(430j) Effect of Different Intermittent Flow Strategies on Mechanotransductive Signaling and Osteoblastic Differentiation of Bone Marrow Stromal Cells

Bone marrow stromal cells (BMSCs) have the potential to be a critical ingredient in engineered bone tissue, but strategies must be developed that direct these multipotent cells to proliferate, differentiate, and form a mineralized collagen matrix. Mechanical forces, such as shear stress, have anabolic effects on bone tissue, activate mechanotransductive signaling pathways, and enhance osteoblastic differentiation of BMSCs. Fundamental studies have shown that shearing flow initiates a temporally coordinated series of biochemical signals that include release of calcium from intercellular stores, synthesis of prostaglandins, activation of MAP kinases ERK-1/2 and p38, and expression of c-fos, COX-2, and Cx43. Evidence has suggested that dynamic flow conditions, such as impulses and rest-inserted flow, are more efficient at initiating mechanotransductive signaling. We postulate that an intermittent shearing flow strategy will modulate cell signaling via autocrine and MAPK pathways and the development of the osteoblastic phenotype.

To test this hypothesis continuous and intermittent fluid flow strategies were compared for their ability to activate biochemical markers of mechanotransduction, maintain cell attachment, and stimulate osteoblastic differentiation. Intermittent flow strategies include varying the recovery time while keeping the duration of flow constant, varying the duration of flow while keeping the recovery time constant, and varying both recovery time and duration of flow. BMSCs were exposed to a shear stress of 0.38 Pa for 24 hrs on day 6 (after the addition of osteogenic supplements) using parallel plate flow chambers. Cell layers were collected for mRNA and analyzed for osteopontin, COX-2, and type I collagen by PCR. Concurrently, the recirculating medium was analyzed for prostaglandin E2. Separately, a set of cell layers were exposed to shearing flow for 24 hrs, then analyzed for cell number.

Preliminary results of this study demonstrate that differing patterns of mechanical stimulus alter mechanotransductive signaling pathways and modulate gene expression and osteoblastic differentiation.