Bone can become brittle when exposed to ionizing radiation across a wide range of clinically relevant doses that span from radiotherapy (accumulative 50 Gy) to sterilization (~35,000 Gy). While irradiation-induced embrittlement has been attributed to changes in the collagen molecular structure, the relative role of collagen fragmentation versus non-enzymatic collagen crosslinking remains unclear. To better understand the effects of radiation on the bone material without cellular activity, we conducted an ex vivo x-ray radiation experiment on excised mouse lumbar vertebrae. Spinal tissue from twenty-week old, female, C57BL/6J mice were randomly assigned to a single x-ray radiation dose of either 0 (control), 50, 1,000, 17,000, or 35,000 Gy. Measurements were made for collagen fragmentation, non-enzymatic collagen crosslinking, and both monotonic and cyclic-loading compressive mechanical properties. We found that the group differences for mechanical properties were more consistent with those for collagen fragmentation than for non-enzymatic collagen crosslinking. Monotonic strength at 17,000 and 35,000 Gy was lower than that of the control by 50% and 73% respectively, (p < 0.001) but at 50 and 1,000 Gy was not different than the control. Consistent with those trends, collagen fragmentation only occurred at 17,000 and 35,000 Gy. By contrast, non-enzymatic collagen crosslinking was greater than control for all radiation doses (p < 0.001). All results were consistent both for monotonic and cyclic loading conditions. We conclude that the reductions in bone compressive monotonic strength and fatigue life due to ex vivo ionizing radiation are more likely caused by fragmentation of the collagen backbone than any increases in non-enzymatic collagen crosslinks.

One potentially important bone quality characteristic is the response of bone to cyclic (repetitive) mechanical loading. In small animals, such as in rats and mice, cyclic loading experiments are particularly challenging to perform in a precise manner due to the small size of the bones and difficult-to-eliminate machine compliance. Addressing this issue, we developed a precise method for ex vivo cyclic compressive loading of isolated mouse vertebral bodies. The method has three key characteristics: 3D-printed support jigs for machining plano-parallel surfaces of the tiny vertebrae; pivotable loading platens to ensure uniform contact and loading of specimen surfaces; and specimen-specific micro-CT-based finite element analysis to measure stiffness to prescribe force levels that produce the same specified level of strain for all test specimens. To demonstrate utility, we measured fatigue life for three groups (n = 5–6 per group) of L5 vertebrae of C57BL/6J male mice, comparing our new method against two methods commonly used in the literature. We found reduced scatter of the mechanical behavior for this new method compared to the literature methods. In particular, for a controlled level of strain, the standard deviation of the measured fatigue life was up to 5-fold lower for the new method (F-ratio = 4.9; p < 0.01). The improved precision for this new method for biomechanical testing of small-animal vertebrae may help elucidate aspects of bone quality.

Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.Alterations in the gravity vector and exposure to ionizing radiation can disrupt skeletal homeostasis in mice 1-3 . There are multiple stressors associated with spaceflight, including microgravity and radiation which are known to cause bone loss 4-6 . Decrements in bone mineral density (BMD) have been observed in astronauts from the Mir missions as well as missions to the International Space Station (ISS) 7-9 . While much research has focused on the detrimental effects of microgravity on skeletal tissue, less is known about the impact of spaceflight radiation. Crewed missions have, to this point, primarily remained within low-Earth orbit (LEO). While sources of ionizing space radiation within LEO include galactic cosmic radiation and charged particles from unpredictable solar particle events (SPE) 10,11 , the presence of the Earth's magnetosphere reduces exposure to ionizing space radiation. Missions beyond LEO pose the greatest risk of radiation exposure and is of significant concern for crew health 12-14 . Spaceflight-relevant radiation includes a mix of low-linear energy transfer (LET) species such as protons and helium ions as well as high-LET species such as iron 15,16 . Beyond LEO, for example, astronauts may be exposed to up to 0.7 Sv of ionizing radiation 12,15,17 during a multi-year mission to the Moon or Mars 14,15,18 .On Earth, bone homeostasis is effectively maintained by the controlled remodeling activity of bone-forming osteoblasts and bone-resorbing osteoclasts. However, exposure to low-LET radiation ( 137 Cs or X-ray, 1-2 Gy) leads to a transient increase in the number of osteoclasts, accompanied by an increase in trabecular separation (Tb.Sp) and decrease in trabecular thickness (Tb.Th), overall leading to a reduction in bone volume fraction (BV/TV) [19][20][21][22] . Together, this early increase in bone resorption and decrease in bone formation due to radiation exposure can result in a state of osteopenia, potentially leading to an increased risk of bone fracture 16,23,24 . A possible mechanism of action responsible for these changes in bone homeostasis is the generation o...

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