Human Cell

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Three-dimensional bone formation including vascular networks derived from dental pulp stem cells in vitro

  • Miho WatanabeEmail author
  • Akihiro Ohyama
  • Hiroshi Ishikawa
  • Akira Tanaka
Research Article


The regeneration of bone tissue is an active area of research, and future clinical applications are expected. Here, to establish new bone graft materials and an experimental bone tissue model, we generated united compact and spongy bone tissues containing vascular networks from human dental pulp stem cells in vitro. We applied the cell bead and cell sheet methods to construct three-dimensional bone tissue, which was cultured using a circumfusion apparatus for 30 days. Using micro-computed tomography, we assessed structural differences between compact and spongy bone. Histological examinations revealed the presence of bone lacunae containing osteocytes, Haversian canal-like structures, and extensive vascularization. Furthermore, tartrate-resistant acid phosphatase (TRAP) staining-positive osteoclast-like cells were also observed. Thus, the bone tissue generated using this method closely resembles native bone tissue and may possess bone remodeling ability. We successfully generated bone tissue containing blood vessel networks in vitro using this method. The generated bone tissue will likely be highly applicable to medical care, the study of osteogenesis, drug-screening assays, and drug development for bone tissue.


Regenerative bone Tissue engineering Three-dimensional culture Angiogenesis Human dental pulp tissue 



We would like to thank Drs. Junko Toyomura (Department of NDU Life Science, School of Life Dentistry, the Nippon Dental University) and Noriko Tominaga (Department of Developmental and Regenerative Density, School of Life Dentistry, the Nippon Dental University) for providing invaluable comments and technical support. We would also like to thank Dr. Yoshiaki Ide (Department of Developmental and Regenerative Density, School of Life Dentistry, the Nippon Dental University), who provided guidance for micro-CT analysis. We are also grateful to Professor Toshiaki Tachibana (Division of Fine Morphology, Core Research Facilities, the Jikei University School of Medicine), who provided guidance for electron microscopy analysis.

Author contributions

HI, AO, MW, and AT conceived and designed the project. MW conducted the experiments, analyzed the results, and wrote the manuscript. All authors discussed the results and commented on the manuscript.


This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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Supplementary material 1 (MPG 5864 KB)
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Supplementary material 2 (DOCX 12 KB)
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Supplementary material 3 (TIF 841 KB)
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Supplementary material 4 (TIF 1013 KB)


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Copyright information

© Japan Human Cell Society and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Oral and Maxillofacial Surgery, Field of Oral and Maxillofacial Surgery and Systemic Medicine, Course of Clinical ScienceNippon Dental University Graduate School of Life Dentistry at NiigataNiigataJapan
  2. 2.Department of NDU Life SciencesNippon Dental University School of Life Dentistry at TokyoTokyoJapan
  3. 3.Laboratory of Clinical Regenerative Medicine, Department of Neurosurgery, Faculty of MedicineUniversity of TsukubaTsukuba-shiJapan
  4. 4.Department of Oral and Maxillofacial SurgeryNippon Dental University School of Life Dentistry at NiigataNiigataJapan

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