Heavy ion irradiation and unloading effects on mouse lumbar vertebral microarchitecture, mechanical properties and tissue stresses.

Astronauts are exposed to both musculoskeletal disuse and heavy ion radiation in space. Disuse alters the magnitude and direction of forces placed upon the skeleton causing bone remodeling, while energy deposited by ionizing radiation causes free radical formation and can lead to DNA strand breaks and oxidative damage to tissues. Radiation and disuse each result in a net loss of mineralized tissue in the adult, although the combined effects, subsequent consequences for mechanical properties and potential for recovery may differ. First, we examined how a high dose (2 Gy) of heavy ion radiation ((56)Fe) causes loss of mineralized tissue in the lumbar vertebrae of skeletally mature (4 months old), male, C57BL/6 mice using microcomputed tomography and determined the influence of structural changes on mechanical properties using whole bone compression tests and finite element analyses. Next, we tested if a low dose (0.5 Gy) of heavy particle radiation prevents skeletal recovery from a 14-day period of hindlimb unloading. Irradiation with a high dose of (56)Fe (2 Gy) caused bone loss (-14%) in the cancellous-rich centrum of the fourth lumbar vertebra (L4) 1 month later, increased trabecular stresses (+27%), increased the propensity for trabecular buckling and shifted stresses to the cortex. As expected, hindlimb unloading (14 days) alone adversely affected microarchitectural and mechanical stiffness of lumbar vertebrae, although the reduction in yield force was not statistically significant (-17%). Irradiation with a low dose of (56)Fe (0.5 Gy) did not affect vertebrae in normally loaded mice, but significantly reduced compressive yield force in vertebrae of unloaded mice relative to sham-irradiated controls (-24%). Irradiation did not impair the recovery of trabecular bone volume fraction that occurs after hindlimb unloaded mice are released to ambulate normally, although microarchitectural differences persisted 28 days later (96% increase in ratio of rod- to plate-like trabeculae). In summary, (56)Fe irradiation (0.5 Gy) of unloaded mice contributed to a reduction in compressive strength and partially prevented recovery of cancellous microarchitecture from adaptive responses of lumbar vertebrae to skeletal unloading. Thus, irradiation with heavy ions may accelerate or worsen the loss of skeletal integrity triggered by musculoskeletal disuse.

Shared oxidative pathways in response to gravity-dependent loading and gamma-irradiation of bone marrow-derived skeletal cell progenitors.

Astronauts are exposed to radiation during space travel under conditions of dramatically reduced weightbearing activity. However, we know little about how gravity-dependent loading affects tissue sensitivity to radiation. We hypothesize gravity-dependent loading and irradiation share common molecular signaling pathways in bone cell progenitors that are sensitive to stress-induced reactive oxygen species (ROS), species capable of impacting skeletal health. To address this, progenitor cells with potential to differentiate into bone-forming osteoblasts were extracted from bone marrow, then cells were centrifuged (from 5-gravity (g) to 50-g for 5-180 min) on day 2 in culture, or were exposed to a single dose (1-5 Gy) of irradiation (137Cs 1 Gy/min) on day 3 or 4. Production of ROS was measured via fluorescence-activated cell sorting (FACS) using an oxidation-sensitive dye. Cell numbers were assessed by measurement of DNA content (CyQUANT). Osteoblastogenesis was estimated by measurement of alkaline phosphatase (ALP) activity and production of mineralized matrix (Alizarin Red staining). Transient centrifugation was a potent stimulus to bone marrow stromal cells, increasing production of ROS (1.2-fold), cell number (1.5-fold to 2.2-fold), and ALP activity (2.7-fold). Radiation also caused dose- and time-dependent increases in ROS production (1.1-fold to 1.4-fold) by bone marrow stromal cells, but inhibited subsequent osteoblast differentiation. In summary, gravity-dependent loading by centrifugation stimulated ROS production and increased numbers of osteoblasts. Although radiation increased production of ROS by bone marrow stromal cells, cell number and differentiation of osteoprogenitors appeared reduced. We conclude gravity-dependent loading and radiation both stimulate production of ROS and affect critical bone cell functions including growth and differentiation.