Mechanical signals are widely accepted as regulators of skeletal homeostasis, such that the addition of exogenous mechanical load enhances osteoblastic bone formation while inhibiting osteoclastic bone resorption (3). Current hypotheses suggest that the deformation of bone in response to an applied load generates substrate strains that drive the movement of interstitial fluid and bone cells sense this fluid movement via changes in chemotransport or shear stress across cell bodies and processes (4-6). The goals of this thesis are three-fold: (1) to examine the effect of oscillatory fluid flow on human mesenchymal stem cell (hMSC) proliferation and to identify candidate signaling cascades involved in this response; (2) to identify the factor that initiates the activation of these signaling cascades; and, (3) to identify the biophysical signal that hMSCs perceive.

One hour of fluid flow exposure induced a significant increase in cellular proliferation over static controls, indicating that like osteoblasts and osteocytes, hMSCs are responsive to fluid flow. Since intracellular calcium is a vital mediator of the processes by which extracellular signals are conveyed to the cell’s interior, we next examined whether calcium signaling pathways contribute to the effect of fluid flow on hMSC proliferation. Fluid flow exposure triggered a robust, but transient increase in intracellular calcium concentration that was partially mediated by the activation of phospholipase C. Increases in intracellular calcium concentration stimulated the activation of calcineurin and the nuclear translocation of its target transcription factor, nuclear factor of activated T cells (NFAT). Fluid flow also stimulated the phosphorylation of the MAP kinases, ERK-1 and -2. Pharmacological inhibition of calcineurin activation or ERK1/2 phosphorylation blocked the effect of fluid flow on hMSC proliferation.

Having determined that fluid flow stimulates hMSC proliferation, we attempted to identify the factor(s) responsible for this response. As previous studies from our laboratory and others suggested a role for extracellular ATP in osteoblastic and osteocytic mechanotransduction (7,10,11), we hypothesized that the release of ATP may also account for the mechanosensitivity of hMSCs. hMSCs actively release ATP in response to fluid flow stimulation and express a number of purinergic receptors necessary to respond to extracellular nucleotides. Treating hMSCs with ATP, but not other nucleotides, induced cellular proliferation at a level similar to that observed with fluid flow. Further, enzymatically degrading extracellular nucleotides with apyrase abolished the effect of fluid flow on hMSC proliferation. Degrading extracellular nucleotides also abrogated the effect of fluid flow on intracellular calcium signaling, the activation of calcineurin, and the nuclear translocation of NFAT. These data strongly suggest that ATP is the factor that mediates the induction of hMSC proliferation in response to fluid flow.

Finally, we examined the contribution of chemotransport and fluid shear stress, two biophysical signals induced by interstitial fluid flow, to the effect of fluid flow on hMSC proliferation. Alterations in chemotransport, but not fluid shear stress, affected the sensitivity of hMSCs to fluid flow. We found that decreasing chemotransport inhibited hMSC proliferation as well as intracellular calcium signaling and ATP release in response to fluid flow. In contrast incrementally increasing fluid shear stress did not alter any of these parameters. These data suggest the clearance of cellular metabolites and replacement of nutrient levels are a prerequisite for hMSC mechanotransduction.

In summary, these studies provide new evidence that mechanical signals regulate the behavior of mesenchymal stem cells and outline for the first time the molecular mechanisms by which fluid flow affects these cells. The similarities between the signaling cascades activated by fluid flow in more mature osteoblastic cells and in hMSCs imply that a common pathway exists by which mechanical signals are translated to cellular responses. These data could be used in the development of new therapeutic techniques designed to enhance the recruitment of mesenchymal stem cells and promote their proliferation and subsequent differentiation into bone-forming osteoblasts. (Abstract shortened by UMI.)