Redifferentiation of Dedifferentiated Bovine Articular Chondrocytes Enhanced by Cyclic Hydrostatic Pressure Under a Gas-Controlled System

Abstract

Hydrostatic pressure is one of the most frequently used mechanical stimuli in chondrocyte experiments. A variety of hydrostatic pressure loading devices have been used in cartilage cell experiments. However, no gas-controlled system with other than a low pressure load was used up to this time. Hence we used a polyolefin bag from which gas penetration was confirmed. Chondrocytes were extracted from bovine normal knee joint cartilage. After 3 passages, dedifferentiated chondrocytes were applied to form a pellet. These pellets were cultured in chemically defined serum-free medium with ITS + Premix for 3 days. Then 5 MPa of cyclic hydrostatic pressure was applied at 0.5 Hz for 4 h per day for 4 days. Semiquantitative reverse transcriptase-polymerase chain reaction showed a 5-fold increase in the levels of aggrecan mRNA due to cyclic hydrostatic pressure load (p < 0.01). Type II collagen mRNA levels were also upregulated 4-fold by a cyclic hydrostatic pressure load (p < 0.01). Type I collagen mRNA levels were similarly reduced in the cyclic hydrostatic pressure load group and in the control group. The partial oxygen pressure (Po₂) and partial carbon dioxide pressure (Pco₂) of the medium in the bag reached equilibrium in 24 h, and no significant change was observed for 3 days afterwards. Po₂ and Pco₂ were very well controlled. The loaded pellet showed better safranin O/fast green staining than did the control pellet. Metachromatic staining by Alcian blue staining was found to be stronger in the loaded than in the control pellets. The extracellular matrices excretion of loaded pellets was higher than that of control pellets. These results suggest that gas-controlled cyclic hydrostatic pressure enhanced the cartilaginous matrix formation of dedifferentiated cells differentiated in vitro.