br Materials and methods br Results br Discussion In these

Materials and methods
Results
Discussion In these experiments, we sought to determine the influence of gravitational mechanical unloading on the regenerative ability of bone marrow progenitors and specifically hypothesized that exposure to microgravity may alter the differentiation of bone marrow mesenchymal and hematopoietic cell lineages. To test this hypothesis, we used a combination of ex-vivo primary cell differentiation, genomics, as well as tissue and cellular analysis of in-situ differentiation to characterize the effects of microgravity unloading on the regenerative cellular physiology of the bone marrow compartment. The choice of the femoral head and neck as a study site is important because this bone region is responsive to impact gravitational load, and because it is distinct from the muscle loading-sensitive ischium region of the pelvis we previously reported in (Blaber et al., 2013). Specifically, bone tissue is exposed to several different types of load, including muscle reaction forces generated by skeletal muscle contractions, ground-reaction forces or weight-bearing forces, and intramedullary pressure gradients, that result in the integration of mechanical signals into architectural alterations commonly known as bone Tubacin (Turner, 1998). The importance of both gravitational forces and muscle forces on skeletal regulation is demonstrated through impact versus no-impact exercise training on bone mineral density (BMD) (Kohrt et al., 2009). Athletes who participate in non-weight bearing exercise such as swimming or cycling are shown to have lower BMD levels (Kohrt et al., 2009; Nikander et al., 2005). On the other hand, those involved in high impact (i.e. volleyball, soccer, squash) and weight-bearing (i.e. weight-lifting, skiing) had higher BMD levels, and furthermore athletes involved in high-impact exercise exhibit uniquely higher section modulus levels (an index of the strength of bone against bending) possibly due to increased cortical thickness of the bone (Kohrt et al., 2009; Nikander et al., 2005, 2009). The amount of load on bone varies daily without significant alterations to bone mass and therefore, a physiological range exists whereby bone is fairly unresponsive to changes in load (Carter, 1984). In spaceflight, when gravitational forces are significantly reduced there is a dramatic impact on the skeleton, approximately 10-fold higher than the accelerated bone loss that occurs at the time of menopause in women (Kohrt et al., 2009). This is because the activities that generate gravitational forces also generate muscle reaction forces and therefore, spaceflight reflects a reduction in both loading types (Kohrt et al., 2009). Although exercise countermeasures have been effective in maintaining muscle mass and strength, bone mineral density has proved challenging to preserve with no-impact exercise and therefore highlights the importance of gravitational impact forces in the maintenance of skeletal mass and integrity (Kohrt et al., 2009; Lang et al., 2004). Increased intramedullary pressure and consequently, increased interstitial fluid flow, has been implicated as a mediator of load-induced bone remodeling. Fluid flow has been shown to stimulate bone cells including osteocytes, osteoblasts, and osteoclasts by shear stress, which may result in the production of signaling molecules known to mediate bone remodeling (Reich and Frangos, 1991; Reich et al., 1990; Stevens et al., 2006). In the absence of bone strain, bone formation has been strongly correlated to fluid pressure gradients and decreased femoral intramedullary pressure during hindlimb suspension may result in decreased cell stimulation and consequently decreased bone formation (Stevens et al., 2006; Qin et al., 2003). The response of bone to alterations in load is site-specific and is dependent on the forces placed on it during normal ambulation and in altered gravity environments. Most non-load bearing bones, including the humerii and ribs, are not altered in response to microgravity exposure (Vailas et al., 1990). On the other hand, the non-load bearing calvarial bones have been reported to exhibit increased bone volume in response to spaceflight, which is possibly due to head-ward fluid redistribution reported both during spaceflight and in rodent hindlimb suspension models (Zhang et al., 2013).