Morphology of the bovine chondrocyte and of its cytoskeleton in isolation and in situ: are chondrocytes ubiquitously paired through the entire layer of articular cartilage?

OBJECTIVES: We compared the morphology and cytoskeleton of chondrocytes seeded in monolayer or in agarose gels with those retained in situ i.e. within their extracellular matrix-the chondrocyte's natural habitat.
METHODS: Cartilage specimens were harvested from adult bovine femora. Chondrocytes were either enzymatically isolated to seed in both monolayer and agarose gel culture conditions or retained in situ. Full thickness cartilage on bone was sliced both parallel and perpendicular to the articular surface. After immunostaining, the morphology of chondrocytes and of their cytoskeletal organization, i.e. distribution of actin and vimentin, in chondrocytes seeded both in monolayer and 3D agarose and those retained in situ, were assessed using confocal laser scanning microscopy.
RESULTS: The general cytoskeletal disposition of chondrocytes in situ was similar to that in agarose gel. Actin was seen to form stress fibres only in 2D culture, but not in 3D culture and in situ. In these latter conditions, actin showed a punctate staining pattern. The vimentin meshwork spanned the cytoplasm from the plasma membrane to the nuclear membrane in all culture conditions. However, the organization of the vimentin had a radiate organization in chondrocytes in monolayer and a more circumferential arrangement both in agarose gel and in situ. We further observed: (i) the prevalence of a bichondral configuration of chondrocytes in situ and (ii) the existence of a vimentin link joining some of the sister cells in situ.
CONCLUSIONS: Bichondral configuration linked with cytoskeletal elements may potentially be significant for the normal function of the chondrocytes, and therefore have implications for approaches to tissue engineering of cartilage.