Mechanical Behavior and Early Molecular Signaling During Mechanotransduction of Fluid Flow in Bone Cells

The capacity for bone cells to sense and respond to skeletal interstitial fluid flow is critical in allowing bone to fulfill its structural functions. However, despite broad efforts over the past two decades to elucidate the molecular mechanisms that transduce the mechanical cues imparted by fluid flow into a biochemical signal, this process remains mostly unknown. Insight into this process can be gained through (1) mechanical analyses of bone cells to interpret their biological responses to fluid flow, and (2) investigations identifying and elucidating early molecular signals that may be generated soon after flow exposure. This dissertation consists of three studies that advance the understanding of cellular mechanical behavior and early molecular signaling events that occur during mechanotransduction of fluid flow in bone cells. In the first study, we quantified time-dependent deformations in bone cells exposed to oscillatory and steady flow profiles by using functionalized fluorescent beads as cellular displacement markers. When exposed to steady flow, the cells exhibited a nearly instantaneous deformation, followed by creep. When exposed to oscillatory flow at frequencies of 0.5-2.0Hz, the cells behaved primarily as elastic bodies. This suggests that differences in the biological responses of bone cells exposed to steady and dynamic flow may be attributable to cellular viscoelasticity. In the second study, we developed a novel class of microstructurally-informed models for the homogenized mechanical response of the actin cytoskeleton, one of the primary determinants of bone cell mechanical behavior. The model accurately captured the mechanical behavior of finite element models of three-dimensional actin networks constructed over a wide range of filament densities and degrees of anisotropy. This class of models is optimally suited for use in image-based mechanical models of cells with realistic actin cytoskeletal microstructures. In the third study, we investigated the capacity of flow to regulate cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger molecule. Bone cells responded to flow with a rapid and transient decrease in intracellular cAMP levels. This response was dependent on the primary cilium, a microtubule-based organelle that we demonstrated to be physically suitable to function as a flow sensor in bone cells. In addition, adenylyl cyclase 6 (AC6), a Ca2+-inhibitable enzyme that catalyzes the synthesis of cAMP, localized to the primary cilium, and flow-induced decreases in cAMP were dependent on AC6 and mechanically-gated ion channels. Finally, short bouts of dynamic flow induced osteogenic transcriptional changes that were mediated by this pathway. These data lead us to a proposed molecular mechanism whereby flow-induced bending of the primary cilium is transduced into a biochemical signal in bone cells.