix of the retained osteochondrotic cartilage (Fig.6b), and mild staining was seen in the chondrocytes in cluster­formations. In this case, hyaluronidase digestion of the cartilage matrix did not modify the reaction.

Binding of anti-Fibronectin was seen in the areas of chon­dronecrosis (Fig. 7) and the proliferative region of the growth cartilage. Mild to moderate immune reaction was observed. A mild to moderate staining was also found in the matrix of the articular cartilage and the rest of the epi­physeal cartilage after hyaluronidase pre-digestion.

Discussion

The composition of the extracellular matrix determines the physical properties of cartilage. A correct composition of the matrix is a prerequisite for normal function. The col­lagen fibrils are important for the maintenance of the form and tensile strength of cartilage; while proteoglycans are responsible for its stiffness and resilience (Kempson, 1970). A few percent of the total collagen content in cartilage

406 S. Ekman et al.

. ,­.. .... .. ... . '

Matrix Proteins in Porcine Growth Cartilage 407

Figs. 3 a-f. Transverse histological sections of joint cartilage immuno-stained for PC-LA (a, b and e) and COMP (c, d and f) by the PAP method using rabbit antibodies. Asterisk = subchondral bone. a) x 30, b) and c) x 300, d) and e) X 40, f) X 30. a-d) from a 3-month old Swedish Landrace-Yorkshire cross-bred pig. Note dark staining in the articular cartilage (arrows). b) Higher magnification of the hypertrophic region from a. A dark staining is present in the pericellular matrix of the hypertrophic chondrocytes (arrowhead). c) Higher magnification of the articular cartilage from d. Dark staining is present in both superficial (arrowhead) and deep articular cartilage. e-f) from a 4-month old Swedish Landrace pig. Empty, acellular vascular channels are present (thick arrow). Note dark staining in the chondronecrosis of e (thin arrows) and f (empty arrows).

Figs. 2 a -c. Transverse Hematoxylin and Eosin (HE)-stained histological sections from; a) a 3-month old Swedish Landrace-Yorkshire cross-bred pig. A focal area of pale degenerated cartilage (thick arrows) is present in the resting region. Note vascular chondrification (thin arrow) and the proliferative region (bent arrow) of the growth cartilage. xso. b) a 4-month old Swedish Landrace pig. An area of degenerated cartilage is present in the basal layer and extending into the subchondral bone (asterisk). The degenerated cartilage is surrounded by clustered chondrocytes (empty arrows). Many acellular vascular channels filled or outlined by debris (black arrows) are present. Growth cartilage (bent arrow) with active ossification is present in adjacent areas. X50. c) a 3-month old Mini-pig. Not the pale articular cartilage (thick arrow), a vascular channel (thin arrow) and active proliferative and hypertrophic regions of the growth cartilage (bent arrow). X50.

408 S. Ekman et al.

••

* 4

Fig. 4. Transverse histological section of joint cartilage from a 4-month old Swedish Landrace pig. The section is immuno-stained for PC-S1 (biglyean) by the PAP method using rabbit antibody to biglycan. Articular cartilage (thick arrows), degenerated vascular channel (bent arrow), chondronecrosis (star) and clustered chondrocytes (empty arrow) can be seen. Note a mild dark stain in the necrotic area. X60.

consists of minor types of collagen (for reference, see Mayne and Irwin, 1986). The cartilage specific collagen type IX may be involved in organizing the fibrillar network of collagen type II. Collagen type XI has been found in hyaline cartilage and may determine the fibril diameter of collagen type II (Mendler et aI., 1989).

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Fig. 5 a-c. Transverse histological sections of joint cartilage immuno-stained for fibromodulin by the PAP method using rabbit antibody to fibromodulin. a) x 40, b) x 300, c) x 30. a-b) from a 3-month old Swedish Landrace-Yorkshire cross-bred pig. Note dark staining in the articular cartilage (arrows). b) is a higher magnification of the articular cartilage seen in a). c) from a 4-month old Swedish Landrace pig. Note the chon­dronecrosis (star) with the clustered chondrocytes (arrows). There is a dark stain in the chondronecrotic area.

A certain composition of the cartilaginous extracellular matrix, where the calcification of the matrix starts, is also crucial in growth cartilage. The calcification is common to

all growth cartilages prior to endochondral ossification. The mechanism of this calcification is, however, not fully

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rI . 6a

Figs. 6 a -b. Transverse histological sections of joint cartilage immuno-stained for CMP (148 kDa protein) by the PAP method using rabbit antibody to CMP. a) from a 3-month old Swedish Landrace-Yorkshire cross-bred pig. Articular cartilage (thick arrow), the resting, the proliferative (empty arrow) and the hypertrophic regions of the growth carti­lage can be seen. No dark stain is found. x 40. b) from a 4-month old Swedish Landrace pig. Note vascular degeneration (thin arrows) and a dark stain in the chondronecrosis (thick arrows). Asterisk = subchondral bone. x 30.

understood (Ali, 1983; Anderson, 1989). Matrix calcifica­tion starts in the hypertrophic region of the cartilage. Here, the hypertrophic chondrocytes have been found to syn­thesize unique macromolecules, e. g. collagen type X (Gib­son and Flint, 1985; Grant et al., 1985). A different mor­phology of the pericellular matrix of the hypertrophic chondrocytes was described by Farnum and Wilsman (1983). They found an electron dense material, only present in this matrix, probably representing a matrix component. Increased amounts of fibronectin in the pericellular matrix and reduced amounts of collagen type II and proteoglycan link protein in the territorial matrix were found in the hypertrophic region compared to the resting and prolifera­tive regions (Horton and Machado, 1988).

Matrix Proteins in Porcine Growth Cartilage 409

~ . • . >~: . ,. ..• -

Fig. 7. Transverse histological section of growth cartilage from a 4-month old Swedish Landrace pig. The section is immuno-stained for Fibronectin by the PAP method using rabbit antibody to fibro­nectin. Note the chondronecrosis (star) with the clustered chondrocytes (black arrows) and the degenerated vascular channel (empty arrow). There is a dark stain in the chandra-necrosis. * = subchondral bone. x 30.

The matrix in the hypertrophic region has a different composition of macromolecules than the rest of the carti­lage. This could facilitate calcification of the cartilage ma­trix by providing nucleation sites, raising the concentration of Ca and P ions in solution locally or remove inhibitory compounds. An abnormal macromolecular composition in the hypertrophic region of the cartilage could lead to a failure of the calcification, followed by an impaired endochondral ossification. Proteoglycan aggregates and high concentrations of proteoglycans have been shown to inhibit mineral growth in vitro (Dziewiatkowski, 1987). The relative amount of aggregating proteoglycans decreased at the time of calcification (Franzen et al., 1982). It has been suggested that changes in the proteoglycan

410 S. Ekman et ai.

structure and organization without a net loss of proteo­glycans (Poole et ai., 1983) may be sufficient for calcifica­tion to occur. In addition, a predominance of proteoglycans with a small hydrodynamic size have been found after onset of osteogenesis (Tian et ai., 1986).

The osteochondrotic cartilage does not calcify properly (Reiland, 1978). Its matrix morphology differs from that of the normal matrix found in the hypertrophic zone (Farnum and Wilsman, 1984). The osteochondrotic cartilage in the epiphyseal growth cartilage is characterized by areas of vascular- and chondronecrosis in the resting, proliferative and hypertrophic regions. Retained chondronecrosis is seen in the subchondral bone (Carlson et ai., 1986).

Farnum and Wilsman (1984) described the osteochon­drotic lesion in the growth plate (metaphyseal growth carti­lage) of the distal ulna, as characterized by having uniformly viable and probably metabolically active chondrocytes. These cells failed to undergo hypertrophic cell maturation. Areas with this type of chondrocyte appear to precede chondronecrosis. These chondrocytes could thus play an important role in the lack of calcification of matrix, by controlling the synthesis and/or removal of select mac­romolecules. Several of the non-collagenous proteins found in the extracellular matrix may play an important role in the endochondral ossification. For example both PG-S2 and fibromodulin bind to collagen and may interfere with mineralization along collagen fibers (for reference, see Heinegard and Oldberg, 1989). The altered composition of the macromolecules in the matrix of the chondronecrosis locus may provide an explanation to the impaired endochondral ossification seen in osteochondrosis.

To determine the location of these proteins a method where the morphology of the cartilage can be visualized simultaneously has to be used, since small osteochondrotic areas and normal cartilage are found intermingled.

The present results suggest that the type of macromole­cules present in the region of chondronecrosis and in the matrix of the growth cartilage differ. The matrix in the chondronecrosis appears to resemble the matrix of the articular cartilage, which does not calcify. Large aggregat­ing proteoglycans, fibromodulin, fibronectin and COMP appeared to be more abundant in the osteochondrotic carti­lage and in the articular cartilage. CMP appeared to be present in the osteochondrotic cartilage, which may be taken to indicate a more fetal cartilage, since CMP is pres­ent throughout the cartilage in the early skeleton (Larsson and Heinegard, unpublished). Fibromodulin modulates the formation of collagen fibrils but it has also been suggested as preventing mineralization along collagen (Heinegard and Oldberg, 1989). The absence of reactivity to the PG-S2 could result from limited species cross-reactivity and/or blocked epitopes.

The chondrocytes produce and degrade the mac­romolecules of the extracellular matrix. The dead chondro­cytes (i. e. cells that lack cell organelles and have disrupted

cell-membranes) in the necrotic areas (Ekman et ai., 1990), however, will not be able to maintain or adequately modify the matrix composition. The cause of the chondrocytic death in the osteochondrotic cartilage is unfortunately not clear. Several authors have suggested that this is an hypoxic insult as a result of vascular degeneration (Woodard et ai., 1987; Carlson et ai., 1989).

A further investigation, at ultrastructural level, of the localization of the macromolecules studied in the present investigation should contribute to a better understanding of the composition of the early osteochondrotic cartilage.

The present study indicates that the chondrocytes in the osteochondrotic cartilage do not synthesize or modify their matrix adequately. Instead the matrix appears to resemble the matrix of the articular cartilage more than the matrix of the growth cartilage.

Acknowledgements

This work was supported by the Swedish Council for Forestry and Agricultural Research (Project no. D288) and the Swedish Medical Research Council. We thank Staffan Johansson, Depart­ment of Medical Chemistry, The Biochemical Centre, Uppsala, Sweden, for the supply of antibodies against fibronectin. We also acknowledge the support from the Swedish University of Agricul­tural Sciences, Uppsala and the Medical Faculty, University of Lund.

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Matrix Proteins in Porcine Growth Cartilage 411

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Dr. Stina Ekman, Department of Anatomy and Histology, Swed­ish University of Agricultural Sciences, Box 7011, S-750 07 Uppsala, Sweden.