Effect of osmolarity on glycosaminoglycan production and cell metabolism of articular chondrocyte under three-dimensional culture system.

OBJECTIVES This study examined how physiological levels of extracellular osmolarity influence proteoglycan accumulation in articular chondrocytes in a three-dimensional culture system. METHODS Cells were obtained from metacarpal phalangeal joints of 18-24 month bovine. They were cultured for 6 days in alginate beads at 4 million cells/ml in DMEM containing 6% FBS under 21% O2. Medium osmolarity was altered by NaCl addition over the range 270-570 mOsm and monitored using a freezing point osmometer. Profiles across intact beads were determined by manual counting using fluorescent probes and transmission electron microscope. Lactate production was measured enzymatically and glycosaminoglycan (GAG) accumulation was measured using a modified dimethylmethylene blue assay. Rate of sulfate GAG synthesis was measured using a standard 35S-sulfate radioactive method. RESULTS The cell viability was similar for the high and low osmolarity cultures. However, confocal microscopy showed that the cells were the largest under 270 mOsm and became smaller with increasing osmotic pressure. GAG production was largest in the 370mOsm, and the capacity for GAG production and cell metabolism (lactate production) was low under hypo-osmolarity and hyper-osmolarity, and cell deaths were often observed on electron microscopy. CONCLUSIONS In our model the prevailing osmolarity was a powerful regulator of GAG accumulation by cultured chondrocytes. These results thus indicate GAG synthesis rates are regulated by GAG concentration, with implications both for the aetiology of osteoarthritis and for tissue engineering.