Osteoporosis International

, Volume 28, Issue 3, pp 1063–1075 | Cite as

Oxygen ultra-fine bubbles water administration prevents bone loss of glucocorticoid-induced osteoporosis in mice by suppressing osteoclast differentiation

  • T. Noguchi
  • K. EbinaEmail author
  • M. Hirao
  • T. Morimoto
  • K. Koizumi
  • K. Kitaguchi
  • H. Matsuoka
  • T. Iwahashi
  • H. Yoshikawa
Original Article



Oxygen ultra-fine bubbles (OUB) saline injection prevents bone loss of glucocorti\coid-induced osteoporosis in mice, and OUB inhibit osteoclastogenesis via RANK-TRAF6-c-Fos-NFATc1 signaling and RANK-p38 MAPK signaling in vitro.


Ultra-fine bubbles (<200 nm in diameter) have several unique properties, and they are tested in various medical fields. The purpose of this study was to investigate the effects of oxygen ultra-fine bubbles (OUB) on glucocorticoid-induced osteoporosis (GIO) model mice.


Prednisolone (PSL, 5 mg) was subcutaneously inserted in 6-month-old male C57BL/6J mice, and 200 μl of saline, OUB-diluted saline, or nitrogen ultra-fine bubbles (NUB)-diluted saline was intraperitoneally injected three times per week for 8 weeks the day after operations. Mice were divided into four groups; (1) control, sham-operation + saline; (2) GIO, PSL + saline; (3) GIO + OUB, PSL + OUB saline; (4) GIO + NUB, PSL + NUB saline. The effects of OUB on osteoblasts and osteoclasts were examined by serially diluted OUB medium in vitro.


Bone mass was significantly decreased in GIO [bone volume/total volume (%): control vs. GIO 12.6 vs. 7.9; p < 0.01] while significantly preserved in GIO + OUB (GIO vs. GIO + OUB 7.9 vs. 12.9; p < 0.05). In addition, tartrate-resistant acid phosphatase (TRAP)-positive cells in the distal femur [mean osteoclasts number/bone surface (mm−1)] was significantly increased in GIO (control vs. GIO 6.8 vs. 11.6; p < 0.01) while suppressed in GIO + OUB (GIO vs. GIO + OUB 11.6 vs. 7.5; p < 0.01). NUB did not affect these parameters. In vitro experiments revealed that OUB significantly inhibited osteoclastogenesis by inhibiting RANK-TRAF6-c-Fos-NFATc1 signaling, RANK-p38 MAPK signaling, and TRAP/Cathepsin K/DC-STAMP mRNA expression in a concentration-dependent manner. OUB did not affect osteoblastogenesis in vitro.


OUB prevent bone loss in GIO mice by inhibiting osteoclastogenesis.


c-Fos Glucocorticoid-induced osteoporosis (GIO) Nuclear factor of activated T-cells 1 (NFATc1) Osteoblasts Osteoclasts Oxygen ultra-fine bubbles (OUB) 



We are grateful to M. Shinkawa and A. Tada for their excellent technical assistance. We thank all the members of Yoshikawa’s laboratory for the helpful discussion and comments.

Compliance with ethical standards

Ethics statement

All experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Osaka University Graduate School of Medicine (permission number 24-022-007).

Authors’ roles

Study design: TN, KE, MH, and HY. Study conduct: TN and KE. Data collection: TN, KE, MH, TM, KK, KK, HM, and TI. Data analysis: TN and KE. Data interpretation: TN, KE, and MH. Drafting the manuscript: TN and KE. Approving the final version of manuscript: TN, KE, MH, TM, KK, KK, HM, TI, and HY. KE takes responsibility for the integrity of the data analysis.


All oxygen and nitrogen ultra-fine bubbles-diluted saline and medium were prepared by Ligaric Company Limited. This research was funded by Nakatomi funding, Health and Labor Sciences Research Grant of Japan, Ligaric Company Limited, and West Nippon Expressway Company. The funders had no role in the study design, decision to publish, or preparation of the manuscript.

Conflict of interest

Osaka University and West Nippon Expressway Company are applying for a patent of the medical use of ultra-fine bubbles.

Supplementary material

198_2016_3830_MOESM1_ESM.pdf (305 kb)
ESM 1 (PDF 304 kb)


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Copyright information

© International Osteoporosis Foundation and National Osteoporosis Foundation 2016

Authors and Affiliations

  • T. Noguchi
    • 1
  • K. Ebina
    • 1
    Email author
  • M. Hirao
    • 1
  • T. Morimoto
    • 1
  • K. Koizumi
    • 1
  • K. Kitaguchi
    • 1
  • H. Matsuoka
    • 1
  • T. Iwahashi
    • 1
  • H. Yoshikawa
    • 1
  1. 1.Department of Orthopaedic Surgery, Graduate School of MedicineOsaka UniversityOsakaJapan

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