In this study, we investigated the bone protective potential of Dried Plum (DP) against independent and combined effects of simulated microgravity (Hindlimb Unloading, HU) and ionizing radiation (IR) on the microarchitecture and mechanical properties of skeletal tissue. A diet supplemented with DP prevented most of the simulated spaceflight-induced damages to both the appendicular (i.e., tibia) and axial (i.e., vertebrae) skeleton. When mice were fed the control diet, a relatively high dose (2 Gy) of low-LET (linear energy transfer) gamma radiation exclusively decreased the bone volume fraction (BV/TV) and trabecular separation (Tb.Sp) of cancellous tissue in the tibia. Based on our results, cancellous bone loss was caused by a thinning, not a decrease in the number, of existing trabeculae, which overall expanded the space between trabeculae. Hindlimb unloading (HU) for 14 days caused bone loss within both cancellous and cortical regions of the tibia and the L4 vertebrae. HU also led to reduced compressive strength of the vertebral body. The independent effects of HU and radiation (IR) remained of similar magnitude in each tissue compartment when mice were exposed to HU and IR simultaneously. Regardless of the treatment (with one exception, Ct.Th proximal tibia), consumption of the dried plum diet prevented detrimental skeletal changes. Mice fed the control diet and exposed solely to IR displayed a 20% decrement in percent bone volume and a 7% increase in the Tb.Sp of the tibia’s cancellous region relative to the sham control. These parameters were unaffected by HU alone. HU groups exhibited a lesser 11% decrement in trabecular thickness (Tb.Th). When IR was combined with HU, BV/TV decreased by another 5%. These results indicate that HU and IR were not clearly additive in this experiment. Among the parameters analyzed, IR only induced changes in Tb.Th and Tb.Sp. Unlike reports by others, there were no significant changes in trabecular number (Tb.N) from any of the treatment groups relative to the sham-irradiated control group. When HU was combined with IR, Tb.Th decreased by 9%.
Our results indicate bone loss was caused by a thinning, not a decrease in the number, of existing trabeculae, which expanded the space between trabeculae. The trabeculae contribute to percent bone volume and microarchitectural integrity. Since HU affects the cortical as well as cancellous tissue, we also determined the extent of cortical bone loss. The cortical region (cortical shell) exhibited a decrease in cortical thickness when mice fed the control diet were exposed to HU, either independently or in combination with IR.
When mice were fed the DP diet, IR-induced cancellous bone loss was entirely prevented, consistent with the radio-protective results reported in our previous study. DP also protected the tibia when IR was combined with HU, which is a novel finding in this report. The decrease in trabecular thickness incurred by HU and HU + IR was entirely prevented by consumption of the DP diet. The DP diet also protected from HU-induced decrease in cortical thickness. Unlike the proximal tibia, the distal tibia did not show changes in cortical structure. In contrast, in the proximal tibia HU and IR together, not each treatment alone, caused a decrease in cortical thickness of the proximal tibia. This finding suggests that DP diet cannot fully prevent from all aspects of bone structural deficits induced by simulated spaceflight.
In order to confirm our results, we examined another skeletal site, the vertebra, which is a representative axial bone. Under conditions of simulated weightlessness and consumption of control diet, BV/TV and Tt.Tb.Th of the vertebral body were reduced accompanied by a reduction in cortical thickness and cortical bone area. Collectively, these findings indicate an overall deterioration of bone via thinning of the vertebrae’s trabeculae and cortical tissue. This deterioration of the microarchitecture directly impacted the overall strength of the tissue, as reflected in the decrease in maximum load tolerable by the vertebral body, as well as the decrease in material stiffness as measured by mechanical compression testing. Taken together, these changes in both microarchitecture and strength have the potential to lead to fracture. However, these changes were not observed when mice were exposed to IR alone. In contrast to other studies where high-LET 56Fe radiation was utilized in combination with HU, our results did not indicate an additive effect of IR on the deterioration of strength and structure of the L4 vertebrae. HU-induced damage to the vertebral body was entirely prevented when mice consumed the DP diet. Interestingly, there was a statistically significant increase in Tt.BV/TV for sham-irradiated, normally loaded (NL) mice fed the DP compared to the sham-irradiated, NL mice fed the control diet (CD). This elevated Tt.BV/TV was consistent for all treatment groups fed DP when compared to NL, CD-fed mice, suggesting a potential anabolic effect of DP.
Treatments that are currently in use to mitigate the effects of mechanical unloading are not without limitations and risks. Exercise in combination with drug treatments such as bisphosphonates have shown beneficial effects in astronauts. However, in patients with osteoporosis, the use of bisphosphonates can increase the risk for atypical femoral fractures possibly due to suppressed bone turnover which may lead to cracks at the microscale and loss of mechanical strength. Although rare, other notable side effects that have been reported to accompany bisphosphonate therapy including osteonecrosis of the jaw and atrial fibrillation. Another limitation of bisphosphonates is that they act mostly on osteoclasts. Osteoblastogenesis from flushed bone marrow cells of the femur was strongly inhibited by exposure to HU, indicating that HU directly damages osteoblast progenitors, potentially affecting in situ bone formation. Mineralization of the osteoblast cells from DP-fed mice was partially restored and taken together with the cell growth data, indicates that DP could prevent the loss of structure and strength by protecting the marrow-derived osteoprogenitors. Ex vivo osteoblastogenesis was only performed on mice exposed to HU because it has been shown previously that low-LET in vivo gamma radiation does not negatively impact osteoblasts.
Overall, the ability of DP to protect osteoblast progenitor cells from HU-induced damage, as shown in this study, holds much promise for development of next generation anti-osteoporotic drugs due to the possibility that DP to act on both osteoblasts and osteoclasts. Our current study is limited to short-duration HU (2 weeks); since astronauts on long-term space missions may require a countermeasure for bone loss throughout the entirety of a multi-year mission beyond low Earth orbit (LEO), it is important in future studies to determine if DP is protective for long-term exposure to simulated, or actual, spaceflight. Further studies also are needed to gain more insight into any potential long-term side effects of consuming a dried plum diet. Dried plum’s potential as a countermeasure against both radiation- and microgravity-induced osteopenia such as loss of bone strength and structure in the tibia and vertebrae has important implications for astronauts in space as well as radiation workers, radiotherapy patients, and individuals with osteoporosis.