An understanding of the processes involved in volume regulation of cartilage cells (chondrocytes) is of interest because changes can have a deleterious effect on matrix metabolism (Urban et al. 1993). There is growing interest in the use of 3-dimensional artificial gels (alginate, agarose) for suspending chondrocytes as this allows more precise control of the chondrocyte’s environment, and prevents changes to phenotype which can occur (Guo et al. 1989). Here, we have compared the volume regulatory response of chondrocytes within gels to hyper- or hypo-osmotic challenge.

Articular chondrocytes isolated into DMEM (380 mosmol, pH 7.4) were obtained from cartilage explants taken from the bovine metacarpal-phalangeal joint of animals slaughtered at the local abattoir. Chondrocytes were entrapped in alginate (0.625 % w/v) or agarose (0.2 %) gels within 2 h and volume regulatory behaviour following osmotic challenges studied using fluorescence imaging of intracellular calcein (Hall & Bush, 2001). A hypotonic challenge (380-180 mosmol) caused rapid cell swelling and regulatory volume decrease (RVD) for cells in all gels (72 ± 15 % recovery within 10 min; for all results, data are means ± S.E.M., n ▓ge│ 3). In contrast, a hypertonic challenge (380-580 mosmol) caused rapid cell shrinkage but no regulatory volume increase (RVI) (P > 0.05, Student’s unpaired t test). When cells were exposed to a hypotonic challenge and then returned to isotonic saline (the ‘post-RVD/RVI protocol’), cells within agarose showed weak post-RVD/RVI (30 ± 5 % recovery in 10 min; n ▓ge│ 3). In contrast, the cells in alginate showed an overshoot in cell volume (140 ± 18 %). REV5901 (50 mM) and bumetanide (75 mM) blockers of the osmolyte channel and Na⁺-K⁺-Cl^– cotransporter, respectively, inhibited RVD and RVI of chondrocytes in alginate by 80 ± 10 and 95 ± 5 %.

The results show that post-RVD/RVI occurred mainly in chondrocytes suspended in alginate, but also in agarose gels. This response is not observed in freshly isolated chondrocytes (Kerrigan et al. 2002), suggesting that changes to chondrocyte physiology occur as a result of gel formation. The encapsulation of chondrocytes within gels is undoubtably a valuable experimental model, although these data suggest that this is not a benign process, and there may be longer-term consequences to chondrocytes which might not accurately model the behaviour of in situ cells.

This work was supported by the Arthritis Research Campaign (H0621), The Wellcome Trust (045925/Z/95/A) and the MRC.