Balk M L, Bray J, Day C, Epperly M, Greenberger J, Evans C H, Niyibizi C
Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Bone. 1997 Jul;21(1):7-15. doi: 10.1016/s8756-3282(97)00075-6.
To understand whether osteogenesis imperfecta (OI) could result from defective differentiation of osteoprogenitor cells, we investigated the osteogenic potential of bone marrow stromal cells from a mouse model of human OI (oim). Bone marrow was flushed from the femurs and tibias of oim and normal littermates using a syringe with Dulbecco's modified Eagle's medium, and cells were allowed to adhere to flasks. Adherent cells were trypsinized and passaged weekly at a 1:4 split. The established stromal cells were assessed for collagen synthesis, alkaline phosphatase, and osteocalcin production in the presence or absence of rhBMP-2. The stromal cells were also assessed for mineralization by Von-Kossa staining and for exogenous gene transfer using adeno-lacZ and a retroviral vector. The bone marrow stromal cells from oim mice synthesized alpha 1(I) homotrimers as expected, whereas the stromal cells from the normal littermates synthesized alpha 1(I)2 alpha 2(I) heterotrimers. The bone marrow stromal cells exhibited low levels of alkaline phosphatase activity under basal conditions: upon treatment with rhBMP-2, the level of the alkaline phosphatase activity increased approximately 40-fold. Cytochemical staining of the cells confirmed the expression of alkaline phosphatase by the oim stromal cells and its augmentation by rhBMP-2. Osteocalcin production in the stromal cells was also enhanced approximately threefold by rhBMP-2. oim stromal cells grown in the presence of beta-glycerophosphate and ascorbic acid demonstrated Von-Kossa-positive solid deposits after 3 weeks in culture. Ten days after infection with adeno-lacZ, approximately 70% of oim stromal cells expressed the transgene product, and after infection with a retrovirus, approximately 20% of the cells expressed the transgene. These data indicate that bone marrow stromal cells, have osteogenic potential, and also the potential to be transduced with exogenous genes. Under basal conditions, however, the stromal cells from oim mice exhibited significantly lower levels of alkaline phosphatase activity than their normal littermates.
为了解成骨不全症（OI）是否可能由骨祖细胞分化缺陷所致，我们研究了来自人类OI小鼠模型（oim）的骨髓基质细胞的成骨潜能。使用装有杜尔贝科改良伊格尔培养基的注射器从oim小鼠和正常同窝小鼠的股骨和胫骨中冲洗出骨髓，使细胞贴附于培养瓶。贴壁细胞用胰蛋白酶消化，并每周以1:4的比例传代。在有或没有重组人骨形态发生蛋白-2（rhBMP-2）的情况下，对已建立的基质细胞进行胶原合成、碱性磷酸酶和骨钙素产生的评估。还通过冯·科萨染色评估基质细胞的矿化情况，并使用腺病毒-β半乳糖苷酶（adeno-lacZ）和逆转录病毒载体评估外源基因转移情况。来自oim小鼠的骨髓基质细胞如预期那样合成α1（I）同三聚体，而来自正常同窝小鼠的基质细胞合成α1（I）2α2（I）异三聚体。在基础条件下，骨髓基质细胞表现出低水平的碱性磷酸酶活性：在用rhBMP-2处理后，碱性磷酸酶活性水平增加了约40倍。细胞化学染色证实oim基质细胞表达碱性磷酸酶，并且rhBMP-2可增强其表达。rhBMP-2还使基质细胞中的骨钙素产生增加了约三倍。在β-甘油磷酸和抗坏血酸存在的情况下培养的oim基质细胞在培养3周后显示出冯·科萨阳性固体沉积物。在用腺病毒-β半乳糖苷酶感染10天后，约70%的oim基质细胞表达转基因产物，在用逆转录病毒感染后，约20%的细胞表达转基因。这些数据表明骨髓基质细胞具有成骨潜能，也具有被外源基因转导的潜能。然而，在基础条件下，来自oim小鼠的基质细胞表现出比其正常同窝小鼠显著更低水平的碱性磷酸酶活性。
