An engineered bone substitute consisting of a bone-like extracellular matrix deposited on the internal pores of a resorbable biomaterial scaffold is postulated to stimulate integration, vascular infiltration, and normal bone remodeling when implanted in vivo. Bone marrow derived mesenchymal stem cells (MSCs) have the potential to synthesize a bone-like extracellular matrix in vitro but culture strategies are needed to facilitate the development of clinically applicable engineered bone graft substitutes. Perfusion bioreactors have been investigated as a component of engineered bone strategies because they supply oxygen and nutrients to cells seeded within a scaffold while applying a mechanical stimulus to the cells. Evidence with planar cell cultures has shown that dynamic flow regimens elicit an enhanced cellular response over steady flow regimens but dynamic perfusion strategies have not yet been translated to 3D scaffold architectures. The objective of this research is to evaluate the effect of frequency of pulsatile flow patterns on the osteogenic differentiation of mesenchymal stem cells seeded in porous scaffolds. We hypothesize that pulsatile flow may be more effective at stimulating osteogenic differentiation of MSCs than continuous perfusion alone and that the cellular response to pulsatile flow may be frequency dependent.

To test the effect of pulsatile flow on osteogenic differentiation we have cultured MSCs in polyurethane foam scaffolds. The scaffolds were either kept under static conditions or subjected to continuous flow or pulsatile flow at frequencies of 0.083, 0.050, and 0.018 Hz. Scaffolds were collected after 14 days of flow to measure proliferation and osteoblastic differentiation of MSCs as indicated by levels of alkaline phosphatase activity and bone extracellular matrix proteins. Preliminary results indicate that all flow conditions enhanced alkaline phosphatase activity and osteopontin expression. Additionally, these markers are preferentially enhanced by pulsatile flow over continuous flow. A trend of increasing alkaline phosphatase activity with decreasing pulse frequency suggests that cells are sensitive to frequency of pulsatile flow. These results indicate that dynamic perfusion may be a useful component of the engineered bone tissue strategy.