Effect of media perfusion rate on cell seeded 3D bone constructs in vitro

Using a tissue culture system we designed that perfuses culture media through 3D porous cellular constructs, this study tested the effects of media perfusion rate on cell viability, proliferation and gene expression within cell-seeded 3D bone scaffolds. Human trabecular bone scaffolds were seeded with MC3T3-E1 osteoblast-like cells and perfused for one week at flow rates of 0.01, 0.1, 0.2 and 1.0 ml/min. Confocal microscopy revealed a strong influence of perfusion rate on cell viability. A flow rate of 1.0 ml/min resulted in substantial cell death throughout the constructs while lowering the flow rate led to an increasing proportion of viable cells, particularly at the construct interior. DNA analysis showed a significant increase of cell proliferation at a flow rate of 0.01 ml/min relative to 0.2 ml/min and static controls. Conversely, mRNA expressions of Runx2, osteocalcin and alkaline phosphatase were upregulated at 0.2 ml/min compared to lower flow rates as quantified by real-time RT-PCR. These data suggest that media perfusion may benefit development of 3D tissues in vitro by enhancing nutrient delivery and waste removal within the constructs and providing flow-mediated mechanical stimuli. However, higher rates of continuous perfusion decrease cell viability perhaps due to excessive shear stresses on cells within the constructs.