Avian Development Facility - Skeletal Development in Embryonic Quail (ADF-Skeletal) - 11.22.16
Under microgravity conditions, bones demineralize, resulting in osteoporosis like (brittle bones) conditions. The study of embryos that develop in microgravity is an important piece of the bone loss puzzle. ADF-Skeletal investigated how the mechanism of bone formation during development of the limbs in quail embryos could provide basic information to help prevent bone loss in astronauts during long duration missions. Science Results for Everyone
Studying bird embryos developing in microgravity is helping scientists figure out why astronauts lose bone mass. Quail eggs on the International Space Station were either spun to reproduce Earth’s gravity or left stationary, with a fixative preserving the embryos at different development stages. Researchers observed no effects on bone mineralization in day 12 embryos, suggesting that space doesn’t affect mineralization in an older embryo. Both egg sets had reduced synthesis of collagen, the proteins that form connective tissue, suggesting that space flight’s effect on collagen production is not corrected by applying 1G. These results could help develop appropriate bone-loss countermeasures for space travelers. Experiment Details
Stephen B. Doty, Ph.D., Hospital for Special Surgery, New York, NY, United States
NASA Ames Research Center, Moffett Field, CA, United States
Space Hardware Optimization Technology Incoporated, Greenville, IN, 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
An earlier version of this experiment, CHIX, flew on Mir and EuroMir.
- The Avian Development Facility (ADF) is an incubator used for the study of quail embryo development (embryogenesis) in space and allowed researchers to study how bone developed in the space flight environment. The data from the ADF experiments provided researchers with a better understanding of the process of bone demineralization and recovery, which allowed scientists the ability to devise methods for mitigating or nullifying bone loss both in space and on Earth. In addition, the embryonic development of long bones mimics the fracture healing process which occurs in fracture repair of adult bone.
- The ADF incubator had two independent carousels that carried the egg holder. For ADF-Skeletal one carousel spun, to create the gravitational force experienced on Earth, and the other carousel remained stationary. This allowed researchers the ability to have an inflight 1g control set of specimens to compare the development with the specimens that remain in microgravity (unspun carousel).
- The ADF had an automated fluid injection system that injected eggs with a fixative to preserve embryos at different developmental stages. A second set of embryos were returned alive but unhatched. This experiment protocol provided researchers with a series of embryos ranging from ages related to very early developmental to very late developmental stages.
- During its flight on STS-108, ADF was used for two investigations: Development and Function of the Avian Otolith System in Normal and Altered Gravity Environments and Skeletal Development in Embryonic Quail on the ISS.
The avian development biology experiment, which is a tool for the study of embryogenesis in space, provides the support hardware needed for researchers to better understand and mitigate or nullify the forces of altered gravity on embryo development. Avian eggs are ideal for studying embryo development since they are self-contained and self-sustaining and can be nurtured without a maternal host. The Avian Development Facility (ADF) allows incubation of avian eggs under controlled conditions (humidity, temperature, and gas environment) on orbit and the fixation of the eggs for study while minimizing the effects of launch and landing. Up to 36 eggs in centrifuge carousels can be exposed to simulated gravity of zero-g to one-g in 0.1-g increments.
During its flight on space shuttle mission STS-108 to the ISS, the ADF housed two investigations: the Development and Function of the Avian Otolith System in Normal and Altered Gravity Environments (ADF-Otolith) and the Skeletal Development in Embryonic Quail on the ISS (ADF Skeletal) investigations.
The objective of this experiment was to define the effects of space flight on embryonic skeletal development. This investigation was a stepping-stone in determining the effect of microgravity on the molecular and cellular biology of bone formation and loss. Many of the biological processes observed in bone formation during embryogenesis (development and growth of an embryo) also occur in the adult skeleton during fracture repair. Furthermore, previous space flight studies identified bone demineralization and bone density loss in embryo quails, which was similar to what is observed in adult humans with osteoporosis. However, up to a certain developmental age embryonic quail bones will rapidly recover from this condition when re-exposed to gravity; whereas humans suffering from bone loss can take years to recover. Therefore, the data gained from this study should provide a foundation of future studies on bone demineralization and density loss and provide insight into the mechanisms involved in the full re-mineralization of bones.
The ADF is the first step to creating new technologies that will support critical biological research in direct support of human space travel. The subsystems used to support the biological specimens can be applied to technologies directed to supporting other animals or the crew. It is a known fact that astronauts lose bone material soon after they enter the weightless environment of space. This loss in bone material is very similar to osteoporosis, which is observed in the elderly population. The loss of bone material poses a significant heath risk because the astronauts bone weaken and become more susceptible to breakage. Also, since they are adults, their body's ability to regenerate the lost bone material once they return to Earth is very limited. Currently, no therapies exist that can stimulate the production of new bone material. Methods designed at preventing bone material loss from the skeleton are still experimental and have not been successful. The space flight microgravity environment is ideal for studying bone formation and material loss because its affects on the skeleton are rapid and occur within days of entry into space. This ADF-Skeletal experiment is designed to investigate the biological processes that are key to stimulating new bone formation. By using the ADF, this experiment can control the embryogenesis environment so that components affected by the space flight environment, especially microgravity, can be identified. By comparing the data to the simulated 1g inflight controls and ground controls, the ADF experiment will be able to identify specific changes to bone formation that are due to the microgravity environment, which in turn will point out the specific biological systems (molecular, cellular, and systemic) that are sensitive to changes in gravity. It is anticipated that the identification of the specific mechanism affected will point where to focus the development of therapeutics. These therapeutics could be used to both stop bone loss or stimulate the generation of bone material while the astronaut is inflight. As a result, long duration space flight and exposure to less than 1g for a prolonged period of time will no longer pose a heath risk to the astronaut while inflight or after returning home.
On Earth, fracture healing consists of cartilage formation, conversion of cartilage to a temporary bony structure, and new bone formation involving osteoblast and collagen synthesis. Furthermore, the healing of a bone fracture in the elderly is very slow and may not result in a fully healed bone. In addition, significant population of elderly adults suffer from loss of bone material due to the onset of osteoporosis. Currently, there is very little understanding of which biological processes are affected that result in these deficiencies. There are no medical interventions that stimulate bone formation, so the only treatments available aim at preventing loss of the bone material that already exists in the skeleton. In contrast, the developing quail is a bone forming system, which allows scientists the ability to clearly investigate and understand the underlying biological processes that drive bone formation. Since the microgravity environment is known to stimulate the loss of bone material in adults in a very short period of time, the space flight microgravity environment is the ideal location to conduct experiments aimed at understanding bone loss. By using the ADF, specimens can be incubated in the exact same environment with the only difference being 1g versus microgravity. This experimental condition is not available on Earth and is critical to specifically isolating the cause of bone material loss. It is anticipated that the data from this ADF study will provide scientists with key information to identify the specific biological processes that contribute to bone loss and the mechanism of this disease process. Overall, the identification of the specific biological processes will provide scientists and pharmaceutical companies candidate targets for the development of therapeutic agents.
Operational Requirements and Protocols
ADF has 36 egg holders, which isolate and cushion the eggs from ambient vibrations that could disturb development. The holders fit into one of the two carousels. One carousel was programmed independently to spin in order to simulate the gravity on Earth. The second carousel remained stationary in order to expose its eggs to microgravity. The egg holders on each carousel were pre-programmed to rotate 180 degrees along the eggs long axis at specific times to simulate the natural egg turning by the mother hen. Prior to launch, the ADF was powered to maintain 13 degrees C in order to keep the fertilized eggs from developing. Once the Shuttle reached space, the ADF was activated by the astronaut to start the incubation mode, which included increasing the temperature to 37 degrees C and starting one of the carousels to spin. Prior to re-entry, the spinning carousel was turned off, which was required to safe the ADF.The ADF provided all life support subsystgems for the developing embryos. The ADF maintained temperature between at 37 degrees C, controlled relative humidity, adjusted the oxygen concentration to 21% during incubation), and removed carbon dioxide (maintained less than 1% during incubation) to provide the optimal incubation environment. When the desired incubation duration was reached, ADF injected a set of pre-selected eggs with a fixative (4% formaldehyde) to preserve the embryo at a specific development stage for study by the researchers after the specimen returned to Earth.
Japanese quail eggs, due to their developmental duration of 15 days, were exposed to either 1g or microgravity during embryogenesis, and the development of their bones were examined using a number of different quantitative methods. Embryonic quail that developed in normal gravity (Earth or space flight experiment), hypergravity (2 - 3g; Ground Experiment), or microgravity conditions were compared. The anatomical development of the receptor cell organization and the afferent innervation patterns of day 4, 7, and 12 quail embryos were collected for comparison across the different gravity conditions and developmental stages.
Prior to launch, the fertilized eggs were maintained in the ADF at 13 degrees C to suspend development. Once on Orbit, the crew activated the ADF to incubation mode, which resulted in a gradual increase of the incubation temperature to 37.5 degrees C. At the same time, one of the carousels was automatically activated to start to spin in order to simulate the Earth's gravitational field. The second carousel remained stationary to expose the eggs on its holder to microgravity. Eggs were rotated in their sample holders once an hour to imitate the turning they would receive if tended by a hen. Per a defined pre-programmed schedule, the ADF automatically injected a pre-defined set of eggs with a fixative. Within hours of landing, the eggs were removed from the ADF and delivered to the researchers for analysis.
Decadal Survey Recommendations
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No space flight effects were observed for osteocalcin levels in the day 12 embryos, based on bone matrix stating. Since osteocalcin reflects the degree of bone mineralization, this would suggest that mineralization is not affected in an older embryo. However, direct mineralization quantitative studies have not been reported for day 7 and day 12 embryos, which should provide definitive evidence for whether osteocalcin-associated processes are affected.
The second finding was that the space flight embryos on the spinning carousel or stationary carousel had a reduced level of collagen-synthesizing activity as compared to the ground control specimens, although the sample size was small. If this trend is validated, it would suggest that space flight has a component that can affect collagen synthesis that is not correctable by an applied one-g force. These insights might be important for the development of appropriate countermeasures for space travel. Final analyses and publication of results are pending (Increment 4 One Year Postflight Report).
Ground Based Results Publications
Doty SB, Vico L, Wronski T, Morey-Holton ER. Use of Animals Models to Study Skeletal Effects of Space Flight. Advances in Space Biology and Medicine. 2005; 10: 209-224.
NASA Fact Sheet
One of two ADF carousels, each featuring 18 sample containers. During space missions, one carousel rotates at 77.3 RPMs to simulate a gravity field, like on Earth. The other carousel remains motionless to provide a microgravity environment for the specimens inside. Image courtesy of NASA, Johnson Space Center.
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Electron micrographs of quail limb bones that formed under the influence of microgravity show decreased mineralization compared to bones formed in normal gravity. The letters "B" and "C" indicate bone and cartilage sides of the sample, respectively, with the arrows marking the junction between bone and cartilage cells. The asterisks indicate where mineralization begins. The bone that developed during spaceflight (top) shows less mineral compared to the control sample (bottom); the control sample clearly shows mineral deposits (dark spots) that are absent in the flight sample. Image courtesy of NASA, Johnson Space Center.
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