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
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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)