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Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system

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Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

Bone remodeling plays a central role in the maintenance of bone homeostasis. Our group has established an in vitro system by which the cellular events during bone remodeling can be observed longitudinally. This study used this system to quantitatively analyze osteoblasts, osteoclasts, and matrices to elucidate their temporal changes and correlations.

Materials and methods

Osteoblasts from EGFP mice were cultured to form calcified nodules, followed by co-culture with bone marrow macrophages from Tnfrsf11aCre/+ x Ai14 mice for 3 weeks (resorption phase). Then cells were cultured with osteoblast differentiation medium for 3 weeks (formation phase). The same sites were observed weekly using 2-photon microscopy. Matrices were detected using second harmonic generation. Parameters related to matrices, osteoblasts, and osteoclasts were quantified and statistically analyzed.

Results

Resorption and replenishment of the matrix were observed at the same sites by 2 photon microscopy. Gross quantification revealed that matrix and osteoblast parameters decreased in the resorption phase and increased in the formation phase, while osteoclast parameters showed the opposite pattern. When one field of view was divided into 16 regions of interest (ROIs) and correlations between parameters were analyzed in each ROI, decreased and increased matrix volumes were moderately correlated. Parameters of matrices and osteoblasts, and those of matrices and osteoclasts exhibited moderate correlations, while those of osteoblasts and osteoclasts were only weakly correlated.

Conclusion

Several correlations between cells and matrix during remodeling were demonstrated quantitatively. This system may be a powerful tool for the research of bone remodeling.

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Acknowledgements

We are grateful to Dr. Yasuhiro Kobayashi (Matsumoto Dental University) for providing the Tnfrsf11aCre/+ mice. This study was supported by JSPS KAKENHI (#18K09718 and #21K10085).

Funding

The work of Atsuhiko Hikita was supported by Japan Society for the Promotion of Science, under Grant 18K09718 and 21K10085.

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Authors and Affiliations

  1. Department of Sensory and Motor System Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, 113‑8655, Japan

    Shuya Oguchi & Kazuto Hoshi

  2. Department of Tissue Engineering, The University of Tokyo Hospital, Tokyo, 113‑8655, Japan

    Tomoaki Sakamoto, Kazuto Hoshi & Atsuhiko Hikita

  3. Department of Oral‑Maxillofacial Surgery, and Orthodontics, The University of Tokyo Hospital, Tokyo, 113‑8655, Japan

    Kazuto Hoshi

Authors
  1. Shuya Oguchi
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  2. Tomoaki Sakamoto
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  3. Kazuto Hoshi
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  4. Atsuhiko Hikita
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Contributions

SO and TS: carried out the experiments. SO and AH: analyzed the data and wrote the draft of the manuscript. AH and KH: supervised the project. All authors contributed to the final version of the manuscript.

Corresponding author

Correspondence to Atsuhiko Hikita.

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Conflicts of interest

Atsuhiko Hikita held an endowed chair supported by FUJISOFT INCORPORATED (until 31 October 2020) and an endowed chair supported by CPC corporation, Kyowa Co., Ltd., Kanto Chemical Co., Inc., and Nichirei Corporation (from 1 July, 2021, to 30 June, 2022), and is affiliated with the social cooperation program of Kohjin Bio Co., Ltd. (since 1 July, 2022).

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Supplementary Information

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774_2022_1381_MOESM1_ESM.docx

Supplemental Fig. 1 a Fluorescence microscopy and bright filed observation of osteoblasts from EGFP mice before and after ALP staining, respectively. Scale bar: 100 μm. b. Fluorescence microscopy and bright filed observation of bone marrow cells from Tnfrsf11aCre/ + x Ai14 mice before and after TRAP staining, respectively. Scale bar: 300 μm (DOCX 81574 kb)

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Oguchi, S., Sakamoto, T., Hoshi, K. et al. Quantitative analyses of matrices, osteoblasts, and osteoclasts during bone remodeling using an in vitro system. J Bone Miner Metab 41, 3–16 (2023). https://doi.org/10.1007/s00774-022-01381-z

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  • DOI: https://doi.org/10.1007/s00774-022-01381-z

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