Characterization of ROCK inhibitor-treated extracellular vesicles and analysis of their effect on osteogenesis

2022 Fiscal Year Research-status Report

• Project/Area Number: 22K17135
• Research Institution: Niigata University
• Principal Investigator: Thant Lay 新潟大学, 医歯学総合病院, 特任助教 (10941867)
• Project Period (FY): 2022-04-01 – 2024-03-31
• Keywords: Extracellular vesicles / ROCK inhibitor / Bone regeneration

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.

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.

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.

Causes of Carryover

We plan to continue both in vitro and in vivo research during next academic year (2023-2024). We will use the finance to purchase the necessary reagents, equipment for this purpose.

Research Products

• [Journal Article] Involvement of Rab11 in osteoblastic differentiation: Its up-regulation during the differentiation and by tensile stress (2022)
  • Author(s): Lay Thant, Yoshito Kakihara, Masaru Kaku, Megumi Kitami, Kohei Kitami, Masaru Mizukoshi, Takeyasu Maeda, Isao Saito, Makio Saeki
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 624 Pages: 16-22
  • DOI: 10.1016/j.bbrc.2022.07.061
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress (2022)
  • Author(s): Lay Thant, Masaru Kaku, Yoshito Kakihara, Masaru Mizukoshi, Megumi Kitami, Moe Arai, Kohei Kitami, Daiki Kobayashi, Yutaka Yoshida, Takeyasu Maeda, Isao Saito, Katsumi Uoshima, Makio Saeki
  • Journal Title: Frontiers in Physiology Volume: 13 Pages: 899699
  • DOI: 10.3389/fphys.2022.899699
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Alteration of the ECM composition and fiber organization in periodontal ligament under mechanical loading (2022)
  • 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
• [Presentation] Extracellular matrix profiling of cultured PDL cells by chemical digestion-assisted proteomics (2022)
  • Author(s): Lay Thant, Masaru Kaku, Azusa Dobashi, Yoshito Kakihara, Isao Saito, Katsumi Uoshima
  • Organizer: 第22回日本再生医療学会総会