Deformation of isolated articular chondrocytes cultured in agarose constructs

Chondrocyte mechanotransduction may be of considerable importance to the development of tissue engineering techniques for cartilage repair. The well characterised isolated chondrocyte/agarose model allows investigation of the influence of cell deformation independent of other possible mechanotransduction pathways.

The current study has optimised a series of confocal microscopy techniques to visualise viable bovine adult articular chondrocytes seeded in 3% agarose constructs. In unstrained agarose, isolated chondrocytes adopted a spherical morphology with diameters of 7-12<IMG WIDTH=8 HEIGHT=14 ALIGN=MIDDLE SRC="/maths/mu.gif">m. This spherical form was maintained throughout a 6 day culture period, although those cells cultured in medium with 20% serum demonstrated a 25% increase in median cell diameter. At day 1 of culture, unconfined compression of agarose constructs up to 30% strain, resulted in cell deformation to an oblate ellipsoid morphology. Deformation was associated with a statistically significant increase in surface area but no significant change in cell volume.

When specimens were compressed after 6 days in culture, a reduction in cell deformation was observed. Biochemical analysis indicated that this was associated with an elaboration of sulphated glycosaminoglycan. Dual scanning confocal microscopy, using a monoclonal antibody to keratan sulphate, indicated the presence of a pericellular shell extending 5-10<IMG WIDTH=8 HEIGHT=14 ALIGN=MIDDLE SRC="/maths/mu.gif">m from the cell membrane. This matrix was stiffer than the surrounding agarose. Removal of the sulphated GAG by digestion with testicular hyaluronidase restored cell deformation, although not to the levels observed at day 1. This suggested the presence of other macromolecules which provide structural importance to the pericellular matrix.

Chondrocytes isolated from the superficial 15% of articular cartilage were smaller and less metabolically active than cells isolated from deeper zones. However, all cells adopted the same spherical morphology in unstrained agarose and deformed to similar levels when compressed at day 1 of culture.

This study provides important insight into the biomechanics of isolated cells in agarose and the possible involvement of chondrocyte deformation in mechanotransduction.