Author(s)

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

Department of Artificial Joint and Regenerative Medicine, Nara Medical University, Nara, Japan.
Department of Orthopedic Surgery, Nara Medical University, Nara, Japan.
Department of Public Health, Health Management and Policy, Nara Medical University School of Medicine, Nara, Japan.

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.

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

Akahane, M. , Ueha, T. , Shimizu, T. , Inagaki, Y. , Kido, A. , Imamura, T. , Kawate, K. and Tanaka, Y. (2012) Increased osteogenesis with hydroxyapatite constructs combined with serially-passaged bone marrow-derived mesenchymal stem cells. Stem Cell Discovery, 2, 133-140. doi: 10.4236/scd.2012.24018.