Bones are exposed to in vivo ionizing radiation exposure in applications of spaceflight, clinical radiotherapy, and ex vivo ionizing radiation exposure in applications of allograft sterilization. Elevated fracture risk has been shown after irradiation at the higher clinical doses, and it is uncertain if there are similar risks with exposure to spaceflight irradiation. Irradiation leads to an early loss of cancellous bone tissue and microarchitecture quality, and reduced matrix quality in the form of increased collagen crosslinks, but it is unclear which of these radiation-induced changes contribute to reduced bone mechanics. While the effects of space- and clinically-related levels of radiation on bone strength have been studied for monotonic (one-time) loading, much less is known about the effects for cyclic (repeated) loading, also referred to as fatigue. The possible increase in collagen crosslinks may have a larger effect on fatigue life by possibly impeding fibrillar sliding, reducing plasticity, and increasing the rate of microcrack growth during cyclic loading. Therefore, the goal of this research was to characterize the cyclic and monotonic mechanical properties after irradiation at spaceflight- and clinically-relevant doses, and to better understand the factors influencing the resulting mechanical behavior.

To address this, we conducted in vivo and ex vivo irradiation experiments using a murine model with a range of radiation doses, including spaceflight (≤ 2 Gy), clinically-relevant (≤ 50 Gy), and sterilization (≤ 35,000 Gy). To compare irradiated bone to healthy tissue, we performed microCT to analyze bone quantity and microarchitecture, biochemical assays to characterize the collagen network and material quality, and cyclic and monotonic compression testing to characterize the mechanical behavior.

To conduct cyclic mechanical testing on experimental tissue, we first developed a precise method for ex vivo cyclic compressive loading of isolated mouse vertebral bodies. In small animals, such as in mice and rats, 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. Our method has three main 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. We found reduced scatter of the mechanical behavior for this new method compared to commonly used methods from literature. We conclude that this new method for cyclic loading of small-animal vertebrae produces highly reproducible measurements of fatigue behavior and an effective tissue elastic modulus, potentially important elements of bone quality.

To investigate effects of in vivo irradiation, we conducted whole-body, acute, gamma-radiation experiments on 17-week old (skeletally-mature) C57BL/6J, male mice at space- and clinicallyrelevant doses (1 and 5 Gy) and collected lumbar vertebrae at 11-days and 12-weeks after exposure. We found with a 5 Gy dose, short-term deficits (i.e. 11-days after exposure) in cancellous microarchitecture (-29.7% Conn.D) that persisted into the long term (i.e. 12-weeks) (- 49.5% Conn.D) and led to reduced fatigue life (-15.1% cycles to failure) in the long term. Results were similar for aging alone. In contrast, short-term, irradiation-induced deficits in bone quantity (-22.3% bone volume fraction) and elevated non-enzymatic collagen crosslinks (+85.5% AGEs) did not have a significant impact on monotonic or cyclic mechanics. We conclude that degraded trabecular microarchitecture with radiation (or aging) can lead to profound deficits in cyclic mechanical properties, more so than molecular changes via collagen crosslinks or reduced bone volume fraction.

Lastly, to assess the non-cellular effects of irradiation on whole-bone and to determine the type of collagen network degradation which primarily impacts the mechanical properties, we conducted an ex vivo x-ray radiation experiment spanning doses from radiotherapy up to sterilization of bone allografts. Excised mouse lumbar vertebrae from 20-week old, female, C57BL/6J mice were irradiated with doses of 0, 50, 1,000, 17,000, and 35,000 Gy. We found lower collagen molecular weight at 17,000 Gy and above (≥ 74%) compared to the control, indicating collagen backbone fragmentation, which coincided with a loss in monotonic strength (≥ 50%) and cyclic mechanical properties at 17,000 Gy and above. In contrast, AGEs, representing non-enzymatic collagen crosslinks, was greater for all radiation doses (67%, 95%, 96%, and 108% for 50, 1,000, 17,000 and 35,000 Gy, respectively), but did not coincide with a reduction in strength or any other mechanical property for 50 and 1000 Gy. We conclude that mechanical properties of ex vivo irradiation are dominated by the direct effects of irradiation through fragmentation of the collagen backbone, and are negligibly influenced by the indirect effects of non-enzymatic crosslinks. Our findings suggest that to maintain mechanical integrity following exposure to ionizing radiation, bone allograft specimens should be sterilized at a dose below 17,000 Gy.

In summary, we have provided the field with a novel and precise methodology for fatigue testing of small animal bone. To our knowledge, this research was the first to conduct fatigue testing after radiation exposure related to spaceflight and clinical radiotherapy. Our in vivo irradiation results highlighted the critical role of bone microarchitecture, compared to bone quantity or collagen crosslinks, in maintaining cyclic mechanical behavior with radiation exposure and aging. Our ex vivo irradiation results clarified primary mechanisms of collagen fragmentation, compared to collagen crosslinks, that led to deficits in monotonic and cyclic mechanics.