Ishikawa Hisato, Kitoh Hiroshi, Sugiura Fumiaki, Ishiguro Naoki
Department of Orthopaedic Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi, 466-8550, Japan.
Acta Orthop. 2007 Apr;78(2):285-92. doi: 10.1080/17453670710013816.
Since the osteogenic potential of bone marrow derived mesenchymal stem cells (BMSCs) becomes reduced with passage, establishment of culture condition that permit the rapid expansion of BMSCs while retaining their potential for differentiation is needed for clinical application. Bone morphogenetic proteins stimulate osteogenic differentiation in mesenchymal progenitor cells as well as increase stem cell numbers. Thus, we analyzed the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteogenic potential of rat BMSCs over several passages.
Osteogenic differentiation in vitro was evaluated in terms of the alkaline phosphatase (ALP) activity and the osteocalcin (OC) concentration in the supernatants, and the expression of ALP and OC mRNA in the cultured cells. For in-vivo osteogenesis, BMSCs cultured with and without rhBMP-2 through all passages were implanted into athymic mice.
The levels of osteogenic markers were significantly higher in the cells of the BMP(+) group than in the cells of the BMP(-) group, although they decreased with passage irrespective of whether or not rhBMP-2 was added. Similar to the in-vitro experiments, there was a greater degree of bone and cartilage tissue formation in the BMP(+) group over all passages.
From our results, osteogenic potential can be maintained even in BMSCs that have been passaged several times in the presence of rhBMP-2. These cells are capable of inducing and participating in bone formation and can be used for clinical applications.
由于骨髓间充质干细胞（BMSCs）的成骨潜能会随着传代而降低，因此需要建立一种培养条件，既能使BMSCs快速扩增，又能保留其分化潜能，以用于临床应用。骨形态发生蛋白可刺激间充质祖细胞的成骨分化，并增加干细胞数量。因此，我们分析了重组人骨形态发生蛋白-2（rhBMP-2）对大鼠BMSCs多代培养后的成骨潜能的影响。
通过检测上清液中碱性磷酸酶（ALP）活性和骨钙素（OC）浓度，以及培养细胞中ALP和OC mRNA的表达，评估体外成骨分化情况。对于体内成骨，将在所有传代过程中添加和未添加rhBMP-2培养的BMSCs植入无胸腺小鼠体内。
BMP（+）组细胞中的成骨标志物水平显著高于BMP（-）组细胞，尽管无论是否添加rhBMP-2，其水平都会随着传代而降低。与体外实验相似，在所有传代过程中，BMP（+）组的骨和软骨组织形成程度更高。
根据我们的结果，即使在rhBMP-2存在的情况下传代多次的BMSCs中，成骨潜能也能得以维持。这些细胞能够诱导并参与骨形成，可用于临床应用。
