Influence of fluid shear stress on the proliferation and differentiation of immature bone cells.

摘要

One of the mysteries of bone physiology is how loads applied to whole bone during normal movement are translated to cellular responses by the osteogenic cells. My hypothesis is that by studying the gene expression profile, proliferation, and expression of differentiation makers over time we will be able to elucidate the response of immature bone cells to fluid forces. While others have looked at fluid shear stress effects in more mature bone cells such as osteoblasts and osteocytes, I have investigated fluid shear stress on the immature bone cells such as osteoprogenitors and pre-osteoblasts. We developed an antibody technique using the STRO-1 antibody to isolate pure human osteoprogenitor cells. Upon stimulation with ascorbic acid, we demonstrated that these cells were viable and able to differentiate into calcium noduleforming osteoblasts. Using a mouse pre-osteoblast cell line (MC3T3E1), we developed a technique to apply fluid shear stress to cells in vitro. We then applied a low level fluid shear stress (0.3 dynes/cm2) to pre-osteoblasts and determined gene expression profile up to three days after stress using a macroarray. The MC3T3E1 pre-osteoblast cells responded to the pulsed, non-sustained fluid shear stress of 0.3 dynes/cm2 by changes in their transcriptional activity. In particular, the stimulus induced the early activation of the genes p57kip2 and egr-1. As a confirmation of the macroarray results, we also demonstrated a decrease in DNA replication in the first 18 hours post-stress followed by an induction of the gene Cbfa-1, a marker of osteoblast differentiation. We also demonstrated that fluid shear stress is able to induce a long-term response, which results in a significant increase in 3H-thymidine incorporation by 72 hours post-stress. Parts of this study were repeated with human osteoprogenitor cells isolated with STRO-1 antibody. Immediately after three hours of fluid shear stress, we observed differential regulation of the early growth response gene, egr-1. At 72 hours post-stress, we observed no differential regulation of a specific set of expressed genes between the stressed and non-stressed cells. Immediately after fluid shear stress we show evidence of relocalization of Rac1, but not RhoA or Cdc42 in stressed cells. RhoA and Rac1 were sequentially upregulated in pre-osteoblasts during the 72 hours post-stress. (Abstract shortened by UMI.)