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
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.
The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module (“Kibo”) on the International Space Station. The CBEF provides “space-based controls” by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.
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.
Bone Proteomics experiment (BOP): the first proteomic analysis of mammalian cells cultured in weightlessness conditions
Purpose. Bone mass loss is a major consequence of extended periods of weightlessness. Many studies performed on astronauts and animals have shown that impaired maturation of osteoblast cells as well as a decrease of their bone-synthesising activity play key roles in microgravity-dependent bone mass loss. Several experiments on single cells and tissues showed that weightlessness can also influence cells cultured in vitro. Many molecular mechanisms are affected, among which the cytoskeleton, signal transduction cascades and gene expression. However, the underlying mechanisms of these changes and their molecular consequences are far from being fully understood. In contrast to weightlessness, dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation, differentiation and matrix production. A growing body of evidence points to extracellular nucleotides (i.e. ATP and UTP) as soluble factors that are released by several cell types in response to mechanical stimulation and that eventually trigger an intracellular signal. We have recently demonstrated that ATP and UTP, as well as mechanical stimulation, can activate two fundamental transcription factors, Runx2 and Egr-1, in the human HOBIT osteoblast cell line [Pines A. et al.,Biochem J. 2003; Costessi A. et al., Bone, 2005]. The purpose of the present study was to investigate the possible role(s) of extracellular nucleotides in the molecular response of osteoblast cells to weightlessness conditions. We focused on two aspects: 1. whether administration of ATP could stimulate osteoblast cells in weightlessness, possibly balancing or overcoming its known negative effects; 2. An analysis of the proteome of osteoblast cells exposed to weightlessness by means of two dimension electrophoresis (2-DE) coupled to mass spectrometry, to identify new molecular targets. Methodology – The BOP experiment. BOP was selected in the Success 2002 Student Contest organized by ESA and awarded to A.C. We developed and produced a new and low-cost dedicated hardware to support the experiment. We decided to use the human osteoblast cell line MG-63, since they had been studied in previous space experiments. The cells were grown in space for about five days in three chambers (control cells and cells treated with ATP for 20’ or 3 hours) and eventually lysed. A parallel ground experiment was performed. BOP flew during the Italian Soyuz Taxi flight in April 2005. Results and conclusions. We developed in less than nine months a new hardware that provided 70 cm2 per chamber and a total of more than 200 cm2. Preliminary analysis indicate that administration of ATP to MG-63 cells cultured in weightlessness conditions is able to increase ERK phosphorylation. Analysis of 2D gels revealed several differentially regulated proteins in response to ATP treatment. The identification of these proteins is in progress. To the best of our knowledge, BOP is the first proteomic study on mammalian cells cultured in space. The conclusion of the analysis will reveal new aspects of osteoblast biology and provide new insights into the molecular responses of human cells to weightlessness.
The levels of 26 minerals in rat body hair were analyzed in control and hindlimb-suspended Wistar Hannover rats (n=5 each). We quantified the levels of 22 minerals in this experiment. However, we were unable to measure the levels of 4 minerals (Be, V, Cd, and Hg) quantitatively because they were below the limit of detection. Of the 22 quantified, the levels of 19 minerals were not significantly different between control and hindlimb-suspended groups. The levels of 3 minerals (Pb, Cr, and Al) tended to be higher in the hindlimb-suspended group than in the control group; however, this difference was not significant. The concentrations of 3 other minerals (I, K, and Mg) were significantly different between the 2 groups. The iodine (I) level was 58.2% higher in the hindlimb-suspended group than in the control group (P<0.05). Potassium (K) and magnesium (Mg) levels were 55.2% and 20.4% lower, respectively, in the experimental group (0.05 in both cases). These results indicate that a physiological change in mineral metabolism resulting from physical or mental stress, such as hindlimb suspension, is reflected in body hair. The Japan Aerospace Exploration Agency (JAXA) has initiated a human research study to investigate the effects of long-term space flight on gene expression and mineral metabolism by analyzing hair samples of astronauts who stayed in the International Space Station (ISS) for 6 months. We believe that hindlimb suspension for 14 days can simulate the effects of an extremely severe environment, such as space flight, because the hindlimb suspension model elicits a rapid physiological change in skeletal muscle, bone, and fluid shift even in the short term. These results also suggest that we can detect various effects on the body by analyzing the human scalp hair shaft.
This review will highlight evidence from crew members flown on space missions >90 days to suggest that the adaptations of the skeletal system to mechanical unloading may predispose crew members to an accelerated onset of osteoporosis after return to Earth. By definition, osteoporosis is a skeletal disorder—characterized by low bone mineral density (BMD) and structural deterioration—that reduces the ability of bones to resist fracture under the loading of normal daily activities. “Involutional” or age-related osteoporosis is readily recognized as a syndrome afflicting the elderly population because of the insipid and asymptomatic nature of bone loss that does not typically manifest as fractures until after age ∼60. It is not the thesis of this review to suggest that spaceflight-induced bone loss is similar to bone loss induced by metabolic bone disease; rather this review draws parallels between the rapid and earlier loss in females that occurs with menopause and the rapid bone loss in middle-aged crew members that occurs with spaceflight unloading and how the cumulative effects of spaceflight and ageing could be detrimental, particularly if skeletal effects are totally or partially irreversible. In brief, this report will provide detailed evidence that long-duration crew members, exposed to the weightlessness of space for the typical long-duration (4–6 months) mission on Mir or the International Space Station, (1) display bone resorption that is aggressive, that targets normally weight-bearing skeletal sites, that is uncoupled to bone formation, and that results in areal BMD deficits that can range between 6 and 20% of preflight BMD; (2) display compartment-specific declines in volumetric BMD in the proximal femur (a skeletal site of clinical interest) that significantly reduces its compressive and bending strength and may account for the loss in hip bone strength (i.e., force to failure); (3) recover BMD over a post-flight time period that exceeds spaceflight exposure but for which the restoration of whole bone strength remains an open issue and may involve structural alteration; and (4) display risk factors for bone loss—such as the negative calcium balance and down-regulated calcium-regulating hormones in response to bone atrophy—that can be compounded by the constraints of conducting mission operations (inability to provide essential nutrients and vitamins). The full characterization of the skeletal response to mechanical unloading in space is not complete. In particular, countermeasures used to date have been inadequate, and it is not yet known whether more appropriate countermeasures can prevent the changes in bone that have been found in previous flights. Knowledge gaps related to the effects of prolonged (≥6 months) space exposure and to partial gravity environments are substantial, and longitudinal measurements on crew members after spaceflight are required to assess the full impact on skeletal recovery.
Characteristics of local human skeleton responses to microgravity and drug treatment for osteoporosis in clinic
Analysis of the results of long-term investigations of bones in cosmonauts on board Mir orbital station(OS) and International Space Station (ISS) (n = 80) was performed. Theoretically predicted (evolutionary predefined) change in mass of different skeleton bones was found to be correlated (r = 0.904) with the position relative to Earth’s gravity vector. Vector dependence of bone loss results from local specificity of expression of bone metabolism genes, which reflects mechanical prehistory of skeleton structures in the evolution of Homo erectus. Genetic polymorphism is accountable for high individual variability of bone loss, which is attested by the dependence of bone loss rate on polymorphism of certain genetic markers of bone metabolism. The type of the orbital vehicle did not affect the individual-specific stability of the bone loss ratio in different segments of the skeleton. This fact is considered as a phenotype fingerprint of local metabolism in the form of a locus-specific spatial structure of distribution of non-collagen proteins responsible for position regulation of endosteal metabolism. Drug treatment of osteoporosis (n = 107) evidences that recovery rate depends on bone location; the most likely reason is different effectiveness of local osteotropic intervention into areas of bustling resorption.
Voxel-based modeling and quantification of the proximal femur using inter-subject registration of quantitative CT images
We have developed a general framework which employs quantitative computed tomography (QCT) imaging and inter-subject image registration to model the three-dimensional structure of the hip, with the goal of quantifying changes in the spatial distribution of bone as it is affected by aging, drug treatment or mechanical unloading. We have adapted rigid and non-rigid inter-subject registration techniques to transform groups of hip QCT scans into a common reference space and to construct composite proximal femoral models. We have applied this technique to a longitudinal study of 16 astronauts who on average, incurred high losses of hip bone density during spaceflights of 4-6 months on the International Space Station (ISS). We compared the pre-flight and post-flight composite hip models, and observed the gradients of the bone loss distribution. We performed paired t-tests, on a voxel by voxel basis, corrected for multiple comparisons using false discovery rate (FDR), and observed regions inside the proximal femur that showed the most significant bone loss. To validate our registration algorithm, we selected the 16 pre-flight scans and manually marked 4 landmarks for each scan. After registration, the average distance between the mapped landmarks and the corresponding landmarks in the target scan was 2.56 mm. The average error due to manual landmark identification was 1.70 mm.