Due to spaceflight, astronauts experience serious, weightlessness-induced bone loss because of an unbalanced process of bone remodeling that involves bone marrow mes- enchymal stem cells (BMSCs), as well as osteoblasts, osteo- cytes, and osteoclasts. The effects of microgravity on osteo- cells have been extensively studied, but it is only recently that consideration has been given to the role of BMSCs. Pre- vious researches indicated that human BMSCs cultured in simulated microgravity (sim-μg) alter their proliferation and differentiation. The spaceflight opportunities for biomedical experiments are rare and suffer from a number of opera- tive constraints that could bias the validity of the experiment itself, but remain a unique opportunity to confirm and explain the effects due to microgravity, that are only par- tially activated/detectable in simulated conditions. For this reason, we carefully prepared the SCD – STEM CELLS DIFFERENTIATION experiment, selected by the European Space Agency (ESA) and now on the International Space Station (ISS). Here we present the preparatory studies per- formed on ground to adapt the project to the spaceflight constraints in terms of culture conditions, fixation and stor- age of human BMSCs in space aiming at satisfying the biological requirements mandatory to retrieve suitable sam- ples for post-flight analyses. We expect to understand better the molecular mechanisms governing human BMSC growth and differentiation hoping to outline new countermeasures against astronaut bone loss.
Research Containing: Bone Remodeling
The effect of weightlessness on the human skeletal system is one of the greatest concerns in safely extending space missions [1–11]. The ability to understand and counteract weightlessness-induced bone mineral loss will be vital to crew health and safety during and after extended-duration space sta- tion and exploration missions [1–7]. Research on bone mineral loss during space flight has gone on for more than half a century, and recent studies have shown significant progress in developing coun- termeasures that have proved to be effective, including good nutrition and exercise. We review the history of this research here and provide a summary of recent and ongoing studies, including efforts to counteract bone and calcium loss resulting from weightlessness. Unfortunately, the most obvious nutritional countermeasure—providing excess calcium—does not protect against bone loss . This result is likely related to the decreased calcium absorption observed in space flight and in ground-based models [13–16]. Phosphate supplementation was also ineffective at reducing calcium excretion . Combination therapy with calcium and phosphorus was also unsuccessful at mitigating bone loss and hypercalciuria . Other nutrients, specifically sodium, protein, potassium, vitamin K, and omega-3 fatty acids, have also been proposed and/or tested as bone loss countermeasures , and are discussed in more detail below.
Bone loss and renal stone risk are longstanding concerns for astronauts. Bone resorption brought on by spaceflight elevates urinary calcium and the risk of renal stone formation. Loss of bone calcium leads to concerns about fracture risk and increased long-term risk of osteoporosis. Bone metabolism involves many factors and is interconnected with muscle metabolism and diet. We report here bone biochemistry and renal stone risk data from astronauts on 4- to 6-month International Space Station missions. All had access to a type of resistive exercise countermeasure hardware, either the Advanced Resistance Exercise Device (ARED) or the Interim Resistance Exercise Device (iRED). A subset of the ARED group also tested the bisphosphonate alendronate as a potential anti-resorptive countermeasure (Bis+ARED). While some of the basic bone marker data have been published, we provide here a more comprehensive evaluation of bone biochemistry with a larger group of astronauts. Regardless of exercise, the risk of renal stone formation increased during spaceflight. A key factor in this increase was urine volume, which was lower during flight in all groups at all time points. Thus, the easiest way to mitigate renal stone risk is to increase fluid consumption. ARED use increased bone formation without changing bone resorption, and mitigated a drop in parathyroid hormone in iRED astronauts. Sclerostin, an osteocyte-derived negative regulator of bone formation, increased 10-15% in both groups of astronauts who used the ARED (p<0.06). IGF-1, which regulates bone growth and formation, increased during flight in all 3 groups (p<0.001). Our results are consistent with the growing body of literature showing that the hyper-resorptive state of bone that is brought on by spaceflight can be countered pharmacologically or mitigated through an exercise-induced increase in bone formation, with nutritional support. Key questions remain about the effect of exercise-induced alterations in bone metabolism on bone strength and fracture risk.
Magnesium is an essential nutrient for muscle, cardiovascular, and bone health on Earth, and during space flight. We sought to evaluate magnesium status in 43 astronauts (34 male, 9 female; 47 +/- 5 years old, mean +/- SD) before, during, and after 4-6-month space missions. We also studied individuals participating in a ground analog of space flight (head-down-tilt bed rest; n = 27 (17 male, 10 female), 35 +/- 7 years old). We evaluated serum concentration and 24-h urinary excretion of magnesium, along with estimates of tissue magnesium status from sublingual cells. Serum magnesium increased late in flight, while urinary magnesium excretion was higher over the course of 180-day space missions. Urinary magnesium increased during flight but decreased significantly at landing. Neither serum nor urinary magnesium changed during bed rest. For flight and bed rest, significant correlations existed between the area under the curve of serum and urinary magnesium and the change in total body bone mineral content. Tissue magnesium concentration was unchanged after flight and bed rest. Increased excretion of magnesium is likely partially from bone and partially from diet, but importantly, it does not come at the expense of muscle tissue stores. While further study is needed to better understand the implications of these findings for longer space exploration missions, magnesium homeostasis and tissue status seem well maintained during 4-6-month space missions.
Osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and increased susceptibility to fractures. The microgravity of space creates an extreme environment that provides a model for osteoporosis in humans. This greatly accelerated form of osteopenia results in a 0.5-2% loss of bone mass per month. Rat models for this osteoporosis have been examined on many occasions, but STS-108 was the first Space Shuttle flight to use mice. Data reported to date indicate that spaceflight experiments with mice hold promise in predicting some spaceflight effects on humans. Due to the cost and infrequency of flights, ground-based models have been developed to mimic the deleterious effects of the microgravity environment. Hindlimb suspension is one such localized model. This model removes gravitational loading from the hindlimbs by suspending the animal by its tail to a guy wire that runs lengthwise across the cage. Because mice had not flown before STS-108, a direct comparison of this model’s ability to predict spaceflight results has not been examined. The objective of this research is to closely repeat the STS- 108 profile, with hindlimb suspension replacing spaceflight. This includes examining the ability of the protein osteoprotegerin, an osteoclast-inhibiting therapeutic, to mitigate the deleterious effects of skeletal unloading. It is expected that the results will lead to better understanding of the mechanisms of mineralization and bone remodeling to aid in development of countermeasures to prevent spaceflight induced osteoporosis and aid the treatment of osteoporosis here on earth.
Effects of spaceflight on the murine mandible: Possible factors mediating skeletal changes in non-weight bearing bones of the head
Spaceflight-induced remodeling of the skull is characterized by greater bone volume, mineral density, and mineral content. To further investigate the effects of spaceflight on other non-weight bearing bones of the head, as well as to gain insight into potential factors mediating the remodeling of the skull, the purpose of the present study was to determine the effects of spaceflight on mandibular bone properties. Female C57BL/6 mice were flown 15d on the STS-131 Space Shuttle mission (n=8) and 13d on the STS-135 mission (n=5) or remained as ground controls (GC). Upon landing, mandibles were collected and analyzed via micro-computed tomography for tissue mineralization, bone volume (BV/TV), and distance from the cemento-enamel junction to the alveolar crest (CEJ-AC). Mandibular mineralization was not different between spaceflight (SF) and GC mice for either the STS-131 or STS-135 missions. Mandibular BV/TV (combined cortical and trabecular bone) was lower in mandibles from SF mice on the STS-131 mission (80.7+/-0.8%) relative to that of GC (n=8) animals (84.2+/-1.2%), whereas BV/TV from STS-135 mice was not different from GC animals (n=7). The CEJ-AC distance was shorter in mandibles from STS-131 mice (0.217+/-0.004mm) compared to GC animals (0.283+/-0.009mm), indicating an anabolic (or anti-catabolic) effect of spaceflight, while CEJ-AC distance was similar between STS-135 and GC mice. These findings demonstrate that mandibular bones undergo skeletal changes during spaceflight and are susceptible to the effects of weightlessness. However, adaptation of the mandible to spaceflight is dissimilar to that of the cranium, at least in terms of changes in BV/TV.
The bone mineral density (BMD) of astronauts decreases specifically in the weight-bearing sites during spaceflight. It seems that osteoclasts would be affected by a change in gravity; however, the molecular mechanism involved remains unclear. Here, we show that the mineral density of the pharyngeal bone and teeth region of TRAP-GFP/Osterix-DsRed double transgenic medaka fish was decreased and that osteoclasts were activated when the fish were reared for 56 days at the international space station. In addition, electron microscopy observation revealed a low degree of roundness of mitochondria in osteoclasts. In the whole transcriptome analysis, fkbp5 and ddit4 genes were strongly up-regulated in the flight group. The fish were filmed for abnormal behavior; and, interestingly, the medaka tended to become motionless in the late stage of exposure. These results reveal impaired physiological function with a change in mechanical force under microgravity, which impairment was accompanied by osteoclast activation.
Osteoblast-osteoclast interaction plays an important role in the bone remodeling. During long duration space flight, astronauts undergo serious bone loss mainly due to the disruption of equivalence between bone formation and bone resorption. Osteoclast precursors often operate under the control of osteoblasts. However, here we show that the osteoclast precursors could in turn influence osteoblasts. RAW264.7 cells, the murine osteoclast precursors, were treated in the simulated weightlessness produced by a Random Positioning Machine (RPM). After 72 h, conditioned mediums (CM) by the RAW264.7 cells from RPM (RCM) or static control (CCM) were collected and were used to culture osteoblastic-like MC3T3-E1 cells. The results showed that the RCM culture inhibited cell viability and slightly altered cell cycle, but the morphology of the MC3T3-E1 cells was not changed by RCM compared to that of CCM. Furthermore, the intracellular ALP level, NO release and expression of osteoblastic marker genes were all down-regulated by RCM culture. These results suggest that osteoclast precursors subjected to RPM exert negative regulation on osteoblasts.
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Reinterpretation of mouse thyroid changes under space conditions: the contribution of confinement to damage
During space missions, astronauts work in a state of separation from their daily social environment and in physical confinement. It has been shown that confinement influences mood and brain cortical activity, but no data has been obtained with regard to its effect on the thyroid gland, the structure and function of which change during spaceflights. Here, we report the results of a study on the effects of confinement on mouse thyroid, which was implemented with the Mice Drawer System Facility maintained on the ground, a system used for spaceflight experiments. The results show that confinement changes the microscopic structure of the thyroid gland and that it exhibits symptoms similar to those that result from physiological and/or pathological hyperfunction. What is left unchanged, however, is the sphingomyelinase-thyrotropin receptor relationship, which is important for thyrotropin response with a consequential production of hormones that act on the metabolism of almost all tissues and reduces the production of calcitonin, a hormone involved in bone metabolism. During space missions, the overexpression of pleiotrophin, a widespread cytokine up-regulated after tissue injury that acts on bone remodeling, attenuates changes to the thyroid that are spaceflight-dependent; therefore we studied the thyroids of pleiotrophin-transgenic mice in the Mice Drawer System Facility. In confinement, pleiotrophin overexpression does not protect from the loss of calcitonin. The contribution of confinement to thyroid damage during spaceflights is discussed.