Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical
deformation and recovery. This loading is essential for chondrocytes to perform their normal function of
maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the
cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional
(3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine
cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular
cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and
labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin
by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin
occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network
beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts
between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep
zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and
beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence
of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were
observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of
cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and
then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected
by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were
placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5,
1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the
vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as
in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the
explants—under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture.
The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate
environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling
pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and
modify their vimentin cytoskeleton in response to changing mechanical conditions.
Functional grading of pericellular matrix surrounding chondrocytes: potential roles in signaling and fluid transport