| Project/Area Number |
22K17135
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 57050:Prosthodontics-related
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| Research Institution | Niigata University |
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Principal Investigator |
Thant Lay 新潟大学, 医歯学系, 特任助教 (10941867)
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| Project Period (FY) |
2022-04-01 – 2024-03-31
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| Project Status |
Discontinued (Fiscal Year 2023)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2023: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2022: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Keywords | Extracellular vesicles / ROCK inhibitor / Bone regeneration |
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| Outline of Research at the Start |
First, we will elucidate the composition of ROCK inhibitor treated extracellular vesicles secreted from mesenchymal cells and its secretory control pathway. Next, we will analyze the mineralization of osteoblasts and bone regeneration by administering ROCKin'-EV to calvarial defects in rats.
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| Outline of Annual Research Achievements |
For the proteomic analysis of ROCK inhibitor-treated extracellular vesicles (ROCKin'-EVs), MagCapture Exosome Isolation Kit was used to prepare Control extracellular vesicles (Control-EVs) and ROCKin'-EVs from the culture medium of MC3T3-E1 cells. Mass spectrometry was performed to elucidate the changes in the proteomic composition. We detected there were changes ROCKin'-EVs protein composition. We identified that a number of Rab GTPase (known as the key regulators of intercellular vesicle transport) were upregulated in ROCKin'-EVs.
For the analysis of osteoblastic mineralization regulated by ROCKin'-EVs, we isolated the Control-EVs and ROCKin'-EVs again from the culture medium of MC3T3-E1 cells on Day3, 6 and 9 of osteoblastic differentiation using the culture filtration device. We measured the insolated proteins in the Control-EVs and ROCKin'-EVs. The protein concentration of the ROCKin'-EVs were 5.9times more than the Control-EVs. The ROCKin'-EVs were diluted to the same concentration as the Control-EVs, and MC3TC-E1 cells were treated with dimethyl sulfoxide (DMSO) (Control), 5 μM ROCK inhibitor (ROCK), 5 μg/ml Control-EVs, and ROCKin'-EVs, with induction of osteoblast differentiation. For the evaluation of mineralization activity, ALP staining and Picrosirus Red staining were performed. ALP and picrosirus red activity were significantly increase in ROCK inhibitor treated cells (ROCK) than in Control, as expected. However, the ALP staining and Picrosirus Red staining of Control-EVs and ROCKin'-EVs treated cells didn’t show significant difference from the Control.
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