Vet Pathol 29:514-520 (1992)
Immunohistochemical Localization of Matrix Proteins in the Femoral Joint Cartilage of Growing Commercial Pigs S.
EKMAN AND
D.
HEINEGARD
Department of Anatomy and Histology, Swedish University of Agricultural Sciences, Uppsala, Sweden (SE); and Department of Physiological Chemistry, University of Lund, Lund, Sweden (DH) Abstract. The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular--epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycanwas more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair. The chondronecrotic areas restricted to the resting region have a matrix that is different from the matrix of the abnormal cartilage extending into the subchondral bone, which resembled the matrix of the proliferative region. Hence the osteochondrotic lesion may not start in the resting region, instead the maturation of chondrocytes seems to stop in the proliferative zone, which would result in impaired bone formation.
Key words: Cartilage; immunohistochemistry; proteoglycan; swine.
Lesions with necrotic chondrocytes in the resting region of the growth cartilage of l2-week-old commercial pigs':" have been referred to as early osteochondrosis. These chondronecrotic areas are thought to ultimately involve the hypertrophic region. The chondronecrotic tissue does not seem to provide a matrix suitable for calcification, thus precluding ossification. The result is persisting necrotic cartilage protruding into the subchondral bone, i.e., osteochon-
drosis." The macromolecular composition of the retained cartilage and the normal articular--epiphyseal growth cartilage from 12- to l6-week-old pigs has previously been studied using polyclonal antibodies against proteoglycans and noncollagenous matrix proteins.' The immunostaining at the light-microscopic level indicated that the matrix in the necrotic cartilage extending into the subchondral bone differed from normal epiphyseal growth cartilage. The macromolecular composition of the extracellular matrix of this necrotic cartilage appeared to resemble the normal articular
cartilage more than the normal growth cartilage, possibly showing an altered processing of matrix constituents. Such a different macromolecular processing may follow death of chondrocytes with ensuing loss of normal synthesis and impaired matrix degradation. If, however, an arrest of chondrocytic maturation in resting or proliferation precedes cell death, deviations in matrix composition may be seen prior to chondronecrosis, A set of different macromolecules were chosen as markers for the composition of the matrix produced by the chondrocytes. Key information on mechanisms can be obtained by establishing whether or not the altered macromolecular composition found in the chondronecrotic retained cartilage, as compared with normal surrounding growth cartilage, could also be detected focally in the morphologically normal resting growth cartilage. Furthermore, the temporal relationship between observed matrix changes and chondrocyte death could provide information on cause and effect.
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Matrix Proteins in Joint Cartilage of Growing Pigs
To detect early changes within the resting region of the growth cartilage, matrix structure was studied at the molecular level using immunocytochemical localization of matrix macromolecules, including collagen type II and proteoglycans, in the femoral articularepiphyseal cartilage complex of young commercial pigs over an extended age range between 6 and 18 weeks.
Materials and Methods Two litters (seven piglets/litter) of purebred Swedish Landrace pigs and one 15-week-old Swedish Landrace-Yorkshire crossbred pig were studied. The pigs received rations made according to Swedish standards,' and the littermates were euthanatized, two at each time, at the age of 6, 8, 10, 12, 14, 16, or 18 weeks. Immediately after stunning and bleeding, 3-5-mm-thick vertical sections covering the entire thickness of the articular and epiphyseal cartilage complexes and a small part of the subchondral bone were prepared. Tissue was taken from medial and lateral condyles of both right and left fernurs. Small pieces (2 x 3 x 4 mm) were cut out from the articular-epiphyseal cartilage complex, frozen in liquid nitrogen, and stored at -70 C. Four to eight pieces were taken from each pig. Four pieces were always cut from macroscopically normal articular-epiphyseal cartilage complex (lateral, right, and left; medial, right, and left). Eight pieces were cut when cartilage retained into subchondral bone was detected macroscopically in all four condyles. The frozen samples were mounted on precooled chucks, cut in a cryostat (Bright Starlet 2212 Cryostat, Huntington, England) into 6-1.@ sections, and put onto poly-L-lysine (Sigma Diagnostics, St. Louis, USA)-covered glass slides. Multiple sections were cut in succession for the full battery of antibodies. The sections were left at room temperature (20-22 C) for I hour and were fixed with acetone for 5 minutes. The sections were washed with 0.03 M phosphate-buffered 0.8% saline solution (PBS) and incubated with 0.3% H 20 , for 15 minutes. Pretreatments with normal swine serum and 4% bovine serum albumin were done to minimize nonspecific binding ofimmunoglobulins. Six different polyclonal primary antibodies raised in rabbits against bovine proteins (cartilage oligomeric matrix protein [COMP], fibromodulin, fibronectin, biglycan [PG-SI], cartilage matrix protein [CMP], and aggrecan [PG-LA]) were used.9.ID.13 Three different monoclonal primary antibodies (lgG) (CII-Cl, Cll-C2, Cll-F4) raised in DBA/l mouse against rat collagen type II were also used. [2 Samples incubated with antibodies were run in duplicate. One set was pretreated with I drop (40 I.d) of bovine testicular hyaluronidase (Sigma I-S, Sigma Chemical Co., St. Louis, MO, USA; 1 mg/ml) in 0.15 M sodium chloride and 5 mM phosphate (pH 7.4) at room temperature for 30 minutes. All antibodies were diluted in PBS containing 4% (w/v) bovine serum albumin. The sections were incubated with antisera at room temperature for 30 to 45 minutes then rinsed in PBS and incubated with an excess of the second antibody (pig anti-rabbit IgG or goat anti-mouse IgG, respectively). A final incubation with peroxidase-antiperoxidase complex was done (rabbit or mouse antiperoxidase and peroxidase, re-
spectively), and the developer 3,3-diaminobenzidine was added to visualize the antibody-antigen complex.' Controls included preincubation with the respective antigens to block the specific antibodies (cartilage oligomeric matrix protein, aggrecan, biglycan, and fibromodulin). A preimmune serum from the same species was also used. As a control for the monoclonal antibodies, an IgG monoclonal antibody (F3; with unknown specificity but nonreactive with collagen type II)" also obtained from the DBA/I mouse was used. Complementary pieces of cartilage were immersed in a 4% aqueous solution of buffered formaldehyde, dehydrated, embedded in paraffin, cut into 6-ttm-thick sections, and stained with hematoxylin and eosin. Selected sections were photographed using a Nikon Microphot-FXA photomicroscope.
Results Light microscopic findings
No pathologic changes were found in the superficial avascular articular cartilage in any of the pigs included in the study. The articular-epiphyseal cartilage complex from all pigs consisted of a thick growth cartilage with many vascular channels comprising intact blood vessels with endothelium and surrounding connective tissue. Apparently occluded vascular channels undergoing chondrification were also observed. In the 6-week-old pigs, neither areas of chondronecrosis nor any signs of disturbed ossification were observed in the growth cartilages. A few areas of chondronecrosis were present in the resting region in almost all sections of the growth cartilage from pigs that were 8 and 10 weeks old; nevertheless, proliferative, hypertrophic, and calcifying regions as well as ossification in underlying layers appeared normal. Often, but not always, areas of chondronecrosis were seen in connection with vascular channels outlined or filled with necrotic debris and erythrocytes. The growth cartilage from the 12-, 14-, 15-, 16-, and 18-week-old pigs presented a much higher abundance of necrotic areas, both in the resting (Fig. I) and in the proliferative, hypertrophic, and calcifying regions, most often in connection with degenerated vascular channels. The necrotic areas involving hypertrophic and calcifying regions caused focally disturbed ossification, creating cartilage extending into the subchondral bone (Fig. 2). Immunocytochemical findings
The immunostaining obtained with the peroxidaseantiperoxidase technique was graded as mild, moderate, or intense (Table 1). No immunostaining was observed when the primary antibody was not included, although cellular staining could be seen when nonimmune rabbit serum replaced the primary antibody.
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Fig. 1. Growth cartilage, femoral condyle; 15-week-old pig. Note the large chondronecrotic area (short arrows) in the resting region and degenerated vascular channels (long arrows). The endochondral ossification is not affected. HE. Fig. 2. Growth cartilage, femoral condyle; 12-week-old pig. Chondronecrotic area extends into the subchondral bone (*) and is surrounded by chondrocytes in cluster formations (short arrows). Note degenerated vascular channels (long arrows) in the area of chondronecrosis. HE. Fig. 3. Articular cartilage, femoral condyle; IO-week-old pig. Immunostained for collagen type II (CI). Intense staining is present in the interterritorial matrix. Peroxidase-antiperoxidase technique.
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Fig. 7. Growth cartilage, femoral condyle, resting region; l5-week-old pig. Immunostained for aggrecan. Two chondronecrotic areas (*) are surrounded by clustered chondrocytes (arrows). There is no staining of the matrix. Nonspecific cellular staining is present. Peroxidase-antiperoxidase technique. Fig. 8. Growth cartilage, femoral condyle; 12-week-old pig. Immunostained for collagen type II (Cl ), Fig. 8a. Note intense staining in the proliferative region (arrows). Peroxidase-anti peroxidase technique. Fig. 8b. Higher magnification of the area in Fig. 8a. Intense staining is seen in the interterritorial matrix (arrows).
This nonspecific cellular staining was also present after blockage of specific antibody by pretreatment with the respective antigen. This cellular stain is of little relevance for the present study, which focused on the matrix where nonspecific stain was never seen. Immunostaining was not seen when antisera-F3 (control) was used, although a mild staining was sometimes observed in the erythrocytes and in the endothelial cells. The normal articular cartilage demonstrated an intense to moderate staining with all the antibodies in 74 (75%) of99 sections (Figs. 3, 4) except against cartilage matrix protein (CMP; Table 1). The reaction was less prominent for biglycan and epitope F4 for collagen
type II. The immune reaction did not differ in different age groups. The immunostaining for all the antibodies, within the matrix of the normal resting region of the growth cartilage was low or nondetectable in all the sections (Fig. 4). The perivascular matrix close to vascular channels undergoing chondrification did not differ, in reaction, from the rest of the surrounding region. Chondronecrotic areas restricted to the resting region were often found in connection with degenerated vascular channels in all pigs ages 8 to 18 weeks. Here, an intense immunostaining with antibodies against fibronectin (Fig. 5) and collagen type II was seen in 61
Fig. 4. Articular cartilage and resting region of the growth cartilage, femoral condyle; l8-week-old pig. Immunostained for fibromodulin. Intense staining is present in the matrix of the articular cartilage. Peroxidase-antiperoxidase technique. Fig. 5. Growth cartilage, femoral condyle; l5-week-old pig. Immunostained for fibronectin. Intense staining (short arrows) is present in the matrix of cells surrounding a degenerated vascular channel (long arrow) within the resting region. Peroxidase-antiperoxidase technique. Fig. 6. Growth cartilage, femoral condyle; IO-week-old pig. Immunostained for fibronectin. Note intense staining in the proliferative region (short arrows) and in the matrix surrounding the clustered chondrocytes (arrows) in the resting region. A degenerated vascular channel (arrowhead) is also seen. Peroxidase-antiperoxidase technique.
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Table 1. Immune reaction in the articular--epiphyseal cartilage complex of 15 growing pigs, aged 6 to 18 weeks, Antisera
Matrix of Articular cartilage Resting region Proliferative region Hypertrophic region Chondronecrosis in Restricted to resting region Extension in subchondral bone
* ++
Cartilage Cartilage Collagen Oligomeric Matrix type II: FibroMatrix Protein Fibronectin Epitope modulin Protein (CMP) CI (COMP)
Aggrecan (PG-LA)
Biglycan (PG-Sl)
++* 0 + +
+* 0 0
++ 0 +
a
++ 0 + +
0* 0 0 0
++ 0 ++ 0
a
a
a
a
a
++
+
++
++
+
a
C2
F4
++ 0 ++
++ 0 ++
+ 0 +
++
++
+
0
++
++
++
++
a
a
a
= moderate to intense; + = mild to moderate; 0 = mild or no staining.
(67%) of 91 sections. The reaction was found mainly around the clustered chondrocytes (Fig. 6). When antisera against aggrecan (Fig. 7), biglycan, fibromodulin, CMP, and cartilage oligomeric matrix protein (COMP) were used, no immune reaction was seen in any of the sections.
Fig. 9. (Left) Growth cartilage, femoral condyle; 14-weekold pig. Immunostained for cartilage oligomeric matrix protein (COMP). Intense staining in the chondronecrosis extends into the subchondral bone. A degenerated vascular channel is present (arrow). Peroxidase-antiperoxidase technique. Fig. 10. (Right) Growth cartilage, femoral condyle; 15week-old pig. Immunostained for collagen type II (CI). Intense staining in the chondronecrotic area. A degenerated vascular channel is present (arrow).
The proliferative, hypertrophic, and calcifying regions of the normal epiphyseal growth cartilage are thin regions compared with the large resting region. This epiphyseal morphology differs markedly from that ofthe growth cartilage in the metaphyseal plate, where the proliferative, hypertrophic, and calcifying regions occupy most of the growth cartilage. A mild to moderate to intense reaction was present in the normal proliferative region of the growth cartilage with the full battery of antibodies except biglycan and CMP (Table 1). The most intense reaction was present when antibodies against fibronectin (Fig. 6) and collagen type II (Fig. 8) were used. The immune reaction against fibromodulin, aggrecan, and COMP was moderate to intense in the older pigs (16 and 18 weeks old) but mild to moderate in all the younger pigs (Table 1). The normal proliferative region showed a stronger immune reaction with antisera against aggrecan and fibromodulin than did the matrix surrounding the clustered cells. The normal hypertrophic and calcifying regions of the growth cartilage were hard to separate. Because the sections were cut undecalcified, much of the calcified matrix as well as the subchondral bone were artifactually lost. Any calcified area that was still present after cutting would only be partially decalcified during the incubation, hence the staining of calcified tissue was not evaluated. Only antisera against COMP and aggrecan showed a mild to moderate reaction in the normal hypertrophic region. None of the other antisera gave any immune reaction in this region (Figs. 6, 8). The chondronecrotic area extending into the subchondral bone stained moderately to intensely with all antibodies. The extensions into subchondral bone were present in all the pigs aged 12 to 18 weeks. All the extensions stained with antisera against aggrecan, biglycan, fibromodulin, COMP (Fig. 9), fibronectin, and
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collagen type II (Fig, 10). Half of the sections stained moderately with antisera against CMP. Discussion
Osteochondrosis appears to be a malfunction of the growth cartilage and is characterized by a focally impaired endochondral ossification." Osteochondrosis has been reported in many domestic animals, including the pig. 8 The ultrastructure of the early osteochondrotic lesi.on in the epiphyseal growth cartilage from growing pigs has been described as chondronecrotic areas in the resting region. 1 The chondronecrosis will ultimately involve the hypertrophic region, causing an insufficient calcification and an impaired ossification. Chondrocytes directly bordering the necrotic area are grouped in cell clusters, especially when present in close proximity to the hypertrophic and proliferative regions. These cells are metabolically active with a prominent rough endoplasmic reticulum and many lipid dropIets.!" Ultrastructural studies of osteochondrosis in the growth plate (metaphyseal growth cartilage) from growing pigs, however, showed that early lesions were accumulations of proliferative chondrocytes not undergoing normal hypertrophic cell maturation prior to chondronecrosis.s A failure ofvascular penetration and subsequent ossification followed. Normal synthesis and degradation of the cartilage extracellular matrix is dependent on the chondrocytes. Many factors could affect the chondrocytes and hence their matrix production. The early osteochondrotic lesions are not inflammatory, and the dead chondrocytes are surrounded by other normal chondrocytes. A change in matrix macromolecular composition should therefore reflect an alteration of the chondrocytic phenotype. The macromolecules in this study were chosen as markers for chondrocyte activity, and their specific function was not considered. The cause of chondrocyte death has been proposed as hypoxia due to necrosis of vascular channels."!" However, a lack of differentiation of the chondrocyte prior to cell death can not be excluded. The chondronecrotic areas in the resting region are probably the earliest lesions, moving basally towards the subchondral bone because of continuing growth in the surrounding areas. However, the matrix in the chondronecrotic areas restricted to the resting region did not resemble the macromolecular composition (when considering antibody staining pattern) of the chondroneerotic areas extending into the subchondral bone. In these areas, the composition is similar to that of the proliferative region with more intense staining for aggrecan, fibronectin, cartilage oligomeric matrix protein, fibromodulin, and collagen type II (Table 1). These results suggest that the early osteochondrotic lesion in 015
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the epiphyseal growth cartilage starts with an accum ulation of proliferative chondrocytes not undergoing hypertrophic differentiation. The chondronecrotic areas restricted to the resting region and often in connection with necrotic vascular channels could be a lesion not associated with the focally disturbed ossification characteristic for osteochondrosis. The pathogenesis of chondronecrosis in the resting or hypertrophic subchondral region may differ. This is also true for the early osteochondrotic lesion in the metaphyseal growth cartilage.' The present results indicate that the matrix surrounding the clustered cells is altered compared with that in normal growth cartilage. Thus, staining for fibronectin and collagen type II (epitope C1) was more prominent in this region than in the resting and hypertrophic regions. Chondrocytes surrounding necrotic vascular channels also stained for fibronectin and collagen type II in their matrix. The apparently increased amount of fibronectin in these domains may indicate a repair response. Alternatively, an altered permeability of the matrix could allow diffusion of plasma fibronectin. Changes in the vascular channels and therefore in the supply of oxygen and nutrients and/or removal of waste metabolites may represent triggering factors. If so, an understanding of the mechanism causing closure and chondrification of the vascular channels should provide important insights into the etiology of chondronecrosis in the resting region. Further information on early metabolic changes and their relation to cell death can be obtained by comparing mRNA levels corresponding to specific proteins in the chondrocytes of normal and osteochondrotic growth cartilage and should allow a more precise demonstration of the altered chondrocyte phenotype and should also allow early identification of dying cells. The resemblance of the matrix in the proliferative region to that in the retained chondronecrotic areas can support the hypothesis that early osteochondrotic changes in the epiphyseal and the metaphyseal growth cartilage primarily result from failure of chondrocyte maturation, which suggests that the pathogenesis for osteochondrosis in the metaphyseal and epiphyseal growth cartilages is similar. Acknowledgements We thank Ms. A Jansson for skilled technical assistance Dr. S. Johansson and Dr. R. Holmdahl for providing us With th~ antibodies against fibronectin and collagen type II, respectively. We also gratefully acknowledge Dr. O. Johnell for introducing us to the peroxidase-antiperoxidase technique used in cartilage. This work was supported by the Swedish Council for Forestry and Agricultural Research (Project No. D288), The Swedish Medical Research Council, a~d
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Alfred Osterlunds Stiftelse, and Axel and Margaret Axison Johnsons Stiftelse. 9
References
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3
4
5 6
7
8
Carlson CS, Hilley HD, Henrikson CK, Meuten DJ: The ultrastructure of osteochondrosis of the articular--epiphyseal cartilage complex in growing swine. Calcif Tissue Int 38:44-51, 1986 Carlson CS, Hilley HD, Meuten DJ: Degeneration of cartilage canal vessels associated with lesions of osteochondrosis in swine. Vet Pathol 26:47-54, 1989 Ekman S, Heinegard D, Johnell 0, Rodriguez-Martinez H: Immunohistochemical localization of proteoglycans and non-collagenous matrix proteins in normal and osteochondrotic porcine articular-epiphyseal cartilage complex. Matrix 10:402-411, 1990 Ekman S, Rodriguez-Martinez H, P16en L: The morphology of normal and osteochondrotic porcine articular-epiphyseal cartilage: a study in the domestic pig and mini-pig of wild hog ancestry. Acta Anat 139:239-253, 1990 Eriksson B, Sanne S, Thomke S: Fodermedlen [Foodstuff], pp. 41-43. Textbook Lt., Stockholm, Sweden, 1972 Farnum CE, Wilsman NJ, Hilley HD: An ultrastructural analysis of osteochondritic growth plate cartilage in growing swine. Vet PathoI21:141-151, 1984 Graham RC, Karnovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14: 291-302,1966 Grondalen T: Osteochondrosis and arthrosis in pigs. 1.
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12
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14 15
16 17
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Incidence in animals up to 120 kg live weight. Acta Vet Scand 15: 1-25, 1974 Heinegard D, Bjorne-Persson A, Coster L, Franzen A, Gardell S, Malmstrom A, Paulsson M, Sandfalk R, Vogel K: The core protein of large and small interstitial proteoglycans from various connective tissues form distinct subgroups. Biochem J 230:421-427,1985 Heinegard D, Oldberg A: Structure and biology of cartilage and bone matrix non-collagenous macromolecules. Fed Am Soc Exp Bioi J 3:2042-2051, 1989 Holmdahl R, Andersson M, Tarkowski A: Origin of the autoreactive anti-type II collagen response. 1. Frequency of specific and multispecific B cells in primed murine lymph node. Immunology 61:369-374, 1987 Holmdahl R, Bailey C, Enander I, Mayer R, Klareskog L, Moran T, Bona C: Origin of the auto reactive antitype II collagen response. II. Specificities, antibody isotypes and usage ofV gene families of anti-type II collagen B cells. J ImmunoI142:1881-1886, 1989 Johansson S, Hook M: Substrate adhesion of rat hepatocytes: on the mechanism of attachment to fibronectin. J Cell Bioi 98:810-817, 1984 Olsson S-E: Osteochondrosis in domestic animals. Acta Radiol SuppI358:7-14, 1978 Reiland S: Osteochondrosis in the pig. A morphologic and experimental investigation with special reference to the leg weakness syndrome. PhD Thesis, University of Stockholm, Sweden, 1975 Reiland S: Morphology of osteochondrosis and sequelae in pigs. Acta Radiol Suppl 358:45-90, 1978 Woodard JC, Becker HN, Poulos PW Jr: Articular cartilage blood vessels in swine osteochondrosis. Vet Pathol 24:118-123,1987
Request reprints from Dr. S. Ekman, Department of Pathology, Box 7028, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, S-750 07 Uppsala (Sweden).
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Immunohistochemical Localization of Matrix Proteins in the Femoral Joint Cartilage of Growing Commercial Pigs S.
EKMAN AND
D.
HEINEGARD
Department of Anatomy and Histology, Swedish University of Agricultural Sciences, Uppsala, Sweden (SE); and Department of Physiological Chemistry, University of Lund, Lund, Sweden (DH) Abstract. The immunocytochemical localization of several matrix macromolecules, including collagen type II and proteoglycans, in the distal femoral articular--epiphyseal cartilage complex of 15 commercial pigs between the age of 6 and 18 weeks was studied. Early osteochondrotic lesions, i.e., chondronecrosis in the resting region of the growth cartilage, as well as extensions of necrotic cartilage into the subchondral bone, were present in all animals, except those 6 weeks old. A battery of antibodies were used for identification of macromolecules in the matrix at different stages of the disease. Chondrocyte involvement in the process could be studied by identifying the sequence of alterations in matrix macromolecules as the lesion developed. The immunostaining for aggrecan (large aggregating proteoglycans), cartilage oligomeric matrix protein, fibronectin, collagen type II, fibromodulin, and biglycanwas more prominent in the areas of chondronecrosis, extending into the subchondral bone, than in the normal resting region. This altered pattern of matrix macromolecules resembled that of the matrix of the proliferative chondrocytes and suggests that the chondrocyte maturation had stopped in the proliferative zone. The matrix in the areas of chondronecrosis in the resting region resembled that in the normal resting region. Thus the chondronecrosis appears to have preceded alterations of the matrix composition. The antibody reactivity pattern was, however, altered in the matrix of the clustered chondrocytes in areas of chondronecrosis. Staining in these regions suggested a more prominent appearance of fibronectin and collagen type II than in the normal matrix of the resting region. These changes are suggestive of attempt to repair. The chondronecrotic areas restricted to the resting region have a matrix that is different from the matrix of the abnormal cartilage extending into the subchondral bone, which resembled the matrix of the proliferative region. Hence the osteochondrotic lesion may not start in the resting region, instead the maturation of chondrocytes seems to stop in the proliferative zone, which would result in impaired bone formation.
Key words: Cartilage; immunohistochemistry; proteoglycan; swine.
Lesions with necrotic chondrocytes in the resting region of the growth cartilage of l2-week-old commercial pigs':" have been referred to as early osteochondrosis. These chondronecrotic areas are thought to ultimately involve the hypertrophic region. The chondronecrotic tissue does not seem to provide a matrix suitable for calcification, thus precluding ossification. The result is persisting necrotic cartilage protruding into the subchondral bone, i.e., osteochon-
drosis." The macromolecular composition of the retained cartilage and the normal articular--epiphyseal growth cartilage from 12- to l6-week-old pigs has previously been studied using polyclonal antibodies against proteoglycans and noncollagenous matrix proteins.' The immunostaining at the light-microscopic level indicated that the matrix in the necrotic cartilage extending into the subchondral bone differed from normal epiphyseal growth cartilage. The macromolecular composition of the extracellular matrix of this necrotic cartilage appeared to resemble the normal articular
cartilage more than the normal growth cartilage, possibly showing an altered processing of matrix constituents. Such a different macromolecular processing may follow death of chondrocytes with ensuing loss of normal synthesis and impaired matrix degradation. If, however, an arrest of chondrocytic maturation in resting or proliferation precedes cell death, deviations in matrix composition may be seen prior to chondronecrosis, A set of different macromolecules were chosen as markers for the composition of the matrix produced by the chondrocytes. Key information on mechanisms can be obtained by establishing whether or not the altered macromolecular composition found in the chondronecrotic retained cartilage, as compared with normal surrounding growth cartilage, could also be detected focally in the morphologically normal resting growth cartilage. Furthermore, the temporal relationship between observed matrix changes and chondrocyte death could provide information on cause and effect.
514 Downloaded from vet.sagepub.com at Monash University on March 6, 2015
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Matrix Proteins in Joint Cartilage of Growing Pigs
To detect early changes within the resting region of the growth cartilage, matrix structure was studied at the molecular level using immunocytochemical localization of matrix macromolecules, including collagen type II and proteoglycans, in the femoral articularepiphyseal cartilage complex of young commercial pigs over an extended age range between 6 and 18 weeks.
Materials and Methods Two litters (seven piglets/litter) of purebred Swedish Landrace pigs and one 15-week-old Swedish Landrace-Yorkshire crossbred pig were studied. The pigs received rations made according to Swedish standards,' and the littermates were euthanatized, two at each time, at the age of 6, 8, 10, 12, 14, 16, or 18 weeks. Immediately after stunning and bleeding, 3-5-mm-thick vertical sections covering the entire thickness of the articular and epiphyseal cartilage complexes and a small part of the subchondral bone were prepared. Tissue was taken from medial and lateral condyles of both right and left fernurs. Small pieces (2 x 3 x 4 mm) were cut out from the articular-epiphyseal cartilage complex, frozen in liquid nitrogen, and stored at -70 C. Four to eight pieces were taken from each pig. Four pieces were always cut from macroscopically normal articular-epiphyseal cartilage complex (lateral, right, and left; medial, right, and left). Eight pieces were cut when cartilage retained into subchondral bone was detected macroscopically in all four condyles. The frozen samples were mounted on precooled chucks, cut in a cryostat (Bright Starlet 2212 Cryostat, Huntington, England) into 6-1.@ sections, and put onto poly-L-lysine (Sigma Diagnostics, St. Louis, USA)-covered glass slides. Multiple sections were cut in succession for the full battery of antibodies. The sections were left at room temperature (20-22 C) for I hour and were fixed with acetone for 5 minutes. The sections were washed with 0.03 M phosphate-buffered 0.8% saline solution (PBS) and incubated with 0.3% H 20 , for 15 minutes. Pretreatments with normal swine serum and 4% bovine serum albumin were done to minimize nonspecific binding ofimmunoglobulins. Six different polyclonal primary antibodies raised in rabbits against bovine proteins (cartilage oligomeric matrix protein [COMP], fibromodulin, fibronectin, biglycan [PG-SI], cartilage matrix protein [CMP], and aggrecan [PG-LA]) were used.9.ID.13 Three different monoclonal primary antibodies (lgG) (CII-Cl, Cll-C2, Cll-F4) raised in DBA/l mouse against rat collagen type II were also used. [2 Samples incubated with antibodies were run in duplicate. One set was pretreated with I drop (40 I.d) of bovine testicular hyaluronidase (Sigma I-S, Sigma Chemical Co., St. Louis, MO, USA; 1 mg/ml) in 0.15 M sodium chloride and 5 mM phosphate (pH 7.4) at room temperature for 30 minutes. All antibodies were diluted in PBS containing 4% (w/v) bovine serum albumin. The sections were incubated with antisera at room temperature for 30 to 45 minutes then rinsed in PBS and incubated with an excess of the second antibody (pig anti-rabbit IgG or goat anti-mouse IgG, respectively). A final incubation with peroxidase-antiperoxidase complex was done (rabbit or mouse antiperoxidase and peroxidase, re-
spectively), and the developer 3,3-diaminobenzidine was added to visualize the antibody-antigen complex.' Controls included preincubation with the respective antigens to block the specific antibodies (cartilage oligomeric matrix protein, aggrecan, biglycan, and fibromodulin). A preimmune serum from the same species was also used. As a control for the monoclonal antibodies, an IgG monoclonal antibody (F3; with unknown specificity but nonreactive with collagen type II)" also obtained from the DBA/I mouse was used. Complementary pieces of cartilage were immersed in a 4% aqueous solution of buffered formaldehyde, dehydrated, embedded in paraffin, cut into 6-ttm-thick sections, and stained with hematoxylin and eosin. Selected sections were photographed using a Nikon Microphot-FXA photomicroscope.
Results Light microscopic findings
No pathologic changes were found in the superficial avascular articular cartilage in any of the pigs included in the study. The articular-epiphyseal cartilage complex from all pigs consisted of a thick growth cartilage with many vascular channels comprising intact blood vessels with endothelium and surrounding connective tissue. Apparently occluded vascular channels undergoing chondrification were also observed. In the 6-week-old pigs, neither areas of chondronecrosis nor any signs of disturbed ossification were observed in the growth cartilages. A few areas of chondronecrosis were present in the resting region in almost all sections of the growth cartilage from pigs that were 8 and 10 weeks old; nevertheless, proliferative, hypertrophic, and calcifying regions as well as ossification in underlying layers appeared normal. Often, but not always, areas of chondronecrosis were seen in connection with vascular channels outlined or filled with necrotic debris and erythrocytes. The growth cartilage from the 12-, 14-, 15-, 16-, and 18-week-old pigs presented a much higher abundance of necrotic areas, both in the resting (Fig. I) and in the proliferative, hypertrophic, and calcifying regions, most often in connection with degenerated vascular channels. The necrotic areas involving hypertrophic and calcifying regions caused focally disturbed ossification, creating cartilage extending into the subchondral bone (Fig. 2). Immunocytochemical findings
The immunostaining obtained with the peroxidaseantiperoxidase technique was graded as mild, moderate, or intense (Table 1). No immunostaining was observed when the primary antibody was not included, although cellular staining could be seen when nonimmune rabbit serum replaced the primary antibody.
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Fig. 1. Growth cartilage, femoral condyle; 15-week-old pig. Note the large chondronecrotic area (short arrows) in the resting region and degenerated vascular channels (long arrows). The endochondral ossification is not affected. HE. Fig. 2. Growth cartilage, femoral condyle; 12-week-old pig. Chondronecrotic area extends into the subchondral bone (*) and is surrounded by chondrocytes in cluster formations (short arrows). Note degenerated vascular channels (long arrows) in the area of chondronecrosis. HE. Fig. 3. Articular cartilage, femoral condyle; IO-week-old pig. Immunostained for collagen type II (CI). Intense staining is present in the interterritorial matrix. Peroxidase-antiperoxidase technique.
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Fig. 7. Growth cartilage, femoral condyle, resting region; l5-week-old pig. Immunostained for aggrecan. Two chondronecrotic areas (*) are surrounded by clustered chondrocytes (arrows). There is no staining of the matrix. Nonspecific cellular staining is present. Peroxidase-antiperoxidase technique. Fig. 8. Growth cartilage, femoral condyle; 12-week-old pig. Immunostained for collagen type II (Cl ), Fig. 8a. Note intense staining in the proliferative region (arrows). Peroxidase-anti peroxidase technique. Fig. 8b. Higher magnification of the area in Fig. 8a. Intense staining is seen in the interterritorial matrix (arrows).
This nonspecific cellular staining was also present after blockage of specific antibody by pretreatment with the respective antigen. This cellular stain is of little relevance for the present study, which focused on the matrix where nonspecific stain was never seen. Immunostaining was not seen when antisera-F3 (control) was used, although a mild staining was sometimes observed in the erythrocytes and in the endothelial cells. The normal articular cartilage demonstrated an intense to moderate staining with all the antibodies in 74 (75%) of99 sections (Figs. 3, 4) except against cartilage matrix protein (CMP; Table 1). The reaction was less prominent for biglycan and epitope F4 for collagen
type II. The immune reaction did not differ in different age groups. The immunostaining for all the antibodies, within the matrix of the normal resting region of the growth cartilage was low or nondetectable in all the sections (Fig. 4). The perivascular matrix close to vascular channels undergoing chondrification did not differ, in reaction, from the rest of the surrounding region. Chondronecrotic areas restricted to the resting region were often found in connection with degenerated vascular channels in all pigs ages 8 to 18 weeks. Here, an intense immunostaining with antibodies against fibronectin (Fig. 5) and collagen type II was seen in 61
Fig. 4. Articular cartilage and resting region of the growth cartilage, femoral condyle; l8-week-old pig. Immunostained for fibromodulin. Intense staining is present in the matrix of the articular cartilage. Peroxidase-antiperoxidase technique. Fig. 5. Growth cartilage, femoral condyle; l5-week-old pig. Immunostained for fibronectin. Intense staining (short arrows) is present in the matrix of cells surrounding a degenerated vascular channel (long arrow) within the resting region. Peroxidase-antiperoxidase technique. Fig. 6. Growth cartilage, femoral condyle; IO-week-old pig. Immunostained for fibronectin. Note intense staining in the proliferative region (short arrows) and in the matrix surrounding the clustered chondrocytes (arrows) in the resting region. A degenerated vascular channel (arrowhead) is also seen. Peroxidase-antiperoxidase technique.
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Table 1. Immune reaction in the articular--epiphyseal cartilage complex of 15 growing pigs, aged 6 to 18 weeks, Antisera
Matrix of Articular cartilage Resting region Proliferative region Hypertrophic region Chondronecrosis in Restricted to resting region Extension in subchondral bone
* ++
Cartilage Cartilage Collagen Oligomeric Matrix type II: FibroMatrix Protein Fibronectin Epitope modulin Protein (CMP) CI (COMP)
Aggrecan (PG-LA)
Biglycan (PG-Sl)
++* 0 + +
+* 0 0
++ 0 +
a
++ 0 + +
0* 0 0 0
++ 0 ++ 0
a
a
a
a
a
++
+
++
++
+
a
C2
F4
++ 0 ++
++ 0 ++
+ 0 +
++
++
+
0
++
++
++
++
a
a
a
= moderate to intense; + = mild to moderate; 0 = mild or no staining.
(67%) of 91 sections. The reaction was found mainly around the clustered chondrocytes (Fig. 6). When antisera against aggrecan (Fig. 7), biglycan, fibromodulin, CMP, and cartilage oligomeric matrix protein (COMP) were used, no immune reaction was seen in any of the sections.
Fig. 9. (Left) Growth cartilage, femoral condyle; 14-weekold pig. Immunostained for cartilage oligomeric matrix protein (COMP). Intense staining in the chondronecrosis extends into the subchondral bone. A degenerated vascular channel is present (arrow). Peroxidase-antiperoxidase technique. Fig. 10. (Right) Growth cartilage, femoral condyle; 15week-old pig. Immunostained for collagen type II (CI). Intense staining in the chondronecrotic area. A degenerated vascular channel is present (arrow).
The proliferative, hypertrophic, and calcifying regions of the normal epiphyseal growth cartilage are thin regions compared with the large resting region. This epiphyseal morphology differs markedly from that ofthe growth cartilage in the metaphyseal plate, where the proliferative, hypertrophic, and calcifying regions occupy most of the growth cartilage. A mild to moderate to intense reaction was present in the normal proliferative region of the growth cartilage with the full battery of antibodies except biglycan and CMP (Table 1). The most intense reaction was present when antibodies against fibronectin (Fig. 6) and collagen type II (Fig. 8) were used. The immune reaction against fibromodulin, aggrecan, and COMP was moderate to intense in the older pigs (16 and 18 weeks old) but mild to moderate in all the younger pigs (Table 1). The normal proliferative region showed a stronger immune reaction with antisera against aggrecan and fibromodulin than did the matrix surrounding the clustered cells. The normal hypertrophic and calcifying regions of the growth cartilage were hard to separate. Because the sections were cut undecalcified, much of the calcified matrix as well as the subchondral bone were artifactually lost. Any calcified area that was still present after cutting would only be partially decalcified during the incubation, hence the staining of calcified tissue was not evaluated. Only antisera against COMP and aggrecan showed a mild to moderate reaction in the normal hypertrophic region. None of the other antisera gave any immune reaction in this region (Figs. 6, 8). The chondronecrotic area extending into the subchondral bone stained moderately to intensely with all antibodies. The extensions into subchondral bone were present in all the pigs aged 12 to 18 weeks. All the extensions stained with antisera against aggrecan, biglycan, fibromodulin, COMP (Fig. 9), fibronectin, and
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collagen type II (Fig, 10). Half of the sections stained moderately with antisera against CMP. Discussion
Osteochondrosis appears to be a malfunction of the growth cartilage and is characterized by a focally impaired endochondral ossification." Osteochondrosis has been reported in many domestic animals, including the pig. 8 The ultrastructure of the early osteochondrotic lesi.on in the epiphyseal growth cartilage from growing pigs has been described as chondronecrotic areas in the resting region. 1 The chondronecrosis will ultimately involve the hypertrophic region, causing an insufficient calcification and an impaired ossification. Chondrocytes directly bordering the necrotic area are grouped in cell clusters, especially when present in close proximity to the hypertrophic and proliferative regions. These cells are metabolically active with a prominent rough endoplasmic reticulum and many lipid dropIets.!" Ultrastructural studies of osteochondrosis in the growth plate (metaphyseal growth cartilage) from growing pigs, however, showed that early lesions were accumulations of proliferative chondrocytes not undergoing normal hypertrophic cell maturation prior to chondronecrosis.s A failure ofvascular penetration and subsequent ossification followed. Normal synthesis and degradation of the cartilage extracellular matrix is dependent on the chondrocytes. Many factors could affect the chondrocytes and hence their matrix production. The early osteochondrotic lesions are not inflammatory, and the dead chondrocytes are surrounded by other normal chondrocytes. A change in matrix macromolecular composition should therefore reflect an alteration of the chondrocytic phenotype. The macromolecules in this study were chosen as markers for chondrocyte activity, and their specific function was not considered. The cause of chondrocyte death has been proposed as hypoxia due to necrosis of vascular channels."!" However, a lack of differentiation of the chondrocyte prior to cell death can not be excluded. The chondronecrotic areas in the resting region are probably the earliest lesions, moving basally towards the subchondral bone because of continuing growth in the surrounding areas. However, the matrix in the chondronecrotic areas restricted to the resting region did not resemble the macromolecular composition (when considering antibody staining pattern) of the chondroneerotic areas extending into the subchondral bone. In these areas, the composition is similar to that of the proliferative region with more intense staining for aggrecan, fibronectin, cartilage oligomeric matrix protein, fibromodulin, and collagen type II (Table 1). These results suggest that the early osteochondrotic lesion in 015
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the epiphyseal growth cartilage starts with an accum ulation of proliferative chondrocytes not undergoing hypertrophic differentiation. The chondronecrotic areas restricted to the resting region and often in connection with necrotic vascular channels could be a lesion not associated with the focally disturbed ossification characteristic for osteochondrosis. The pathogenesis of chondronecrosis in the resting or hypertrophic subchondral region may differ. This is also true for the early osteochondrotic lesion in the metaphyseal growth cartilage.' The present results indicate that the matrix surrounding the clustered cells is altered compared with that in normal growth cartilage. Thus, staining for fibronectin and collagen type II (epitope C1) was more prominent in this region than in the resting and hypertrophic regions. Chondrocytes surrounding necrotic vascular channels also stained for fibronectin and collagen type II in their matrix. The apparently increased amount of fibronectin in these domains may indicate a repair response. Alternatively, an altered permeability of the matrix could allow diffusion of plasma fibronectin. Changes in the vascular channels and therefore in the supply of oxygen and nutrients and/or removal of waste metabolites may represent triggering factors. If so, an understanding of the mechanism causing closure and chondrification of the vascular channels should provide important insights into the etiology of chondronecrosis in the resting region. Further information on early metabolic changes and their relation to cell death can be obtained by comparing mRNA levels corresponding to specific proteins in the chondrocytes of normal and osteochondrotic growth cartilage and should allow a more precise demonstration of the altered chondrocyte phenotype and should also allow early identification of dying cells. The resemblance of the matrix in the proliferative region to that in the retained chondronecrotic areas can support the hypothesis that early osteochondrotic changes in the epiphyseal and the metaphyseal growth cartilage primarily result from failure of chondrocyte maturation, which suggests that the pathogenesis for osteochondrosis in the metaphyseal and epiphyseal growth cartilages is similar. Acknowledgements We thank Ms. A Jansson for skilled technical assistance Dr. S. Johansson and Dr. R. Holmdahl for providing us With th~ antibodies against fibronectin and collagen type II, respectively. We also gratefully acknowledge Dr. O. Johnell for introducing us to the peroxidase-antiperoxidase technique used in cartilage. This work was supported by the Swedish Council for Forestry and Agricultural Research (Project No. D288), The Swedish Medical Research Council, a~d
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Request reprints from Dr. S. Ekman, Department of Pathology, Box 7028, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, S-750 07 Uppsala (Sweden).
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