Commercial Biomedical Testing Module: Effects of Osteoprotegerin on Bone Maintenance in Microgravity (CBTM) - 11.22.16
Commercial Biomedical Testing Module: Effects of Osteoprotegerin on Bone Maintenance in Microgravity (CBTM) provides the capability to use the microgravity environment for evaluation of new pharmaceutical candidates in small mammals. Results may expedite the review of new pharmaceuticals for allowing immediate access to new disease treatments. Science Results for Everyone
Twenty-four tiny, whiskered astronauts – female mice – helped researchers evaluate bone loss in space. The mice lost femur elastic strength, experienced significant decrease in bone formation, and had decline in muscle fiber diameter size. Data also indicated altered immune functions; significantly higher levels but decreased functionality of platelets, which promote blood clotting; and changes in some small non-coding genetic molecules (miRNA), including those associated with muscle growth and fiber type. Some of the mice were injected pre-flight with a bone-resorption-inhibiting protein and, after landing, had greater bone mineral density than untreated mice, suggesting this therapy could help preserve bone health during spaceflight. Experiment Details
Ted A. Bateman, Ph.D., University of North Carolina, Chapel Hill, NC, United States
Paul J. Kostenuik, Amgen, Thousand Oaks, CA, United States
Brooke C. Harrison, Ph.D., University of Colorado, Boulder, CO, United States
David L. Allen, Ph.D., University of Colorado, Boulder, CO, United States
Beverly E. Girten, Ph.D., Ames Research Center, Moffett Field, CA, United States
Virginia L. Ferguson, Ph.D., University of Colorado, Boulder, CO, United States
Alan Boyde, University of London, London, United Kingdom
Timothy G. Hammond, M.B.B.S., Durham Veterans' Affairs Medical Center, Durham, NC, United States
Leslie L. Leinwand, University of Colorado, Boulder, CO, United States
BioServe Space Technologies, University of Colorado, Boulder, CO, United States
Sponsoring Space Agency
National Aeronautics and Space Administration (NASA)
Human Exploration and Operations Mission Directorate (HEOMD)
ISS Expedition Duration
December 2001 - June 2002
Similiar investigations to CBTM were conducted on STS-60, STS-63 and STS-77 as Immune-01, Immune-02 and Immune-03, respectively.
- Commercial Biomedical Testing Module: Effects of Osteoprotegerin on Bone Maintenance in Microgravity (CBTM) helps determine the efficacy of the novel protein osteoprotegerin in a complete unloading environment and examines spaceflight as a unique pre-clinical model for osteoporosis. Osteoprotegerin is a recently discovered, naturally circulating protein that increases bone density by inhibiting bone resorption.
- Osteoporosis is a debilitating disease that afflicts millions of people worldwide. One of the physiological changes experienced by astronauts during spaceflight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton. This bone loss experienced by astronauts is similar to osteoporosis in the elderly population. Osteoprotegerin (OPG), a bone metabolism regulator, is being considered by the Food and Drug Administration (FDA) as a new treatment for osteoporosis.
- Laboratory mice are treated with either OPG or a placebo before launch of Expedition 4. The mice are housed in an animal enclosure module designed specifically for spaceflight. This experiment provides a preclinical trial model to determine the effectiveness of OPG in treating bone loss.
Osteoporosis is a debilitating disease that afflicts millions worldwide. One of the physiological changes experienced by space crews during spaceflight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton; a loss that is similar to that experienced by the elderly population on Earth. Osteoprotegerin (OPG), which is a bone metabolism regulator, is being evaluated by the Food and Drug Administration (FDA) as a new treatment for osteoporosis.
Commercial Biomedical Testing Module: Effects of Osteoprotegerin on Bone Maintenance in Microgravity (CBTM) examines the effects of OPG on bone maintenance in space using aged mice (older than nine months) as test subjects. The bone changes observed in older mice more closely reflect the bone changes observed in older humans. The mice are housed in three animal enclosure modules (AEMs), which provide the animal subjects with everything necessary to maintain health. Half of the mice are treated with OPG, a novel protein that regulates bone resorption, and half are treated with a placebo.
The investigations resulting from the CBTM tissue sharing program are as follows:
- Skeletal muscle adaptations to microgravity exposure in the mouse
Brooke C. Harrison, Ph.D., University of Colorado, Boulder, CO
David L. Allen, Ph.D., University of Colorado, Boulder, CO
Beverly E. Girten, Ph.D., Ames Research Center, Moffett Field, CA
- The effects of microgravity on murine caudal vertebrae
Virginia L. Ferguson, Ph.D., University of Colorado, Boulder, CO
Alan Boyde, Ph.D., University of London, London, UK
- The effect of microgravity on the expression levels of mouse genes found in the kidney, liver and gut tissue
Timothy Hammond, M.D., Tulane University, New Orleans, LA
This investigation examines kidney, liver, and gut tissue from mice flown in microgravity. The investigations isolate RNA from these tissues for gene array analysis and measure the expression levels of more than 36,000 mouse genes and ESTs using Affymetrix Genchip Murine Genome U7f4 Set.
- Effects of spaceflight on murine skeletal muscle gene expression
Leslie Leinwand, Ph.D., University of Colorado, Boulder, CO
This investigation aims to study the changes in gene expression and adaptations of murine skeletal muscle in spaceflight. First, the effects of spaceflight on mRNA expression in gastrocnemius muscle is studied by utilizing both a global microarray approach and a candidate gene approach using quantitative real-time polymerase chain reaction (QRT-PCR). Second, the effects of spaceflight on global mRNA expression to those of hindlimb suspension for a comparable length of time are assessed. Third, because all spaceflight studies contain the possible confound of several hours of reloading upon re-entry and landing, the effects of the 3.5 hours of reloading on mRNA expression of suspended mouse muscle are evaluated. ^ back to top
Astronauts suffer from a significant loss of bone mass during space flight, the ISS Medical Project office has developed some countermeasures to hinder the rapid loss of bone mass. Despite these countermeasures bone mass loss continues to be a problem for astronauts. Finding additional countermeasures will increase the overall health of astronauts on long duration missions.
Earth Applications^ back to top
In microgravity, the messages received by the osteoblasts and osteoclasts are altered. Specifically, without the stresses caused by the Earth's gravitational pull, osteoclasts remove more bone and osteoblasts deposit less new bone. Understanding how these signals change and how OPG mitigates these changes will give scientists insight in how to fight bone loss in astronauts during long duration space flight and in osteoporosis patients on Earth.
Osteoporosis is a major public health threat for an estimated 44 million people worldwide. Space flight induces a systematic, accelerated bone loss, hence, this investigation will provide a good model for osteoporosis and potential treatments. It will provide scientists further insight into skeletal loss from microgravity and the role of OPG as a potential treatment for osteoporosis.
Operational Requirements and Protocols^ back to top
CBTM is a sortie, meaning that CBTM will launch and return on STS-108. The AEMs require power from an external source to operate the fans and lighting, but no computer support is needed. Crew interaction is minimal.
During the flight, crew will check the AEMs daily to make sure the equipment is functioning nominally and to make sure there is water in the Water Refill Box. The crew will probably need to add more water, using the water refill line, three times during the flight. After the flight, the AEMs will be returned to the research team.
Decadal Survey Recommendations
Information Pending^ back to top
Results/More InformationDuring ISS Expedition 4, 24 female mice were flown to ISS on shuttle flight STS-108 in three AEMs. The AEMs remained on STS-108 throughout the 12-day mission.
Mice exposed to microgravity exhibited a 15 - 20 percent decline in femur elastic strength and a 40 - 60 percent decrease in bone formation when compared to the controls. The femur elastic strength decline was caused by three mechanisms: reduced bone formation, increased bone resorption, and inhibition of mineralization. OPG treatment in mice exposed to microgravity nearly reversed the decline in strength and the increase in bone resorption found in untreated mice (Bateman 2004).
Mechanical testing data were complimented by serum, messenger ribonucleic acid (mRNA), and histological analyses that indicated a decline in bone formation and an increase in bone resorption in addition to an inhibition of mineralization. OPG mitigated the decline in mechanical strength by preventing increase in resorption and maintaining mineralization. In addition to this detailed analysis of skeletal properties, a secondary analysis of calf muscles from placebo-treated specimens was performed to collect baseline data to validate space-flown mice as an appropriate model for sarcopenia (age-related muscle loss). Space flight caused a 15 - 30 percent decline in muscle fiber diameter size compared to appropriate ground controls (Harrison et al. 2003).
Data obtained from the mice following return to Earth indicated some alternations in immune functions. Analysis of the spleenocytes (immune cells produced by the spleen) indicated an increase in B-cell (white blood cell that matures in the bone marrow and, when stimulated by an antigen, differentiates into plasma cells) production compared to T-cells (white blood cells that complete maturation in the thymus and have various roles in the immune system). A slightly lower white blood-cell count in the flight animals compared to the controls was not statistically significant. The spleen mass was 18 - 28 percent lower in flight mice compared to controls. Results also indicated that flight mice weighed 10 - 12 percent less than ground controls (Pecaut et al. 2003).
The ability to survive a major physical trauma in microgravity may be compromised due to an altered immune system. Platelets (constituent of blood that promotes clotting at the site of injury) are the primary cells involved in the wound healing process. The animals studied had significantly higher platelet levels but low volume compared to the controls. This indicates that the lack of platelets in the wound healing process is not a problem, but that platelets formed in microgravity have a decreased functionality in the wound healing process. Data indicated that a short stay in microgravity can induce significant changes in immune defense mechanisms, hematopoiesis (blood cell formation), and other aspects of health (Gridley et al. 2003).
Analysis of microarray data revealed that 272 mRNAs were significantly altered by space flight, the majority of which displayed similar responses to hindlimb suspension, while reloading tended to counteract these responses. Several mRNAs altered by space flight were associated with muscle growth, including the PI3 kinase regulatory subunit p85 alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase while mRNA levels of the myostatin inhibitor FSTL3 tended to decrease in response to space flight. In addition, mRNA levels of the slow-oxidative fiber associated transcriptional co-activator peroxisome proliferator associated receptor-(PPAR) gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in space flight gastrocnemius. Finally, space flight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that space flight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type (Allen et al. 2008).Over the course of 10 years or so, the skeleton in the human body is completely replaced. This lifelong process involves a balance between removal of old and adding new bone tissue. But when bone resorption becomes greater than bone building in the skeleton, as with aging and during space mission, it leads to decreased bone strength and density, with increased fracture risk along with formation of kidney stone due to excess calcium from dissolved bone. The discovery, in the mid to late 1990s, of new proteins, labeled receptor activator of nuclear factor-κB ligand (RANKL)/RANK/osteoprotegerin (OPG), and their role in the regulation of bone resorption provides promising new treatment methods for osteoporosis and similar disorders. This study demonstrates that that many negative effects of space-induced bone loss can be greatly reduced with the antiresorptive osteoprotegerin–immunoglobulin Fc segment complex (OPG–Fc) - a protein that causes sustained inhibition of bone resorption after a single injection. After landing, space-flown mice treated with OPG–Fc, had greater bone mineral density (BMD) than untreated spaceflight or ground control mice. Osteoclast (bone cell that break down bone tissue) numbers were markedly reduced by OPG–Fc in spaceflight animals, indicating that inhibition of bone resorption explained the positive changes in bone mass, geometry, architecture and strength. In addition, OPG-Fc was found to increase tibial trabecular bone volume and volumetric BMD in all groups. The effects of treatment provide a plausible mean for preserving bone health during spaceflight. However, inhibition of bone formation appeared to be a more significant mechanism for spaceflight-induced osteopenia (bone loss less severe than osteoporosis), and OPG-Fc did not result in any reversal of spaceflight-related changes in bone formation. These observations suggest that the beneficial effects of OPG-Fc on mouse bone during spaceflight are due to dramatic and sustained suppression of bone resorption, and that a single pre-flight treatment with OPG-Fc effectively controls the destructive effects of spaceflight on mouse bone (Lloyd et al. 2015).^ back to top
Zawieja DC, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA, Morony SE, Stodieck LS, Lacey DL, Simske SJ, Delp MD. Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets. Journal of Applied Physiology. 2003; 94(5): 2095-2103. DOI: 10.1152/japplphysiol.01052.2002. PMID: 12514166.
Harrison BC, Allen DL, Girten BE, Stodieck LS, Kostenuik PJ, Bateman TA, Morony SE, Lacey DL, Leinwand LL. Skeletal muscle adaptations to microgravity exposure in the mouse. Journal of Applied Physiology. 2003; 95(6): 2462-2470. DOI: 10.1152/japplphysiol.00603.2003. PMID: 12882990.
Lloyd SA, Morony SE, Ferguson VL, Simske SJ, Stodieck LS, Warmington KS, Livingston EW, Lacey DL, Kostenuik PJ, Bateman TA. Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice. Bone. 2015 December; 81: 562-72. DOI: 10.1016/j.bone.2015.08.021.
Pecaut MJ, Nelson GA, Peters LL, Kostenuik PJ, Bateman TA, Morony SE, Stodieck LS, Lacey DL, Simske SJ, Gridley DS. Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions. Journal of Applied Physiology. 2003 May; 94(5): 2085-2094. DOI: 10.1152/japplphysiol.01052.2002.
Bateman TA, Bandstra ER. Chapter 20: How animal models inform the debate. Cleveland, OH: Bone Loss During Spaceflight: Etiology, Countermeasures, and Implications for Bone Health on Earth; 2007.
Allen DL, Bandstra ER, Harrison BC, Thorng S, Stodieck LS, Kostenuik PJ, Morony SE, Lacey DL, Hammond TG, Leinwand LL, Argraves WS, Bateman TA, Barth JL. Effects of Spaceflight on Murine Skeletal Muscle Gene Expression. Journal of Applied Physiology. 2008. DOI: 10.1152/japplphysiol.90780.2008. PMID: 19074574.
Bateman TA. Molecular therapies for disuse osteoporosis. Gravitational and Space Biology. 2004; 17(2): 83-89.
Ground Based Results Publications
Adams GR, Caiozzo VJ, Baldwin KM. Skeletal muscle unweighting: spaceflight and ground-based models. Journal of Applied Physiology. 2003; 95: 2185-2201.
Sieck GC. Commentary. Journal of Applied Physiology. 2003; 94: 2084.
Dalton P, Gould M, Girten BE, Stodieck LS, Bateman TA. Preventing annoyance from odors in spaceflight: a method for evaluating the sensory impact of rodent housing. Journal of Applied Physiology. 2003; 95(5): 2113-2121.
BioServe Space Technologies
NASA Fact Sheet
Fluorescent image of femur diaphysis from spaceflight placebo treated mouse, indicating greatly decreased bone formation (calcein label indicates where bone was forming at the time of launch, allowing quantification of bone formation rates during flight. Courtesy image from Marshall Space Center.
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Fluorescent image of femur diaphysis from ground control placebo treated mouse, indicating greatly decreased bone formation (calcein label indicates where bone was forming at the time of launch, allowing quantification of bone formation rates during flight. Courtesy image from Marshall Space Center.
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Image on the left shows a microCT image of trabecular bone from proximal tibia from spaceflight mouse compared to ground control mouse on right. Courtesy image from Marshall Space Center.
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BioServe Post-doctoral fellow and engineer, Ted Bateman (on left) and a NASA engineer (right) prepare CBTM for launch on board STS-108. Image courtesy of Marshall Space Center.
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This study investigates the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity using female C57BL/6J mice aged 64 days. The mice are divided into baseline, animal enclosure module (AEM) control, and spaceflight treatment groups. The spaceflight group is flown on the Space Shuttle Endeavour for approximately 12 days of microgravity exposure.
This investigation examines the effect of microgravity on the mouse caudal vertebrae. The primary tool used is quantitative backscattered electron (qBSE) imaging. In addition, the investigators examine the tails using a PIXImusTM Mouse Densitometer (pixel size of 0.18 x 0.18 mm). Vertebrae are similar in structure to long bones. They have growth plates at the ends of each bone and grow longitudinally. BSE imaging provides a unique means to examine these bones in 3-D. Surfaces are exposed by using a treatment to remove any non-osseous tissue. The periosteal and endosteal surfaces are analysed for morphology. Growth plates and articular calcified cartilage (ACC) are examined to determine the condition of the mineralizing front. The surface state determines the extent of formation, resting, or resorption. These results will be correlated with the densitometry data.