ARTICLE |
Correspondence to: S. Milz, Anatomische Anstalt, Ludwig-Maximilians-Universität, Pettenkoferstr. 11, D-80336 München, Germany. E-mail: stefan.milz@anat.med.uni-muenchen.de
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Summary |
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The rabbit suprapatella is a sesamoid fibrocartilage in the deep surface of the tendon of vastus intermedius and an integral part of the knee joint. We report the presence of a variety of proteoglycans (aggrecan and versican), glycosaminoglycans (chondroitin 4 and 6 sulfate, dermatan sulfate, keratan sulfate) and glycoproteins (tenascin) in its extracellular matrix and the intermediate filament vimentin in the fibrocartilage cells. The most significant finding is the presence of aggrecan in the extracellular matrix, along with its associated link protein and several of its integral glycosaminoglycans. Aggrecan probably enables the suprapatella to withstand compression. Although it can be assumed that aggrecan metabolites detected in synovial fluid from some human joints are predominantly associated with articular hyaline cartilage, the presence of aggrecan in the rabbit suprapatella means that this cannot be assumed for all animal knee joints. We conclude that it is important for orthopedic researchers who use animal models for arthritis research to check for the presence of a suprapatella when joint fluid analyses are interpreted.
(J Histochem Cytochem 50:955960, 2002)
Key Words: fibrocartilage, aggrecan, link protein, collagens, extracellular matrix, vimentin
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Introduction |
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A NUMBER OF ANIMALS, including the rabbit, have been used as experimental models in studies of osteoarthritis or rheumatoid arthritis (
Wrap-around tendons are frequently characterized by their content of aggrecan and Type II collagen (
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Materials and Methods |
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The suprapatellae were dissected out from the hind limbs of six mature female Chinchilla-bastard rabbits (mean body weight 4500 g) that were sacrificed by IV injection of ketamine, xylazine, and thiopental for control purposes associated with a separate and unrelated study by other researchers. The tissue was fixed in 90% methanol at 4C for at least 24 hr, infiltrated overnight in a 5% sucrose solution in PBS at pH 7.4, mounted on chucks in Jung tissue embedding medium (Leica; Oberkochen, Germany), frozen in an HM 500 OMV cryostat (Microm; Walldorf, Germany), and cryosectioned at 12 µm. Sections were stained with toluidine blue for metachromasia and labeled with a panel of monoclonal antibodies directed against PGS, GAGs, glycoproteins, and proteins (Table 1). All sections were treated with 0.3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity, and nonspecific binding of the secondary antibodies was reduced by blocking with normal horse serum for 60 min. Control sections were incubated with normal mouse immunoglobulins (10 µg/ml) or the primary antibody was omitted and sections were treated with PBS alone. Antibody binding was detected with a Vectastain ABC Elite avidinbiotin kit (Vector Labs; Burlingame, CA) and sections were counterstained with Mayer's hematoxylin.
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Many of the primary antibodies required some enzymatic predigestion step or other chemical pretreatment (Table 1). This served either to expose epitopes hidden by the extracellular matrix (ECM; antibodies to tenascin and versican), or to generate stubs carrying neoepitopes (antibodies to the chondroitin sulfates, link protein, and aggrecan). It is important to note that enzyme pretreatment is critical for the subtle distinctions among the different GAGs (
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Results |
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The suprapatella appeared as an intensely metachromatic sesamoid fibrocartilage in the deep part of the tendon of vastus intermedius (Fig 1A). However, near the articular surface of the suprapatella, there was a thin layer of poorly staining fibrous tissue (Fig 1C) in which the cells were flattened and arranged with their long axes parallel to the surface. Immediately deep to this fibrous zone were large chondrocytes separated by an ECM with a more hyaline appearance (Fig 1C). Deeper still, the majority of the metachromatic region of the suprapatella was characterized by a basketweave-like arrangement of collagen fibers. The immunohistological staining characteristics of the fibrocartilage are summarized in Table 1.
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Aggrecan was highly characteristic of the suprapatella and strong labeling was detected in most parts of the tissue except for its superficial fibrous zone, where labeling was much weaker (Fig 1B and Fig 1D). It was also present in the deep part of the tendon of vastus intermedius, although not in its superficial part (Fig 1B). Adjacent sections labeled for link protein, although the labeling was patchy (Fig 1E). The distribution of versican was broadly complementary to that of aggrecan. Therefore, whereas strong labeling characterized both parts of the tendon of vastus intermedius (Fig 1I), labeling in the suprapatella was largely restricted to the superficial fibrous zone, although some labeling was also seen in the larger fibrocartilage cells (Fig 1J). Several of the GAGs that form part of aggrecan (i.e., keratan sulfate, chondroitin 4 and 6 sulfates, and dermatan sulfate) were also detected in the suprapatella (Fig 1F1H; Table 1).
Tenascin labeling was rather variable, but most of the suprapatella was generally devoid of label. Where staining was detected, it was usually in the superficial fibrous zone and in both parts of the tendon of vastus intermedius. Fibrocartilage cells in the suprapatella labeled strongly for vimentin (Fig 1K), but there were only a few strands of label in tendon cells.
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Discussion |
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Aggrecan is a large aggregating PG commonly associated with the water-trapping properties of articular cartilage. The profusion of its negatively charged GAG side-chains creates domains with a high osmotic pressure and great potential to suck water into the tissue (
Our results are of general interest in relation to animal model studies of osteoarthritis and rheumatoid arthritis, because a suprapatella is present in the knee joints of several laboratory animals used for this purpose. These include not only the rabbit but also the guinea pig, mouse, rat, cat, and dog (
The distribution of versican in the suprapatella is the reciprocal of that of aggrecan. Therefore, it is more conspicuous in the overlying quadriceps tendon than in the suprapatella itself, and within the latter it is most prominent in the superficial fibrous zone, i.e., where aggrecan labeling is much weaker. Such a restriction of labeling largely to the periphery of cartilaginous tissue has been reported previously for versican in developmental studies (e.g.,
It is intriguing that some intracellular signal for versican was evident in the fibrocartilage cells of the suprapatella. This suggests either that the cells can synthesize versican without releasing it to the ECM or that it is destined only for the immediate pericellular matrix. It is therefore pertinent to note that the molecule can bind to cell membranes (
Tenascin labeling in the suprapatella was highly variable, even though large rounded fibrocartilage cells were always present. Although labeling was occasionally seen around these cells, it was sometimes more obvious in the more flattened cells near the surface of the patella. Therefore, our results do not fit easily with the suggestion of
Vimentin expression has also been associated with a fibrocartilage phenotype (
We therefore conclude that it is important for orthopedic researchers to be aware of the suprapatella and to consider its presence when joint fluid analyses in animals widely used for arthritis research are interpreted. This structure is commonly neglected or overlooked, but it can be as prominent as the patella itself. It contains a wide range of molecules that are also expressed in articular cartilage and is present in a number of animals that are highly significant in orthopedic research.
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Acknowledgments |
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Supported by the Friedrich Baur Stiftung Munich. The monoclonal antibodies 12/21/1-C-6, 9/30/8A4, 12C5 and Amf-17b were obtained from the Developmental Studies Hybridoma Bank maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, under contract NO1-HD-7-3263 from the NICDH.
The practical work was performed at the Anatomy Department, Munich.
Received for publication October 10, 2001; accepted January 23, 2002.
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