ARTICLE |
Correspondence to: Doreen E. Ashhurst, Dept. of Anatomy, St George's Hospital Medical School, Cramner Terrace, Tooting, London SW17 ORE, UK.
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Summary |
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We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:16031615, 1999)
Key Words: articular cartilage, biglycan, decorin, matrilin-1, development, aging, knee joint, mRNA, in situ hybridization, immunohistochemistry
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Introduction |
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Articular cartilage matrix is composed of a number of components which include collagens, proteoglycans, and glycoproteins. The major proteoglycan (PG) is aggrecan, which forms aggregates with hyaluronan via its core protein. Other PGs include the family of small, leucine-rich PGs: biglycan, decorin, fibromodulin, and lumican. Decorin, biglycan, and fibromodulin comprise about 12% of the total dry mass of articular cartilage (
Biglycan and decorin have very similar core proteins which are substituted with two and one glycosaminoglycan (GAG) chains, respectively (
Decorin may have a role in collagen fibrillogenesis. It binds to the fibrils in specific regions (
The function of biglycan is less well understood. It may be involved in the promotion of matrix mineralization (
Biglycan and decorin in human, bovine, and marsupial adult articular cartilages are mainly confined to the upper region (
Matrilin-1 is a 148-kD glycoprotein with three disulfide-bonded monomers (
Matrilin-1 is not found in normal human and chicken articular cartilage at any stage in development (
In both osteoarthritic and rheumatoid cartilage there is increased degradation of biglycan and decorin compared to age-matched control tissue (
In the rabbit knee joint, precise changes with age in the fibrillar collagens indicate that the articular cartilage is unique and distinguished from the underlying epiphyseal cartilage from the earliest stages of joint development (
The aims of this study were, therefore, first to investigate the changes in the distribution of the small PGs during development and aging, second, to determine whether the results provide any further evidence for the uniqueness of articular cartilage, and finally to provide data that will enable a better understanding of the changes that occur with the onset of osteoarthritis.
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Materials and Methods |
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All reagents and labeled antibodies were obtained from Sigma (Poole, UK) and all restriction enzymes and polymerases were obtained from Roche (Lewes, UK) unless otherwise stated.
Preparation of Tissue
Hindlimbs distal to the mid-femoral region were removed from New Zealand White rabbit fetuses aged 17, 20, and 25 days. Knee joints were dissected from newborn and 1-week-old rabbits. The tibial plateau and underlying bone were taken from 3-, 6-, and 1214-week, 8-month, and 2-year-old rabbits. At least two rabbits were used at each stage. The tissues were fixed in 4% paraformaldehyde in 0.05 M Tris-Hcl buffer, pH 7.3, for 18 hr, then washed extensively in buffer. With the exception of 17-day joints, the tissue was decalcified in 14.3% EDTA, pH 7.0, until radiographically free from calcium. After washing, the tissue was dehydrated in graded ethanols, cleared in methyl salicylate, and embedded in paraffin. Sagittal longitudinal sections were cut at 7 µm. For histological observations, sections were stained with hematoxylin and eosin.
Immunohistochemistry
Antibodies to Biglycan and Decorin.
Chicken polyclonal antibodies to purified bovine biglycan and purified bovine decorin (a kind gift from Prof. D. Heinegård) were used. The total IgG was purified as described by
Sections were dewaxed and rehydrated before the following standard pretreatments: (a) 0.1% trypsin in 0.05 M Tris-saline, pH 7.8, with 0.1% CaCl2 at 37C for 1 hr (to reduce crosslinking and aid tissue permeability); (b) 2% hyaluronidase in PBS, pH 7.3, at 37C for 1 hr (to remove GAG chains and unmask antigenic sites); and (c) 2% L-lysine in PBS for 15 min (to reduce charged sites). The sections were washed in PBS between each treatment. To block nonspecific binding, the sections were incubated in heat-inactivated normal rabbit serum (Harlem Sera Labs; Loughborough, UK) with the addition of 4% bovine serum albumin (BSA) and 0.3% Triton X-100 for 30 min. This was drained from the slide and the primary antibody was added at a dilution of 1:1000 in 1% BSA. The sections were incubated overnight at 4C. After washing, the sections were incubated in alkaline phosphatase-labeled rabbit anti-chicken IgG antibodies for 90 min. The alkaline phosphatase label was localized using the following substrate: 5 µg naphthol AS-BI phosphate was dissolved in 1 drop dimethylformamide and added to 5 µg Fast Red TR in 10 ml veronal acetate buffer, pH 9.2. Levamisole (1 µg/ml) was added to inhibit endogenous alkaline phosphatase activity. The sections were incubated in the substrate for 20 min before being washed and mounted in glycerin jelly.
For negative controls, the primary antibody was replaced with normal chicken serum; at no time was there any binding (see Figure 6). For positive controls, using the same protocol, biglycan and decorin antibody binding was localized in skin and ligaments, which are known to contain biglycan and decorin (not shown).
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Antibodies to Matrilin-1.
Rabbit polyclonal antibodies to purified bovine tracheal matrilin-1 (a kind gift from Prof. M. Paulsson) were used. They were prepared as described by
Anti-matrilin-1 antibody (4 µl) and goat anti-rabbit IgG (1 µl) were mixed together in 400 µl PBS and stirred overnight at 4C. One µl of heat-inactivated normal rabbit serum was added to remove the excess anti-rabbit IgG and stirred at 4C for at least 2 hr.
After the same pretreatments used for biglycan and decorin antibodies, the complexes were placed on the sections and incubated at room temperature for 4 hr. The sections were then washed with PBS and the labeled antibodies were localized as above. The sections were mounted in glycerin jelly.
For negative controls, the primary antibody was replaced with normal rabbit serum. At no time was there any binding in the negative controls. Matrilin-1 is found only in cartilage. The binding of the complex by all other appropiate cartilaginous regions, e.g., growth plate and axial skeleton, served as a positive control. At no time was there any binding in noncartilaginous tissues.
In Situ Hybridization
Generation of Riboprobes.
cDNA probes to human biglycan and decorin were a gift from Dr. L. Fisher (NIH; Bethesda, MD). These probes recognized the appropiate rabbit mRNAs on Northern blots (
Procedure.
The sections were dewaxed and rehydrated. The following pretreatments were performed: (a) 0.2 N HCl for 20 min; (b) 6% H2O2 for 30 min; (c) proteinase K (20 µg/ml) for 10 min at 37C; (d) 4% paraformaldehyde in PBS for 20 min at 4C; (e) 0.1 M glycine twice for 10 min; and (f) 0.25% acetic anhydride in triethanolamine (pH 8) for 10 min. Between each treatment, the sections were washed in PBS. They were then dehydrated through graded ethanols and allowed to dry. Fifteen µl of hybridization solution was applied to each section. The hybridization solution (low stringency) contained 50% formamide, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 x Denhardt's, 0.5% SDS, 600 mM NaCl, 10% dextran sulfate, 0.5 µg/ml yeast tRNA (Roche), and labeled antisense or sense probe at a final concentration of 1.7 ng/ml. Hybridization was for 18 hr at 45C for decorin mRNA and 50C for biglycan mRNA. After hybridization, the coverslips were removed in 2 x SSC before rinsing in TrisEDTA buffer. The sections were then treated with 20 µg/ml RNase in TrisEDTANaCl buffer, pH 8.0, for 20 min at 37C. The sections were washed twice in 2 x SSC for 15 min, then 1 x SSC for 10 min at 55C; a final wash in 1 x SSC was at room temperature. The digoxigenin label was detected using the Roche kit, except that 0.3% Triton X-100 was added to the antibody solution. This blocks the nonspecific binding by cartilaginous matrices that arises after treatment with proteolytic enzymes (
At no time was there any reaction with the sense, i.e., negative control, probes (see Figure 13). For positive controls, hybridization was performed on rabbit tissues, including ligaments, skin, and blood vessels. The cells in all these tissues are positive with both probes.
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Results |
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Nomenclature
The term "epiphyseal cartilage" is used here to denote the cartilage in the epiphysis that will be removed by ossification to leave the articular and growth plate cartilages. In 17- and 20-day fetuses, the interzone comprises the two chondrogenous layers and the thin intermediate layer (
Fetal Knee Joints
Cavitation has not yet occurred in the 17- and 20-day fetal knee joints (Figure 1). Antibodies to biglycan are not bound by the matrix of the interzone. The antibodies are bound quite strongly by the hypertrophic cartilage in the diaphysis, but towards and into the epiphysis the antibody binding becomes progressively weaker until, in the region adjacent to the interzone, there is no detectable binding (not shown; Table 1). Many chondrocytes in the epiphyseal cartilage, but few in the interzone, express the mRNA for biglycan (Figure 2). The binding of decorin antibodies follows an identical pattern to that of biglycan (not shown; Table 1). Most chondrocytes in the epiphyseal cartilage, but only very few in the interzone, express decorin mRNA (Figure 3). Antibodies to matrilin-1 are not bound by the interzone or the epiphyseal cartilage immediately below, but they are bound strongly in the rest of the epiphyseal cartilage (Figure 4; Table 1).
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By 25 days, cavitation has occurred. The chondrocytes in the future articular cartilage are more densely packed than those of the epiphyseal cartilage (Figure 5). The chondrocytes on the surface are flattened. Antibodies to biglycan bind weakly to the matrix of the developing articular cartilage but more strongly to the matrix of the epiphyseal cartilage below (Figure 7; Table 1). Many chondrocytes in both the articular and epiphyseal cartilages express the mRNA for biglycan (Figure 8). Decorin antibodies are bound very weakly in the developing articular cartilage, whereas the epiphyseal cartilage matrix binds the antibodies more strongly (Figure 9; Table 1). Many chondrocytes in both regions are expressing the mRNA for decorin (Figure 10). Antibodies to matrilin-1 are not bound by the matrix of the developing articular cartilage. The epiphyseal cartilage binds the antibodies very strongly (Figure 11; Table 1).
Neonatal Articular Cartilage
At 1 postnatal week the chondrocytes in the articular cartilage are still closer together than those in the epiphyseal cartilage. There is a layer, about two cells thick, of flattened cells on the surface (Figure 12). The secondary ossification center is developing in the center of the epiphysis. Biglycan antibodies are bound by both the articular and epiphyseal matrices (Figure 14; Table 1) and most chondrocytes are expressing the mRNA for biglycan (Figure 15). Antibodies to decorin are now bound by the articular and epiphyseal matrices, except in the surface layer (Figure 16; Table 1). Most chondrocytes express the mRNA for decorin (Figure 17). Antibodies to matrilin-1 are not bound by the articular cartilage but they are bound throughout the epiphyseal cartilage matrix (Figure 18; Table 1).
At 3 weeks the articular cartilage, with some underlying epiphyseal cartilage, forms a discrete layer, and by 6 weeks ossification is almost complete (Figure 19). Immediately below the surface layer the cells are randomly arranged, but below this they are beginning to form columns. The surface layer is that containing the flattened cells (one to two cells deep); the upper, middle, and deep regions are indicated in Figure 19 (
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Juvenile Cartilage
At 1214 weeks the articular cartilage lies on a layer of compact subchondral bone and the tidemark can now be distinguished (Figure 25). Below the flattened surface cells there is a region in which the cells are irregularly arranged. Below this the cells are arranged in columns of four to six cells. Under the tidemark, the arrangement is again irregular. Biglycan antibody binding is variable at the surface but is stronger below, especially in the territorial matrix. Below the tidemark, binding is pericellular (Figure 26; Table 2). A few chondrocytes express the mRNA for biglycan (not shown). Decorin antibody binding is variable, especially in the upper region of the articular cartilage; it is weak in the surface layer. Pericellular binding occurs throughout the cartilage (Figure 27; Table 2). A few chondrocytes express the mRNA for decorin (not shown). Antibodies to matrilin-1 are not bound by the matrix of the articular cartilage (not shown; Table 2).
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Adult Articular Cartilage
By 8 months the growth plates have closed and the animal is skeletally mature. The articular cartilage is very similar to that at 1214 weeks (Figure 28) and it does not change appreciably between 8 months and 2 years. Biglycan antibody binding above the tidemark is highly variable and decreases with depth, although there is strong pericellular binding below the tidemark (Figure 29; Table 2). Decorin antibody binding is confined to the interterritorial matrix of the middle and upper regions of the cartilage. Below this region, pericellular binding is seen around some chondrocytes (Figure 30; Table 2). A few chondrocytes still express biglycan and decorin mRNAs to 2 years (not shown). Antibodies to matrilin-1 are not bound by the articular cartilage (not shown; Table 2).
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Discussion |
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The distributions of the small PGs biglycan and decorin and the glycoprotein matrilin-1, during development and aging of the rabbit knee joint have been examined.
Biglycan
Before cavitation, biglycan could not be detected in the interzone, i.e., the region from which the articular cartilage develops. Biglycan is present in the deeper regions of the epiphyseal cartilage. A similar distribution was observed in Monodelphis (
At 6 postnatal weeks the articular cartilage forms a discrete layer on the subchondral bone but ossification is incomplete. Biglycan is found throughout the cartilage but antibody binding is stronger near the hypertrophic cells. As the skeleton matures, biglycan becomes increasingly confined to the pericellular matrix, a distribution similar to that reported previously in bovine and human articular cartilage (
In the 20-day fetus the expression of biglycan mRNA in the epiphyseal and presumptive articular cartilages suggests that the synthesis of biglycan is just beginning. Few chondrocytes in the interzone express the mRNA at this stage, which correlates with the weak antibody binding in the 25-day fetal articular cartilage. The number of articular chondrocytes that express the mRNA for biglycan is higher in the 25-day fetus and in the 1- and 6-week neonate, but the number gradually decreases to 2 years. This suggests that synthesis reaches a maximum in the late fetus and neonate and then declines with age. Studies on isolated human chondrocytes showed that the level of mRNA expression for biglycan decreased with age (
The pericellular localization of biglycan in the hypertrophic region of the juvenile rabbit (6 weeks) may be associated with its possible role in the initiation of mineralization (
Decorin
Before cavitation, decorin is not detected in the matrix or cells of the interzone, but some decorin is present in the deeper regions of the epiphyseal cartilage of the rabbit. In contrast, decorin was found in the chondrocytes and matrix of the interzone of Monodelphis (
In the 20-day fetus, the expression of decorin mRNA by cells in the epiphyseal cartilage and interzone suggests that the synthesis of decorin is just beginning. Very few chondrocytes in the interzone express the mRNA at this stage, which correlates with the very weak antibody binding in the presumptive articular cartilage of the 25-day fetus. The number of articular chondrocytes that express the mRNA is higher from the 25-day fetus to the 6-week neonate, but they then decrease in number to 2 years. Thus, synthesis reaches a maximum in the late fetus and neonate and then declines with age.
Decorin binds to Type II collagen and may be involved in the control of fibrillogenesis (
Matrilin-1
Matrilin-1 is absent from the interzone and therefore from the presumptive articular cartilage, but it is widely distributed in the epiphyseal and diaphyseal cartilages at 20 days. Its presence in this cartilage precedes that of the small PGs. At 6 postnatal weeks it is present only in the pericellular matrix of the hypertrophic layer, i.e., the remnants of the epiphyseal cartilage. On completion of endochondral ossification, only articular cartilage, which never contained matrilin-1, remains. This distribution agrees with that in developing human and mouse articular cartilage (
Matrilin-1 protein and mRNA accumulate during skeletogenesis in the growth plate of human, mouse, and chicken (
The distributions of matrilin-1 and Type II collagen are very similar before cavitation in the chicken and rabbit (
Matrilin-1 enables the articular and epiphyseal cartilages to be distinguished in a manner analogous to the pericellular distribution of Type V collagen in the interzone and in developing and aging articular cartilage (
Conclusions
Neither biglycan nor decorin was detected in the interzone of the developing rabbit knee joint. Therefore, the chondroitin sulfate found in the interzone of developing human knee joints by
In the adult articular cartilage, the variable distributions of biglycan and decorin could reflect the different weight-bearing properties in different regions of the knee joint. Both biglycan and decorin synthesis are increased in load-bearing regions of canine, equine, and ovine articular cartilage (
Loss of biglycan and decorin occurs in human osteoarthritis (
The results presented here and those of
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Acknowledgments |
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Supported by the Arthritis Research Campaign (grant AO526).
We wish to acknowledge Prof D. Heinegård and Prof M. Paulsson for their kind gifts of antibodies. The technical expertise of Ms Y. Bland has been invaluable.
Received for publication March 15, 1999; accepted July 13, 1999.
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