Effect of cell density on the rate of glycosaminoglycan accumulation by disc and cartilage cells in vitro.

Aggrecan concentration falls markedly during cartilage and disc degeneration with unfortunate biomechanical and physiological consequences. There is thus now an increasing interest in developing biological methods for its replacement. One approach is to stimulate aggrecan and hence glycosaminoglycan (GAG) production by resident cells through growth factor or genetic engineering. Another approach is to implant autologous cells into the cartilage or disc to enhance GAG production. In both instances GAG accumulation depends both on the number of active cells in the cartilage or disc and the rate of aggrecan synthesis per cell. Here we examine how cell density influences the rate of glycosaminoglycan accumulation in a three-dimensional cell culture system. In the results, at cell densities found in vivo (standard condition) in the articular cartilage and the disc nucleus viz. 4 million cells/ml and at 21% oxygen the concentration of GAG in the bead reached 520.9+/-62.4 microg/ml and 649.0+/-40.1 microg/ml, respectively, in 5 days. These concentrations could be increased to 2-4-fold by raising cell density to 25 million cells/ml. However, rates of GAG production per cell decreased by 50-60%. These results showed that rate of accumulation of glycosaminoglycan in this culture system in vitro is slow and is limited both by the rate of production of GAG per cell and by the cell density which can be maintained in a viable state. Although GAG production can be increased somewhat by use of higher cell densities, the consequent fall in concentration of oxygen and other nutrients in the center of three-dimensional constructs slows metabolism and leads to apoptosis and cell death, which again limits the rate that the cells can produce matrix.