Characterization of ROCK inhibitor-treated extracellular vesicles and analysis of their effect on osteogenesis
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 |
Review Section |
Basic Section 57050:Prosthodontics-related
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Research Institution | Niigata University |
Principal Investigator |
Thant Lay 新潟大学, 医歯学総合病院, 特任助教 (10941867)
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Project Period (FY) |
2022-04-01 – 2024-03-31
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Project Status |
Granted (Fiscal Year 2022)
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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 |
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 |
MagCapture Exosome Isolation Kit was used to prepare Control extracellular vesicles (Control-EVs) and ROCK inhibitor-treated extracellular vesicles (ROCKin'-EVs) from the culture medium of BMSCs. 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. We isolated the Control-EVs and ROCKin'-EVs from the culture medium of BMSCs again using total exosome isolation aiming to collect large amount of EVs. Western blotting of Control-EVs and ROCKin'-EVs was performed. The quantity of isolated EVs was inconsistent and necessary to be normalized.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In order to collect large amount of EVs, total exosome isolation (Invitrogen) was used. However, normalizing the amount of EVs by this protocol is challenging. In order to overcome this, we will isolate and repeat the investigation of mineralizing activity of ROCKin'-EVs on osteoblasts using MagCapture Exosome Isolation Kit.
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Strategy for Future Research Activity |
We will investigate the mineralizing activity of ROCKin'-EVs on osteoblasts using MagCapture Exosome Isolation Kit. To evaluate the mineralizing activity, ALP staining, Alizarin Red S staining will be performed. To evaluate the expression levels of osteogenic genes (RUNX2, ALP), western blotting will be performed. Examination of bone regeneration ability of ROCKin'-EVs in rat model will be performed after reconfirming the In Vivo mineralization activity.
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Report
(1 results)
Research Products
(4 results)
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[Presentation] Alteration of the ECM composition and fiber organization in periodontal ligament under mechanical loading2022
Author(s)
Lay Thant, Masaru Kaku, Yoshito Kakihara, Masaru Mizukoshi, Megumi Kitami, Kohei Kitami, Daiki Kobayashi, Yutaka Yoshida, Takeyasu Maeda, Katsumi Uoshima, Isao Saito, Makio Saeki
Organizer
第54回日本結合組織学会学術大会
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