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Kawate, K., Yajima, H., Ohgushi, H., Kotobuki, N., Sugimoto, K., Ohmura, T., Kobata, Y., Shigematsu, K., Kawamura, K., Tamai, K. and Takakura, Y. (2006) Tissue-engineered approach for the treatment of steroid-induced osteonecrosis of the femoral head: transplantation of autologous mesenchymal stem cells cultured with beta-tricalcium phosphate ceramics and free vascularized fibula. Artifical Organs, 30, 960-962. doi:10.1111/j.1525-1594.2006.00333.x

has been cited by the following article:

• TITLE: Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells

  AUTHORS: Manabu Akahane, Tomoyuki Ueha, Takamasa Shimizu, Yusuke Inagaki, Akira Kido, Tomoaki Imamura, Kenji Kawate, Yasuhito Tanaka

  KEYWORDS: Osteogenesis; Mesenchymal Stem Cells; Serial Passaging; Hydroxyapatite; Tissue Engineering; Bone Reconstruction

  JOURNAL NAME: Stem Cell Discovery, Vol.2 No.4, October 30, 2012

  ABSTRACT: We have previously reported on both the osteogenic potential of hydroxyapatite (HA) combined with bone marrow-derived mesenchymal stem cells (BMSCs) and a method involving osteogenic matrix cell sheet transplantation of BMSCs. In the present study, we assessed the osteogenic potential of serially-passaged BMSCs, both in vitro and in vivo. We also assessed whether an additional cell-loading technique can regain the osteogenic potential of the constructs combined with serially-passaged BMSCs. The present study revealed that passage (P) 1 cells cultured in osteogenic-induced medium showed strong positive staining for alkaline phosphatase (ALP) and Alizarin Red S, whereas P3 cells showed faint staining for ALP, with no Alizarin Red S staining. Staining of P1, P2 and P3 cells were progressively weaker, indicating that the osteogenic potential of the serially-passaged rat BMSCs is lost after P3 in vitro. The in vivo study showed that little bone formation was observed in the HA constructs seeded with P3 cells, 4 weeks after subcutaneous implantation. However, P3 cell/HA constructs which had increased cell-loading showed abundant bone formation within the pores of the HA construct. ALP and osteocalcin mRNA expression in these constructs was significantly higher than that of constructs with regular cell-seeding. The present study indicates that the osteogenic potential of the constructs with serially-passaged BMSCs is increased by additional cell-loading. This method can be applied to cases requiring hard tissue reconstruction, where BMSCs require serial expansion of cells.