The strong correlation between a bone's architectural properties and the mechanical forces that it experiences has long been attributed to the existence of a cell that not only detects mechanical load but also structurally adapts the bone matrix to counter it. One of the most likely cellular candidates for such a "mechanostat" is the osteocyte, which resides within the mineralized bone matrix and is perfectly situated to detect mechanically induced signals. However, as osteocytes can neither form nor resorb bone, it has been hypothesized that they orchestrate mechanically induced bone remodeling by coordinating the actions of cells residing on the bone surface, such as osteoblasts. To investigate this hypothesis, we developed a novel osteocyte-osteoblast coculture model that mimics in vivo systems by permitting us to expose osteocytes to physiological levels of fluid shear while shielding osteoblasts from it. Our results show that osteocytes exposed to a fluid shear rate of 4.4 dyn/cm 2 rapidly increase the alkaline phosphatase activity of the shielded osteoblasts and that osteocytic-osteoblastic physical contact is a prerequisite. Furthermore, both functional gap junctional intercellular communication and the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 signaling pathway are essential components in the osteoblastic response to osteocyte communicated mechanical signals. By utilizing other nonosteocytic coculture models, we also show that the ability to mediate osteoblastic alkaline phosphatase levels in response to the application of fluid shear is a phenomena unique to osteocytes and is not reproduced by other mesenchymal cell types.osteocyte; osteoblast; fluid-flow; coculture; mechanical stimulation; gap junction; intercellular communication ONE OF THE major tenets of bone biology is that mechanical load perturbs interstitial fluid and that, upon detecting these perturbations, osteocytes embedded deep within mineralized bone remotely coordinate the adaptive response by directing the actions of effector cells such as bone-forming osteoblasts and bone-resorbing osteoclasts (4, 12). Although this theory is widely accepted, there is little experimental evidence that, in response to fluid shear, embedded osteocytes directly alter the cellular behavior of surface-residing osteoblasts or osteoclasts. In vivo, osteocytes pass dendritic-like cellular processes through channels in the mineralized matrix (canaliculi) and form physical connections, specifically gap junctions, with both neighboring osteocytes and surface-residing bone cells (11,23). We hypothesize that these intercellular gap junctions provide the means by which mechanically induced fluid shear signals may be communicated from osteocytes to effector cells such as osteoblasts and osteoclasts.There is abundant evidence suggesting that gap junctional intercellular communication (GJIC) contributes to mechanotransduction in the musculoskeletal system. Banes et al. (3) demonstrated that equibiaxial strain upregulates connexin 43 (C...

Although the mechanisms by which osteoblasts and osteocytes respond to fluid flow are being elucidated, little is known about the mechanisms by which bone marrow-derived mesenchymal stem cells respond to such stimuli. Here we show that the intracellular signaling cascades activated in human mesenchymal stem cells by fluid flow are similar to those activated in osteoblastic cells. Oscillatory fluid flow inducing shear stresses of 5, 10, and 20 dyn/cm 2 triggered rapid, flow rate-dependent increases in intracellular calcium that pharmacological studies suggest are inositol trisphosphate mediated. The application of fluid flow also induced the phosphorylation of extracellular signal-regulated kinase-1 and -2 as well as the activation of the calcium-sensitive protein phosphatase calcineurin in mesenchymal stem cells. Activation of these signaling pathways combined to induce a robust increase in cellular proliferation. These data suggest that mechanically induced fluid flow regulates not only osteoblastic behavior but also that of mesenchymal precursors, implying that the observed osteogenic response to mechanical loading may be mediated by alterations in the cellular behavior of multiple members of the osteoblast lineage, perhaps by a common signaling pathway. mechanotransduction; bone; marrow MAINTENANCE OF APPROPRIATE bone mass requires the coordination of bone resorption by osteoclasts and bone deposition by osteoblasts, and it is well established that mechanical stimuli can regulate the balance between bone formation and resorption. The addition of exogenous mechanical load is believed to stimulate new bone formation through increases in osteoblastic activity and concomitant decreases in osteoclastic activity (20). Conversely, removal of mechanical load, as is the case during spaceflight and disuse, leads to decreased osteoblastic activity and loss of bone mass (42,56).Accumulating evidence suggests that individual bone cells, including osteocytes and osteoblasts, are responsible for perceiving and responding to mechanical signals. Such signals, which may include streaming potentials, mechanical strain, and fluid shear stress, elicit a host of biochemical responses including mobilization of second messengers such as calcium (22,72), nitric oxide (26, 30), prostaglandins, and inositol trisphosphate (IP 3 ) (49); activation of kinase cascades including the MAP kinase and PKC pathways (44, 50); and modulation of gene expression (5, 46, 73). Strain-induced oscillatory fluid flow has been shown by our laboratory (73) and others (45) to be a potent biophysical stimulus. In MC3T3-E1 preosteoblasts, fluid flow induces the mobilization of intracellular calcium (Ca i 2ϩ ), activates ERK1/2, and increases osteopontin mRNA levels (11,72,73). Recent evidence has shown that exposing the osteocytic cell line MLO-Y4 to physiological levels of fluid flow induces similar responses (1, 6, 52).Although the mechanisms by which osteoblasts and osteocytes respond to mechanical stimuli are being elucidated, little is known about how bo...

Oscillatory fluid flow induced the vesicular release of ATP from human BMSCs that directly contributes to the induction of BMSC proliferation. Degrading extracellular nucleotides prevents fluid flowinduced increases in intracellular calcium concentration, the activation of calcineurin, and the nuclear translocation of NFAT.Introduction: Regulation of bone cell activity by autocrine/paracrine factors is a well-established mechanism by which skeletal homeostasis is regulated by mechanical signals. The release of extracellular nucleotides in particular has been shown to induce many of the responses thought to be necessary for load-induced bone formation. In these studies, we examined the effect of oscillatory fluid flow on the release of ATP from bone marrow stromal cells (BMSCs) and the effect of ATP release on BMSC proliferation and intracellular calcium signaling pathways. Materials and Methods: BMSCs were exposed to oscillatory fluid flow, and the concentration of ATP in conditioned media samples was determined using a luciferin:luciferase-based reaction. Western blot analysis was used to examine the expression of purinergic receptors. Using pharmacological antagonists of gap junction hemichannels and vesicular trafficking, we studied the mechanism of ATP release from BMSCs. Apyrase was used to study the effect of extracellular nucleotides on intracellular calcium concentration, calcineurin activity, and nuclear factor of activated T cells (NFAT) nuclear translocation. Results and Conclusions: Fluid flow exposure induced the flow rate-dependent release of ATP from BMSCs that was attenuated by treatment with monensin and N-ethylmaleimide, suggesting a vesicular mechanism. Treating BMSCs with ATP, but not other nucleotides, increased cellular proliferation. Moreover, extracellular ATP was a prerequisite for fluid flow-induced increases in intracellular calcium concentration, activation of calcineurin, the nuclear translocation of NFATc1, and proliferation. These data indicate that ATP regulates not only osteoblastic and osteocytic cell behavior but also that of mesenchymal precursors and support our hypothesis that similar mechanotransduction mechanisms are activated by fluid flow in these cell types.