| The mechanical loading of bone during locomotion is essential in maintaining bone mass and the structural integrity of the skeleton [1]. Excessive loss of bone mass associated with osteoporosis is suggested to affect over 28 million Americans and cause over 70% of all bone fractures in the United States of America alone [2, 3]. As a result, there is a profound interest in understanding the underlying mechanisms that stimulate bone formation to enhance its structural integrity. Mechanical loads applied to bone generate a variety of mechanical stimuli, such as substrate strain, interstitial fluid flow induced shear stress (FSS), and hydraulic pressure, that can trigger an anabolic response in osteoblasts [4--9]. Although the short and long term anabolic response of osteoblasts to FSS has been well documented [7, 10--23], little attention has been given to the response induced by pressure [24--29].;The goal of this dissertation is to provide further insight to the influence that pressure may have on the anabolic responses of osteoblasts using innovative techniques to mimic physiological conditions measured during dynamic loading of bone. The overall hypothesis is that hydraulic pressure stimulates an anabolic response in osteoblasts and plays a key role in regulating the anabolic response during fluid flow stimulation.;Exogenous loading of bone with hydraulic pressure has been shown to increase bone formation at the tissue level [30--33]; however, there is limited understanding of the underlying mechanisms that translate pressure into an anabolic response [4, 5, 24, 26]. Extensive literature has shown that FSS stimulates an anabolic response through specific pathways involving purinergic signaling of ATP, cyclooxygenase-2 (COX-2) production and reorganization of the cytoskeleton [4, 5, 10, 13, 15, 34]. The first study of this dissertation tested the hypothesis that cyclic hydraulic pressure (CHP) can induce an anabolic response in osteoblasts to the same degree as FSS. Overall, CHP induces a similar magnitude of ATP release and COX-2 production as FSS, along with changes in cell stiffness. Contrary to our expectations, FSS caused a significant depolymerization of the microtubule organization, while CHP appeared to enhance the microtubule network as well as the actin cytoskeleton.;Previous studies have shown that the magnitude and frequency of mechanical stimuli applied to osteoblasts can significantly influence their anabolic response [7, 24, 25, 27, 35]. The magnitude of hydraulic pressure needed to elicit an anabolic response was on the order of 500 mmHg. The magnitude of hydraulic pressure and interstitial fluid flow generated under physiological loading is often estimated using various models [36--41]. A key parameter used to estimate the mechanical environment experienced by bone cells is the permeability of the vascular and lacunar-canalicular system (LCS) [36]. In-vitro measurement of the intramedullary pressure (IMP) generated by axial loading would provide experimental permeability measures and increase the accuracy of such models. The second study of this dissertation established the IMP response to static and dynamic loading of bone in order to measure the permeability of the LCS independent of vascular porosity. Given the pressure diffusion characteristics of the IMP throughout the LCS during quasi-instantaneous loading, the permeability of the LCS was measured on the order of 2.3 x 10-23 m2. Based on this permeability the hydraulic pressure within the LCS can reach magnitudes of 5 MPa. In addition, the IMP response to dynamic and static loading exhibited a linear relationship with increasing magnitudes of load.;Lastly, the addition of hydraulic pressure during FSS has the potential to enhance the anabolic response of osteoblasts, such as nuclear factor kappa B (NFkappaB) nuclear translocation associated with COX-2 production. Understanding how the mechanisms specific to FSS or hydraulic pressure may interact when osteoblasts are subjected to both stimuli simultaneously is unclear, but resembles a more physiological response. The hypothesis stated that hydraulic pressure would enhance the anabolic response, specifically ATP release and NFkappaB translocation to the nucleus. However, a biphasic response in ATP release was observed with respect to increasing magnitudes of static and cyclic hydraulic pressure. In addition, the absence of pressure had no effect on NFkappaB translocation to the nucleus, but inhibits actin stress fiber formation and reorganization. As a result, FSS is the dominant stimuli of the anabolic response of osteoblasts, while hydraulic pressure regulates purinergic signaling.;In light of each study, hydraulic pressure can in fact generate an anabolic response in osteoblasts at high magnitudes, while physiological magnitudes of pressure during FSS enables osteoblasts to regulate their mechanosensitivity. Overall, each of these studies provides an extensive look at the contribution hydraulic pressure can have on the anabolic response of bone. |
