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TNFα-dependent mTOR activity is required for tenotomy-induced ectopic ossification in mice

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Ectopic ossifications often occur in skeletal muscles or tendons following local trauma or internal hemorrhage, and occasionally cause severe pain that limits activities of daily living. However, mechanisms underlying their development remain unknown.

Materials and methods

The right Achilles tendon in 8-week-old female or male mice was dissected. Some mice were injected intraperitoneally either with phosphate-buffered saline, dimethyl sulfoxide, cimetidine, rapamycin, celecoxib or loxoprofen for 10 weeks. One week after surgery, immunohistochemical analysis was performed for mTOR, TNFα or F4/80. Ten weeks after surgery, ectopic ossification at the tenotomy site was detected by 3D micro-CT.

Results

Ectopic ossification was seen at dissection sites in all wild-type mice by dissection of the Achilles tendon. mTOR activation was detected at dissection sites, and development of ectopic ossification was significantly inhibited by administration of rapamycin, an mTOR inhibitor, to wild-type mice. Moreover, administration of the histamine 2 blocker cimetidine, which reportedly inhibits ectopic ossification in tendons, was not effective in inhibiting ectopic ossification in our models. TNFα-expressing F4/80-positive macrophages accumulate at dissection sites and that ectopic ossification of the Achilles tendon dissection was significantly inhibited in TNFα-deficient mice in vivo. Ectopic ossification is significantly inhibited by administration of either celecoxib or loxoprofen, both anti-inflammatory agents, in wild-type mice. mTOR activation by Achilles tendon tenotomy is inhibited in TNFα-deficient mice.

Conclusion

The TNFα-mTOR axis could be targeted therapeutically to prevent trauma-induced ectopic ossification in tendons.

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Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

T. Miyamoto was supported by a grant-in-aid for Scientific Research in Japan and a grant from the Japan Agency for Medical Research and Development.

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Authors and Affiliations

  1. Department of Orthopedic Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Yu Kushima, Yuiko Sato, Tami Kobayashi, Morio Matsumoto, Masaya Nakamura, Takuji Iwamoto & Takeshi Miyamoto

  2. Department of Orthopedic Surgery, National Defense Medical College, Namiki 3-2, Tokorozawa, Saitama, 359-8513, Japan

    Yu Kushima

  3. Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Yuiko Sato, Tami Kobayashi & Takeshi Miyamoto

  4. Department of Orthopedic Surgery, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan

    Yuko Fukuma & Takeshi Miyamoto

Authors
  1. Yu Kushima
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  2. Yuiko Sato
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  3. Tami Kobayashi
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  4. Yuko Fukuma
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  5. Morio Matsumoto
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  6. Masaya Nakamura
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  7. Takuji Iwamoto
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  8. Takeshi Miyamoto
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Contributions

Investigation: YK, YS, TK; conceptualization: TM; data curation: TI; funding acquisition: TM; supervision: MM, MN, TI, and TM; writing: TM.

Corresponding authors

Correspondence to Takuji Iwamoto or Takeshi Miyamoto.

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Kushima, Y., Sato, Y., Kobayashi, T. et al. TNFα-dependent mTOR activity is required for tenotomy-induced ectopic ossification in mice. J Bone Miner Metab 41, 583–591 (2023). https://doi.org/10.1007/s00774-023-01437-8

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  • DOI: https://doi.org/10.1007/s00774-023-01437-8

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