Examination of the "Osteopontin Hypothesis"
(OSTEOPONTIN) - 11.12.14
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Examination of the "Osteopontin Hypothesis" (Osteopontin) is a Japan Aerospace Exploration Agency experiment that explores the role of this complex protein in controlling bone loss in the microgravity environment. Bone marrow cells from knockout mice engineered to lack osteopontin are placed onboard the International Space Station (ISS) for exposure to microgravity and artificial gravity, along with bone marrow cells from normal mice, in the Kibo module's centrifuge for 21 days. The bone marrow cells are then preserved for analysis on Earth.
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Japan Aerospace Exploration Agency (JAXA), Tsukuba, , Japan
Sponsoring Space Agency
Japan Aerospace Exploration Agency (JAXA)
ISS Expedition Duration
March 2015 - September 2015
Previous ISS Missions
- The mechanisms of microgravity-induced osteoporosis are not well understood. Osteopontin (OPN), one of the abundant proteins in the bone matrix, is believed to be involved in this process. Thus, the goal of this investigation is to examine the influence of microgravity on the mineralization of cultured bone marrow cells derived from osteopontin-deficient mice and control mice, onboard the ISS.
- Bone marrow stromal cells are prepared and frozen on the ground, launched to the ISS, and cultured under microgravity for 3 weeks. The culture medium is changed at day 10. After 21 days of cultivation, the culture medium is again collected, RNA later reagent is added to the cells for preservation in the freezer, and the chemically fixed cells are stored at 4°C. After return to earth, the samples are analyzed for minerals, proteins, RNA expressing, and image analysis for Alizarin Red staining of the mineralization nodules.
- This study is beneficial for the development of novel drugs, which are expected to provide great value to many people facing immobility induced osteoporosis.
Osteopontin plays an important but poorly understood role as a chemical bridge in remodeling bones, particularly in the function of osteoclasts, cells that tear down bone. Bone loss is a significant health hazard that becomes greater for astronauts with longer stays in space, and which has resisted exercise and other countermeasures. Proving that osteopontin is an important regulator of this may contribute to effective therapeutics for osteoporosis caused by microgravity.
Osteoporosis is a significant health hazard for the elderly, and other patients, suffering from various heart disease, stroke, and neurodegenerative diseases. Understanding the role of osteopontin in bone loss under microgravity should help in studying its role on Earth and developing novel therapies for treating it.
This experiment requires launching 24-lines (included 8-line backups) of frozen bone marrow stromal cells. For culturing these cells, the CBEF and five sets of the PFK-III are required. For sample storage after the experiment, freezer space is required for used culture medium from day 10 of the culture, and for the cells soaked with the RNAlater reagent after culture completion at day 21. It also requires Storage space in the refrigerator is required for the paraformaldehyde fixed cells after completion of the culture at day 21.
In this experiment, 16-lines of frozen bone marrow stromal cells from Osteopontin-deficient mice or control mice are launched. Crew tasks are required at three time points of experiment:
- The frozen syringes are unstowed and used to plate the cells on the Measurement Experiment Culture Chamber, by adding warmed culture medium. Each cell-suspension is plated on two chambers, and the cells in a total of 32 chambers are incubated in the Measurement Experiment Culture Cage at 37°C using CBEF. Each half of the duplicated chambers is cultured in microgravity aboard ISS, and the other half is cultured under the artificial 1-gravity conditions.
- At 10 day of the culture, the culture medium is changed.
- At 21 day of the culture, 12 chambers are frozen in the freezer, after exchange of the culture medium to RNAlater for preserving RNA. The remaining 20 chambers are stored in the refrigerator after exchanging the culture medium to 0.99% paraformaldehyde. These specimens are stained by alizarin red later. The used culture medium is collected and stored in the MELFI. These samples are used for calcium and phosphorous measurement studies.