Chondrons from articular cartilage. V. Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy

Journal of Cell Science, Dec 1992

C.A. Poole, S. Ayad, R.T. Gilbert

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Chondrons from articular cartilage. V. Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy

C. ANTHONY POOLE 1 SHIRLEY AYAD 0 RAYMOND T. GILBERT 1 0 Department of Biochemistry and Molecular Biology, University of Manchester Medical School , Manchester, M13 NPT, England 1 Department of Anatomy, University of Auckland, School of Medicine , Private Bag 92019, Auckland , New Zealand V.* Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy - The pericellular microenvironment around articular cartilage chondrocytes must play a key role in regulating the interaction between the cell and its extracellular matrix. The potential contribution of type VI collagen to this interaction was investigated in this study using isolated canine tibial chondrons embedded in agarose monolayers. The immunohistochemical distribution of an anti-type VI collagen antibody was assessed in these preparations using fluorescence, peroxidase and gold particle probes in combination with light, confocal and transmission electron microscopy. Light and confocal microscopy both showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte with reduced staining in the tail region and the interconnecting segments between adjacent chondrons. Minimal staining was recorded in the territorial and interterritorial matrices. At higher resolution, type VI collagen appeared both as microfibrils and as amorphous deposits that accumulated at the junction of intersecting capsular fibres and microfibrils. Electron microscopy also showed type VI collagen anchored to the chondrocyte membrane at the articular pole of the pericellular capsule and tethered to the radial collagen network through the tail at the basal pole of the capsule. We suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the col lagens, proteoglycans and glycoproteins of the pericellular microenvironment. Secondly, specific cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. In this way type VI collagen could provide a close functional interrelationship between the chondrocyte, its pericellular microenvironment and the load bearing extracellular matrix of adult articular cartilage. The interaction between the chondrocyte and its extracellular matrix is of critical importance in regulating the development, maintenance and repair of articular cartilage. However, it is now generally accepted that the chondrocytes in adult articular cartilage are surrounded by a specialised microenvironment, which effectively insulates the chondrocyte and physically separates the cell from direct interaction with the bulk of the load-bearing matrix. The pericellular microenvironment must therefore play an important role in mediating the interaction between the chondrocyte and its extracellular matrix. Collectively, the chondrocyte and its pericellular microenvironment are thought to represent the chondron, arguably the primary functional and metabolic unit of hyaline cartilages (for review see Poole, 1992). Slow-speed homogenisation techniques have now been developed to separate significant numbers of chondrons from the bulk extracellular matrix of a variety of normal mammalian articular cartilages (Poole et al. 1988a,b; Poole, 1992). With the identification of the chondron as a true microstructure of articular cartilage, current studies have focused on defining the composition and organisation of the chondron, and its role in chondrocyte - matrix interactions. Using a variety of immunohistochemical techniques, the chondron has been shown to contain collagen types II, VI and IX (Poole et al. 1988a,c), the aggrecan components chondroitin 4-sulphate, chondroitin 6-sulphate, keratan sulphate, core protein and hyaluronan binding region (Poole et al. 1991), and the glycoprotein fibronectin (Poole, 1990). Type VI collagen was originally identified as a highly disulphide bonded aggregate in pepsin digests of aortic intima (Chung et al. 1976), but is now considered a component of most, if not all, connective tissues (for reviews see Engel et al. 1985; Rauterberg et al. 1986; Timpl and Engel, 1987). In bovine articular cartilage, type VI collagen accounts for only 1-2% of the total collagens present (Eyre et al. 1987), but its preferential localisation in the chondron (Poole et al. 1988a) suggests it represents a significant component of the pericellular microenvironment. The type VI collagen monomer consists of a short triplehelical domain (105 nm) at each end of which is a large globular domain (Engel et al. 1985: Rauterberg et al. 1986; Timpl and Engel, 1987). Biosynthetic studies have demonstrated that monomers assemble intracellularly into dimers and tetrameres, which are then secreted into the extracellular matrix and assemble (...truncated)


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C.A. Poole, S. Ayad, R.T. Gilbert. Chondrons from articular cartilage. V. Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy, Journal of Cell Science, 1992, pp. 1101-1110, 103/4,