Introduction Mechanical loading of cells and tissues by grav

Introduction Mechanical loading of guanidine hydrochloride and tissues by gravity-generated forces has broad effects on mammalian cell and tissue physiology, such as in promoting the normal function and regeneration of bone, muscle, blood, and other tissues. In a healthy organism, many cell types, including osteoblasts, osteoclasts, leukocytes, and erythrocytes, are lost through a variety of mechanisms such as differentiation, senescence, apoptosis, and lysis. Cell loss, however, is balanced by proliferation and differentiation of cells from reservoirs of multi- and pluripotent somatic stem cells such as in the marrow of long bones, maintaining tissue mass homeostasis, and ultimately tissue regenerative health (Gao et al., 2012; Musaro et al., 2007; Olsson et al., 2007; Stephens and Genever, 2007; Torella et al., 2007; Zawadzka and Franklin, 2007). Strong evidence supports the idea that in-vivo tissue regenerative health is stimulated by mechanical loading related to gravity, such as in skeletal weight bearing, muscle action, and ambulation (Angevaren et al., 2008; Galloway et al., 2013; Ksiezopolska-Orlowska, 2010; Yokota et al., 2011). In addition, in-vitro studies of mechanical stimulation from load or hypergravity, show that it can promote progenitor cell proliferation and differentiation (Dvorochkin et al., 2011; Fitzgerald and Hughes-Fulford, 1996). Conversely when organisms are exposed to microgravity, they experience significant mechanical unloading of tissues, leading to various degenerative conditions. Specifically, in bone tissue, exposure to microgravity is known to cause bone loss and have significant effects on mineral homeostasis, due to a loss of balance between osteoclast bone resorption and osteoblast bone formation (Bucaro et al., 2007; Dai et al., 2007; Tamma et al., 2009; Vico et al., 2000). In order for bone mineral homeostasis to occur, there must be a tightly regulated balance between bone formation by osteoblasts and bone resorption by osteoclasts (Datta et al., 2008). Uncoupling of these processes results in either osteoporosis, or excessive bone resorption, such as in quadriplegia, age-related disuse, and exposure to microgravity, or osteopetrosis (excessive bone formation) (Tamma et al., 2009; Vico et al., 2000). Rapid bone loss has been documented within the first several days of exposure to microgravity, due to an initial increase in osteoclast numbers and activity, and is thought to be adaptive in nature with respect to the ratio of bone mass to strain (Tamma et al., 2009; Berezovska et al., 1998; Blaber et al., 2013; Saxena et al., 2011; Nabavi et al., 2011). However, decreased osteoblast numbers and function (Nabavi et al., 2011; Landis et al., 2000; Carmeliet et al., 1997; Hughes-Fulford and Lewis, 1996) and decreased numbers and differentiation capacity of mesenchymal stem cells (MSCs) (Dai et al., 2007) have also been reported indicating that increased osteoclastogenesis may be a rapid and short or mid-term response to mechanical unloading. Furthermore, widespread alterations have been noted in the hematopoietic system after exposure to microgravity, including decreased differentiation of white blood cells from hematopoietic precursors, and decreased numbers and function of T-lymphocytes, reduced number and activity of natural killer (NK cells), reduction in red blood cell mass (spaceflight anemia), and increased platelet formation (Gridley et al., 2009; Rizzo et al., 2012; Udden et al., 1995; Cogoli, 1996; Woods and Chapes, 1994; Sonnenfeld, 2002). Because of the widespread physiological effects that reductions in gravity loading have on tissue regenerative processes, we have hypothesized that mechanical unloading in microgravity may cause a reduction in somatic stem cell proliferation and differentiation, resulting in a reduced ability of tissues to repair and regenerate. To test this hypothesis, we used the femoral head and proximal shaft of mice exposed to microgravity to model in-vivo mechanical unloading of bone and marrow tissue regenerative stem cells and progenitor lineages. Specifically, we first characterized the degenerative effects of unloading in the femoral head, and then conducted ex-vivo post-microgravity cell culture assays of bone marrow proliferation and differentiation into hematopoietic and mesenchymal cell lineages. Finally, we performed bone marrow gene expression analysis, and in-situ bone marrow cellular and tissue analysis of erythrocyte and megakaryocyte differentiation. Our results show that mechanical unloading of the bone marrow compartment in microgravity has profound effects on cellular and molecular aspects of stem cell regenerative osteogenesis and hematopoiesis. Furthermore, these results begin to elucidate cellular mechanisms leading to microgravity-induced anemia and immune deficiencies, as well as reduced bone formation and increased bone resorption.