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 loss
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.
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 sta tion(OS) and International Space Station (ISS) (n = 80) was performed. Theoretically predicted (evolution ary 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 evo lution 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 posi tion 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 interven tion into areas of bustling resorption.
Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n=12/group) were injected subcutaneously with OPG-Fc (20mg/kg) or an inert vehicle (VEH), 24h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P<0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.
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.
Astronauts experience bone loss after the long spaceflight missions. Identifying specific regions that undergo the greatest losses (e.g. the proximal femur) could reveal information about the processes of bone loss in disuse and disease. Methods for detecting such regions, however, remains an open problem. This paper focuses on statistical methods to detect such regions. We perform statistical parametric mapping to get t-maps of changes in images, and propose a new cross-validation method to select an optimum suprathreshold for forming clusters of pixels. Once these candidate clusters are formed, we use permutation testing of longitudinal labels to derive significant changes.
Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?
Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.
Simulated microgravity alters multipotential differentiation of rat mesenchymal stem cells in association with reduced telomerase activity
Microgravity is one of the most important characteristics in space flight. Exposure to microgravity results in extensive physiological changes in humans. Bone loss is one of the changes with serious consequences: however. the mechanism retains unclear. As the origin of osteoprogenitors, mesenchymal stein cells (MSCs) may play an important role in it. After cultured under simulated microgravity (in a rotary cell culture system, RCCS), MSCs were stained using oil red O to identify adipocytes. The mRNA level of bone morphogenetic protein (BMP)-2 and peroxisome proliferators-activated receptor (PPAR) gamma 2 was determined by RT-PCR. Otherwise, MSCs were induced to osteogenic differentiation after microgravity culture, and then the activity of alkaline phosphatase (ALP) was determined by PNPP and the content of osteocalcin (OC) by ELISA. Furthermore, the telomerase activity in MSCs was measured by TRAP. The results showed that simulated microgravity inhibited osteoblastic differentiation and induced adipogenic differentiation accompanied by the change of gene expression of BMP-2 and PPAR gamma 2 in MSCs. Meanwhile, the telomerase activity decreased significantly in MSCs under simulated microgravity. The reduced bone formation in space flight may partly be due to the altered potential differentiation of MSCs associated with telomerase activity which plays a key role in regulating the lifespan of cell proliferation and differentiation. Therefore. telomerase activation/replacement may act as a potential countermeasure for microgravity-induced bone loss. (C) 2008 Elsevier Ltd. All rights reserved.
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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.
Recovery of spaceflight-induced bone loss: Bone mineral density after long-duration missions as fitted with an exponential function
The loss of bone mineral in NASA astronauts during spaceflight has been investigated throughout the more than 40 years of space travel. Consequently, it is a medical requirement at NASA Johnson Space Center (JSC) that changes in bone mass be monitored in crew members by measuring bone mineral density (BMD), with dual-energy X-ray absorptiometry (DXA) before and after flight, of astronauts who serve on long-duration missions (4?6 months). We evaluated this repository of medical data to track whether there is recovery of bone mineral that was lost during spaceflight.Our analysis was supplemented by BMD data from cosmonauts (by convention, a space traveler formally employed by the Russia Aviation and Space Agency or by the previous Soviet Union) who had also flown on long-duration missions. Data from a total of 45 individual crew members a small number of whom flew on more than one mission were used in this analysis. Changes in BMD (between 56 different sets of pre- and postflight measurements) were plotted as a function of time (days after landing). Plotted BMD changes were fitted to an exponential mathematical function that estimated: (i) BMD change on landing day (day 0) and (ii) the number of days after landing when 50% of the lost bone would be recovered (50% recovery time) in the lumbar spine, trochanter, pelvis, femoral neck and calcaneus. In sum, averaged losses of bone mineral after long-duration spaceflight ranged between 2% and 9% across all sites with our recovery model predicting a 50% restoration of bone loss for all sites to be within 9 months.