In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene
In vitro and in vivo induction of bone formation using a recombinant adenoviral vector carrying the human BMP-2 gene
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DOI：
10.1007/bf02678146
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发表时间：
2001-02-01
影响因子：
4.2
通讯作者：
Riew, KD
中科院分区：
文献类型：
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作者：
Cheng, SL;Lou, J;Riew, KD
骨形态发生蛋白-2（BMP-2）在体内和体外均可诱导骨形成。尽管需要高浓度（高达毫克）的BMP-2来在体内实现这种效果。此外，临床应用通常限于植入时的单次剂量。为了延长BMP-2的转化作用，我们使用携带人BMP-2基因的重组腺病毒载体（AdV-BMP 2）来培养成熟雄性新西兰白色兔的骨髓间充质干细胞（MSC）。将多能MSC与AdV-BMP 2孵育过夜，然后在生长培养基中培养1周。对组织培养物的分析表明，这些AdV-BMP-2转导的MSC产生BMP-2蛋白，分化成骨祖细胞系，并在体外诱导骨形成。与未转导的细胞和用对照腺病毒构建体转导的细胞相比，这些MSC具有增加的碱性磷酸酶活性，增加的I型胶原蛋白、骨桥蛋白和骨钙素mRNA的表达，并诱导基质矿化。为了分析体内成骨潜能，将Adv-BMP 2转导的MSC自体植入供体兔L5和L 6之间的横突间间隙中。4周后，在植入用AdV-BMP 2转导的细胞的区域中，通过放射学检查证实了新骨的产生，而在植入用对照腺病毒构建体转导的细胞的部位没有明显的骨。组织学检查进一步证实了新骨形成的存在。这些积累的数据表明，有可能用携带BMP-2基因的重组腺病毒载体成功地转染间充质干细胞，使得这些细胞将产生BMP-2，分化成骨祖细胞系，并在体外和体内诱导骨形成。此外，与我们以前的报道（one out five，Calcif Tissue Int 63：357-360，1998）相比，在移植前将AdV-BMP 2转导的细胞在培养物中再孵育7天提高了骨形成的成功率（三分之三）。
It has been well established that bone morphogenetic protein-2 (BMP-2) can induce bone formation both in vivo and in vitro. although high concentrations (up to milligrams) of BMP-2 have been required to achieve this effect in vivo. Further, clinical applications are usually limited to a single dose at the time of implantation. In an attempt to prolong the transforming effect of BMP-2 we used a recombinant adenoviral vector carrying the human BMP-2 gene (AdV-BMP2) to transduce marrow-derived mesenchymal stem cells (MSC) of skeletally mature male New Zealand white rabbits. The pluripotential MSC were incubated with AdV-BMP2 overnight followed by culture in growth medium for 1 week. Assays on tissue cultures demonstrated that these AdV-BMP2 transduced MSC produced BMP-2 protein, differentiated into an osteoprogenitor line, and induced bone formation in vitro. These MSC had increased alkaline phosphatase activity, increased expression of type I collagen, osteopontin, and osteocalcin mRNA, and induced matrix mineralization compared with both non-transduced cells and cells transduced with a control adenoviral construct. To analyze the osteogenic potential in vivo, Adv-BMP2-transduced MSC were autologously implanted into the intertransverse process space between L5 and L6 of the donor rabbits. The production of new bone was demonstrated by radiographic examination 4 weeks later in areas implanted with cells transduced with AdV-BMP2, whereas no bone was evident at sites implanted with cells transduced with the control adenoviral construct. Histological examination further confirmed the presence of new bone formation. These accumulated data indicate that it is possible to successfully transduce mesenchymal stem cells with a recombinant adenoviral vector carrying the gene for BMP-2 such that these cells will produce BMP-2, differentiate into an osteoprogenitor line, and induce bone formation both in vitro and in vivo. Moreover, incubation of the AdV-BMP2-transduced cells for an additional 7 days in culture before transplantation enhances the success rate in bone formation (three out of three) as compared with our previous report (one out of five, Calcif Tissue Int 63:357-360, 1998).