Three-dimensional in vitro effects of compression and time in culture on aggregate modulus and on gene expression and protein content of collagen type II in murine chondrocytes.

The objectives of this study were to determine how culture time and dynamic compression, applied to murine chondrocyte-agarose constructs, influence construct stiffness, expression of col2 and type II collagen. Chondrocytes were harvested from the ribs of six newborn double transgenic mice carrying transgenes that use enhanced cyan fluorescent protein (ECFP) and green fluorescent protein (GFP-T) as reporters for expression from the col2a1 and col1a1 promoters, respectively. Sixty-three constructs (8mm diameter x 3mm thick) per animal were created by seeding chondrocytes (10 x 10(6) per mL) in agarose gel (2% w/v). Twenty-eight constructs from each animal were stimulated for 7, 14, 21, or 28 days in a custom bioreactor housed in an electromagnetic system. Twenty-eight constructs exposed to identical culture conditions but without mechanical stimulation served as nonstimulated controls for 7, 14, 21, and 28 days. The remaining seven constructs served as day 0 controls. Fluorescing cells with rounded morphology were present in all constructs at all five time points. Seven, 14, 21, and 28 days of stimulation significantly increased col2 expression according to ECFP fluorescence and messenger RNA expression according to quantitative reverse transcriptase polymerase chain reaction. Col2 gene expression in stimulated and nonstimulated constructs showed initial increases up to day 14 and then showed decreases by day 28. Stimulation significantly increased type II collagen content at 21 and 28 days and aggregate modulus only at 28 days. There was a significant increase in aggregate modulus in stimulated constructs between day 0 and 7 and between day 21 and day 28. This study reveals that compressive mechanical stimulation is a potent stimulator of col2 gene expression that leads to measurable but delayed increases in protein (type II collagen) and then biomechanical stiffness. Future studies will examine the effects of components of the mechanical signal in culture and address the question of whether such in vitro improvements in tissue-engineered constructs enhance repair outcomes after surgery.