The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats
The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats
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DOI：
10.1097/bsd.0b013e31814cf51d
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发表时间：
2008-07-01
影响因子：
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通讯作者：
Wang, Jeffrey C.
中科院分区：
文献类型：
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作者：
Miyazaki, Masashi;Sugiyama, Osamu;Wang, Jeffrey C.
研究设计：大鼠脊柱融合模型：目的：本研究旨在评估用含骨形态发生蛋白（BMP）-2的慢病毒转染的达特骨髓细胞（RBC）在大鼠模型中诱导后外侧脊柱融合的能力。背景资料概要：脊柱关节固定术是一种常见的脊柱手术，自体移植物仍然是实现脊柱融合的标准。然而，它的采购与显着的发病率，和假关节的发生率已报告为5%至43%。骨不连经常导致临床症状的不满意解决，并且通常导致高医疗成本和发病率以及需要额外的手术。这些问题导致外科医生寻找替代方案来刺激骨形成。重组BMP也已成功用于临床试验。然而，需要大剂量的BMP来诱导充分的骨修复。区域基因治疗的发展可能是一种更有效的方法，将蛋白质递送到特定的解剖部位。此外，产生腺BMP-2的大鼠骨髓源性细胞已成功用于诱导后路脊柱融合。近年来，基于人类免疫缺陷病毒的慢病毒载体已被开发用于基因治疗。慢病毒能够插入宿主基因组，确保延长基因表达。然而，安全性问题是一个主要问题，当采用这些载体用于临床use.Methods：在体外研究中，我们使用的红细胞转染慢病毒载体编码BMP-2（慢-BMP-2），红细胞转染慢病毒载体编码的绿色荧光蛋白（GFP）（慢-GFP），和未转染的红细胞;后2个被用作对照。比较各组之间的碱性磷酸酶（ALP）染色和ALP活性，以评估Lenti-BMP-2转染的RBC刺激成骨细胞分化的能力。在大鼠脊柱后外侧融合模型中，实验研究包括4组。组I包括6只动物，植入含有5百万个用Lenti-BMP-2转染的RBC的胶原海绵。第2组包括3只动物，植入含有5百万个转染有Lenti-GFP的RBC的胶原海绵。第3组包括植入含有5百万个未转染RBC的胶原海绵的6只动物。第4组包括仅植入胶原海绵的3只动物。通过在第4、6和8周获得的X线片对大鼠进行评估。死亡后，他们的脊柱被切除，并通过手动触诊，高分辨率的微计算机断层扫描，和组织学analysis.Results：ALP染色显着大于在Lenti-BMP-2转染的红细胞比未转染的红细胞和Lenti-GFP转染的红细胞。ALP活性在Lenti-BMP-2转染的RBC中比在未转染的RBC和Lenti-GFP转染的RBC中高3倍。在大鼠脊柱融合模型中，影像学评估、高分辨率微型计算机断层扫描和手动触诊显示第1组所有大鼠在8周时均实现脊柱融合。第2、3和4组为对照组。对照组大鼠L4-L5均未发生融合。结论：本研究证明，通过慢病毒基因转移产生BMP 2的红细胞可诱导充分的脊柱融合。使用含有BMP-2 cDNA的慢病毒载体将是脊柱融合策略的一种新的和有前途的方法。
Study Design: Rat spinal fusion model.Objective: This study aimed to assess the ability of tat bone marrow cells (RBMCs) transfected with bone morphogenetic protein (BMP)-2-containing lentivirus to induce a posterolateral spinal fusion in a rat model.Summary of Background Data: Spinal arthrodesis is a commonly performed spinal procedure and autograft remains the standard for achieving spinal fusion. However, its procurement is associated with significant morbidity, and the rate of pseudoarthrosis has been reported to be 5% to 43%. Nonunion frequently leads to an unsatisfactory resolution of clinical symptoms and usually results in high medical costs and morbidity as well as the need for additional surgeries. These problems have led surgeons to search for alternative solutions to stimulate bone formation. Recombinant BMPs have also been used successfully in clinical trials. However, large doses of BMPs were required to induce adequate bone repair. The development of a regional gene therapy may be a more efficient method to deliver proteins to a specific anatomic site. Furthermore, adeno-BMP-2-producing rat bone marrow-derived cells have been used successfully to induce posterior spinal fusion. Recently, lentiviral vectors on the basis of human immunodeficiency virus have been developed for gene therapy. Lentiviruses are capable of insertion into the host genome, ensuring a prolonged gene expression. However, safety issues are a major concern when adopting these vectors for clinical use.Methods: In vitro study, we used RBMCs transfected with lentivirus vectors encoding BMP-2 (Lenti-BMP-2), RBMCs transfected with lentivirus vectors encoding the green fluorescent protein (GFP) (Lenti-GFP), and untransfected RBMCs; the latter 2 were used as controls. Alkaline phosphatase (ALP) staining and ALP activity were compared between the groups to assess the ability of the Lenti-BMP-2-transfected RBMCs to stimulate osteoblastic differentiation. In the rat posterolateral spine fusion model, the experimental study comprised 4 groups. Group I comprised 6 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-BMP-2. Group 2 comprised 3 animals that were implanted with a collagen sponge containing 5 million RBMCs transfected with Lenti-GFP. Group 3 comprised 6 animals that were implanted with a collagen sponge containing 5 million untransfected RBMCs. Group 4 comprised 3 animals that were implanted with a collagen sponge alone. The rats were assessed by radiographs obtained at 4, 6, and 8 weeks. After death, their spines were explanted and assessed by manual palpation, high-resolution microcomputerized tomography, and histologic analysis.Results: The ALP staining was significantly greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. The ALP activity was 3-fold greater in the Lenti-BMP-2-transfected RBMCs than in the untransfected RBMCs and the Lenti-GFP-transfected RBMCs. In the rat spine fusion model, radiographic evaluation, high-resolution microcomputerized tomography, and manual palpation revealed spinal fusion in all the rats in Group 1 at 8 weeks. Groups 2, 3, and 4 comprised the control group. None of the rats in the control group (0 of 12) developed fusion at L4-L5.Conclusions: The present study demonstrated that BMP2-producing RBMCs, created through lentiviral gene transfer, induced sufficient spinal fusion. The use of lentiviral vectors that contain the cDNA for BMP-2 will be a novel and promising approach for a spinal fusion strategy.