Bone regeneration from mouse compact bone-derived cells

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  • YAMACHIKA Eiki
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • MATSUBARA Masakazu
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • KITA Kenichiro
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • TAKABATAKE Kiyofumi
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • FUJITA Yuuki
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • MATSUMURA Tatsushi
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • HIRATA Yasuhisa
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
  • IIDA Seiji
    Department of Oral and Maxillofacial Reconstructive Surgery, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences

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Other Title
  • マウス皮質骨由来細胞による骨再生
  • マウス ヒシツコツ ユライ サイボウ ニ ヨル ホネ サイセイ

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Abstract

Substantial progress has been made in the development of cell therapy; however, there have been no practical applications related to bone regeneration. We analyzed mouse bone-marrow-derived lineage (-) cells and compact bone-derived lineage (-) cells as candidate mesenchymal stem cells (MSCs), and confirmed bone regeneration in vivo. Materials and Methods: Bone marrow-derived cells were collected from the femurs of 5-week-old C57-GFP male mice. Bone marrow-derived cells were harvested by removing the epiphyses and flushing with medium, after which the femurs were broken into approximately 1 mm3 fragments and treated with collagenase II. Cells released from the compact bone were aspirated and cultured with the bone fragments. These cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum at 37℃ in a 5% CO2 humidified incubator for 3 days, and the difference between bone marrow-derived cells and compact bonederived cells was investigated. After 3 days of incubation, cells were labeled with allophycocyanin (APC) antilineage antibody cocktail, analyzed by flow cytometry, and sorted into lineage (+) or lineage (-) populations. Cells that had acquired lineage (-) were then induced to differentiate into osteogenic lineages by incubation with osteogenic media for 3 days. Differentiated cells and 50 mg of beta-TCP were mixed to form an osteogenic pellet. Pellet samples were implanted into subcutaneous pouches of SCID mice and retrieved after 4 weeks. Tissue samples were fixed in 1.5% paraformaldehyde and embedded in paraffin. Sections were stained with hematoxylin and eosin, and immunohistochemical analysis were performed using anti-GFP antibody. Results: Approximately 90% of bone marrow-derived cells expressed lineage markers. However, only 40% of compact bone-derived cells expressed these markers. Both bone marrow-derived and compact bone-derived cells sorted as lineage (-) showed fibroblastic shapes and plastic-adhesion ability. The number of lineage (-) cells from an individual mouse femur was about 10,000 from bone marrow and about 400,000 from compact bone. Moreover, we found that cells isolated from compact bone and sorted as lineage (-) cells were capable of supporting bone formation in vivo. The methods and results described here may facilitate understanding of MSC biology and lead to clinical applications.

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