人骨形态发生蛋白2基因活化纳米骨浆的成骨能力***☆

背景：纳米骨浆在骨缺损修复过程中仅起支架和骨传导作用，不具备骨诱导能力。目的：观察人骨形态发生蛋白2(human bone morphogenetic protein -2，hBMP-2)基因活化纳米骨浆的异位诱导成骨能力和修复骨缺损效果。时间及地点：实验于2006-06/2007-03在华中科技大学同济医学院分子生物中心实验室和协和医院骨科实验室完成。材料：将纳米骨浆与hBMP-2基因真核表达质粒相复合，形成hBMP-2基因局部释放系统。方法：实验一：昆明种小鼠24只右侧大腿后群肌袋注射纳米骨浆+hBMP-2质粒作为实验侧，其中12只小鼠左侧大腿后群肌袋注射纳米骨浆+空白质粒为对照侧1，另12只左侧注射纳米骨浆为对照侧2。实验二：新西兰白兔54只，其中6只作左桡骨中段骨缺损，不植入材料，为空白对照；另48只制成双侧桡骨中段15 mm骨缺损模型，随机分成3组，缺损处分别植入 hBMP-2+纳米骨浆、空白质粒+纳米骨浆、纳米骨。主要观察指标：实验一：采用放射学、分子生物学和组织形态学等方法检测成骨基因hBMP-2表达和诱导成骨效应。②实验二：取桡骨标本作影像学检查、组织学观察和生物力学检测。结果：实验一：小鼠术后2，4周实验侧有hBMP-2表达。实验侧术后2周出现大量软骨组织和呈岛状散在分布的骨组织，术后4周可见大量成骨组织，新生骨相互融合生长并形成较为成熟的板层骨和骨小梁结构；对照侧未见骨组织形成。实验侧碱性磷酸酶活性明显高于对照侧(P < 0.01)。实验二：术后12周空白对照组骨缺损无愈合，其余3组骨缺损均修复。植入hBMP-2+纳米骨浆组的碱性磷酸酶水平、成骨速度及新生骨力学强度均明显优于植入空白质粒+纳米骨浆或纳米骨组（P < 0.05）。 结论：经组织学、影像学及生物力学的综合检测验证，纳米骨浆复合hBMP-2质粒后，具有一定的骨诱导作用，植入体内后成骨速度、质量及力学强度较单纯的纳米骨浆明显增强，能够有效修复骨缺损。

Osteogenic potential of human bone morphogenetic protein 2 gene-activated nanobone putty

BACKGROUND: Nanobone putty plays a scaffold and bone conduction role in the repair of bone defects, but it lacks the capacity of bone induction.OBJECTIVE: To investigate the osteogenic potential of human bone morphogenetic protein 2 (hBMP2) gene activated nanobone putty in inducing ectopic bone formation, and to study the effects of the hBMP2 gene activated nanobone putty on repairing bone defects. TIME AND SETTING: Experiments were performed at the bio-molecular central laboratory in Tongji Medical College and the orthopaedic laboratory of Union Hospital, Huazhong University of Science and Technology from June 2006 to March 2007.MATERIALS: Nanobone putty was compounded with hBMP-2 gene eukaryotic expression plasmid to prepare hBMP-2 gene local release system. METHODS: Test one: Twenty-four Kunming mice were used. The nanobone putty + hBMP2 plasmid was injected into the right thigh muscle pouches of the mice (experiment side). The nanobone putty + blank plasmid or nanobone putty was injected into the left thigh muscle pouches of 12 mice (control side 1), whereas nanobone putty into the left thigh muscle pouches of remaining 12 mice (control side 2). Test two: A total of 54 New Zealand rabbits were used. Bilateral 15 mm radial defects were made in forty-eight rabbits. These rabbits were randomly divided into three groups and implanted with nanobone putty + hBMP2 plasmid, putty + blank plasmid, and nanobone putty, respectively. Other 6 rabbits with left radial defects served as blank controls. MAIN OUTCOME MEASURES: Test one: The expression of hBMP2 and ectopic bone formation were evaluated by radiography, molecular biology and histomorphology. Test two: Radial specimen was tested by imaging detection, morphology and biomechanics measurement. RESULTS: Test one: The tissue from the experimental side of the mice expressed hBMP2 at postoperative 2 and 4 weeks. Obvious cartilage and island-distributed immature bone formation in implants of the experiment side were observed at 2 weeks after operation, and massive mature bone observed at 4 weeks. Newly formed bone had fused and formed mature lamellar bone and trabecularism. No bone formation was observed in the control side of the mice. The alkaline phosphatase activity in the experiment side of the mice was higher than that in the control side (P < 0.01). Test two: The defects in blank controls were not healed at postoperative 12 weeks, whereas bone defect in other groups was all healed. The alkaline phosphatase level, new bone formation rate and mechanical strength of nanobone putty + hBMP2 plasmid group were A was significantly higher than those of putty + blank plasmid group and nanobone putty group (P < 0.05). CONCLUSION: By means of histology, imaging and biological mechanics, the hBMP2 gene can activate nanobone putty exhibit osteoinductive ability, and has a better bone defect repair capability than nanobone putty used only regarding the bone formation rate, quality and mechanical strength.