The volume of in situ chondrocytes within the superficial and mid-zones increased with cartilage degeneration. Cell swelling was greater than that expected from the increased hydration in OA, suggesting that an increase in chondrocyte volume might play a role in the changes to matrix metabolism occurring in OA.

Articular chondrocytes experience changes to matrix hydration during both physiological (static load) and pathophysiological (osteoarthrosis, OA) conditions. Such changes should alter chondrocytes' volume, which has been shown to modify matrix metabolism. However, the osmometric behaviour of chondrocytes is not well understood. Here, using confocal laser scanning microscopy (CLSM ), we have investigated the 'passive' osmotic responses of fluorescent-labelled chondrocytes within, and isolated from, the matrix. The volume-regulatory pathways normally activated by cell shrinkage/swelling, were blocked by bumetanidel REV5901, respectively. Chondrocytes in situ were broadly grouped in1.o superficial (SZ), mid (MZ) and deep (DZ) zones, and there was a significant increase in resting cell volume with depth into the cartilage. Variation in medium osmolarity (range 0-530 mOsm; corresponding to an extracellular osmolarity of -150 to -600 mOsm) caused a rapid and sustained change to in situ MZ chondrocytes' volume. Over the range 180-380 mOsm, the change to in situ or isolated chondrocytes' volume was similar. For MZ chondrocytes, ideal osmometric (Boyle-van't HofF) behaviour was apparent over the extracellular osmolarity range of -250 to -600 niOsm. Chondrocytes within the SZ appeared to be more sensitive to reduced osmolarity, swelling more for a given reduction in osmolarity, than M Z or D Z chondrocytes. These data show that over wide variations in osmolarity, articular chondrocytes in situ were osmotically sensitive, and for MZ chondrocytes behaved as perfect osmometers with the extracellular matrix (ECM) not restraining cell volume changes. Changes to matrix hydration may therefore alter passive chondrocytes' volume and unless compensated by volume-regulatory pathways, could lead to changes in cell volume, and hence matrix metabolism.

Articular chondrocytes in vivo are exposed to a changing osmotic environment under both physiological (static load) and pathological (osteoarthritis) conditions. Such changes to matrix hydration could alter cell volume in situ and influence matrix metabolism. However the ability of chondrocytes to regulate their volume in the face of osmotic perturbations have not been studied in detail. We have investigated the regulatory volume decrease (RVD) capacity of bovine articular chondrocytes within, and isolated from the matrix, before and following acute hypotonic challenge. Cell volumes were determined by visualising fluorescently-labelled chondrocytes using confocal laser scanning microscopy (CLSM) at 21 degrees C. Chondrocytes in situ were grouped into superficial (SZ), mid (MZ), and deep zones (DZ). When exposed to 180mOsm or 250mOsm hypotonic challenge, cells in situ swelled rapidly (within approximately 90 sec). Chondrocytes then exhibited rapid RVD (t(1/2) approximately 8 min), with cells from all zones returning to approximately 3% of their initial volume after 20 min. There was no significant difference in the rates of RVD between chondrocytes in the three zones. Similarly, no difference in the rate of RVD was observed for an osmotic shock from 280 to 250 or 180mOsm. Chondrocytes isolated from the matrix into medium of 380mOsm and then exposed to 280mOsm showed an identical RVD response to that of in situ cells. The RVD response of in situ cells was inhibited by REV 5901. The results suggested that the signalling pathways involved in RVD remained intact after chondrocyte isolation from cartilage and thus it was likely that there was no role for cell-matrix interactions in mediating RVD.

A single impact caused temporal and spatial changes to in situ chondrocyte viability with cell shrinkage occurring in the majority of cells. However, chondrocyte shrinkage by raising medium osmolarity at the time of impact protected the cells from injury, whereas swollen chondrocytes were markedly more sensitive. These data showed that chondrocyte volume could be an important determinant of the sensitivity and response of in situ chondrocytes to mechanical stress.

Medium osmolarity significantly affects chondrocyte death in wounded articular cartilage. The greatest chondrocyte death occurs at 0 mOsm. Conversely, increased medium osmolarity (480 mOsm) is chondroprotective. The majority of cell death occurs within 2.5 hours, with no significant increase over seven days.