Iida Jin, Yoshikawa Takafumi, Akahane Manabu, Ohgushi Hajime, Dohi Yoshiko, Takakura Yoshinori, Nonomura Akitaka
Department of Orthopaedic Surgery, Nara Medical University, Kashihara City, Nara 634-8522, Japan.
Cell Transplant. 2004;13(4):357-65. doi: 10.3727/000000004783983873.
Osteogenesis occurs in porous hydroxyapatite (HA) when porous HA blocks combined with marrow mesenchymal cells are grafted in vivo. In vitro bone formation occurs in HA pores when HA combined with marrow cells is cultured in osteogenic medium containing dexamethasone. This cultured bone/HA construct possesses higher osteogenic ability when it is grafted in vivo. In the present study, we compared the osteogenic potential of a cultured bone/HA construct with that of a marrow mesenchymal cell/HA composite. Marrow cells were obtained from the femoral bone shaft of 7-week-old, male Fischer 344 rats and were cultured in T-75 flasks. Cells were concentrated, then frozen and stored in liquid nitrogen for 6 months. The cryopreserved cells were then thawed and prepared for subculture in porous HA (5 x 5 x 5 mm, Interpore 500) and for implantation with porous HA. After 2 weeks of subculture, three cultured bone/HA constructs were separately implanted in the right side of the back of each syngeneic 7-week-old male Fischer rat, and three thawed cell/HA composites (without subculture) were separately implanted in the left side. These implants were harvested at 2 or 4 weeks postimplantation, and prepared for histological, biochemical, and genetic analysis. Alkaline phosphatase activity and osteocalcin content of cultured bone/HA constructs were much higher than those of the cell/HA composites at 2 and 4 weeks postimplantation. Histological examination and gene expression data agreed with these findings. The culture technique discussed herein should facilitate the development of biosynthetic bone implants with higher osteogenic capacity.
当将多孔羟基磷灰石（HA）块与骨髓间充质细胞结合后移植到体内时，会发生骨生成。当HA与骨髓细胞结合并在含有地塞米松的成骨培养基中培养时，会在HA孔隙中发生体外骨形成。这种培养的骨/HA构建体在移植到体内时具有更高的成骨能力。在本研究中，我们比较了培养的骨/HA构建体与骨髓间充质细胞/HA复合物的成骨潜力。从7周龄雄性Fischer 344大鼠的股骨干中获取骨髓细胞，并在T-75培养瓶中培养。将细胞浓缩，然后冷冻并在液氮中保存6个月。然后将冷冻保存的细胞解冻，准备在多孔HA（5×5×5mm，Interpore 500）中传代培养并与多孔HA一起植入。传代培养2周后，将三个培养的骨/HA构建体分别植入每只同基因7周龄雄性Fischer大鼠背部右侧，将三个解冻的细胞/HA复合物（未传代培养）分别植入左侧。在植入后2周或4周收获这些植入物，并准备进行组织学、生化和基因分析。植入后2周和4周时，培养的骨/HA构建体的碱性磷酸酶活性和骨钙素含量远高于细胞/HA复合物。组织学检查和基因表达数据与这些结果一致。本文讨论的培养技术应有助于开发具有更高成骨能力的生物合成骨植入物。
