Repair of large-segmental femoral defects using lentivirus-mediated bone morphogenetic protein 2/bone marrow mesenchymal stem cells/demineralized bone matrix

doi:10.3969/j.issn.2095-4344.0462

Abstract

Abstract:

BACKGROUND: Our research team has confirmed that compared to the adenoviral vector, transfection by lentiviral vector to rabbit bone marrow mesenchymal stem cells (BMSCs) is more effective that the expression of enhanced green fluorescent protein (EGFP)/bone morphogenetic protein 2 (BMP-2) can be persistent for a longer term. But further investigations are needed to explore whether BMSCs transfected with hBMP-2 through lentivirus combined with demineralized bone matrix (DBM) can promote bone defect repair.
OBJECTIVE: To evaluate the effect of lentivirus-mediated hBMP-2/BMSCs/DBM (LV-hBMP-2/BMSCs/DBM) on the repair of large-segmental femoral defects and to explore the new treatments for large-segmental femoral defects.
METHODS: Large-segmental bone defect models were made in the right femur of 48 New Zealand white rabbits by cutting the middle femoral bone and steel plate fixation. Then, animal models were randomly divided into four groups (n=12 per group) and implanted with nothing (control), DBM, hBMP-2/DBM, and LV-hBMP-2/BMSCs/DBM. Three rabbits from each group were sacrificed at 2, 4, 8 and 12 weeks after surgery to evaluate the repairing effect of femoral defects through hematoxylin-eosin staining, biomechanical analysis and radiological examination.
RESULTS AND CONCLUSION: X-ray results revealed that osteotylus formed in all the four groups to different extents, and Lane - Sandhu X-ray scores were ranked as follows: control group < DBM group < hBMP-2/DBM group < LV-hBMP-2/BMSCs/DBM group (P < 0.05). Findings from the three-point bending test showed that the maximum load of the LV-hBMP-2/BMSCs/DBM group was significantly higher than that of the hBMP-2/DBM group, but is still lower than that of the normal femur at 8 and 12 weeks after modeling (P < 0.05). Hematoxylin-eosin staining results revealed that a few trabecular bones arranged disorderedly and a large amount of fibrous tissues in the control group; the DBM scaffold was basically degraded in the DBM group presenting with partially disordered trabecular bones and a large amount of fibrous tissues; the trabecular bones in the bone defect area were basically connected into line to start the shaping of the cortical bone, and recanalization of the medullary cavity was insignificant in the hBMP-2/DBM group; new cortical bone formed in the bone defect area and the medullary cavity was recanalized in the LV-hBMP-2/BMSCs/DBM group. These findings indicate that LV-hBMP-2/BMSCs/DBM can produce a large amount of calluses, promote formation of new cortical bone, and promote bone conduction, bone induction and osteogenesis after implantation into the bone defect; and this material has good repairing effect on large-segmental femoral defects of rabbits.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Lentivirus, Bone Morphogenetic Proteins, Mesenchymal Stem Cells, Tissue Engineering

CLC Number:

R394.2

Tao Xuan, Li Qiang, Li Shi-peng, Shi Zheng-song, Zhou Zhen-jie. Repair of large-segmental femoral defects using lentivirus-mediated bone morphogenetic protein 2/bone marrow mesenchymal stem cells/demineralized bone matrix[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(9): 1338-1343.

Figures/Tables 2

2.1 实验动物数量分析 48只新西兰大白兔均进入结果分析。 2.2 各组兔左侧股骨X射线观察见图2。各组在各时间点的钢板螺钉固定牢固。造模后2周时，A、B、C组在骨缺损区域均只有少量骨痂出现，D组新生骨痂较多，未形成完整骨桥。 4周时，A组骨缺损区域近远端新生骨痂已向中间延伸；B组钢板对侧骨痂密度影较术后2周时增高，骨缺损区域也有新生骨痂；C组骨痂已基本形成骨桥，截骨面稍模糊；D组骨痂已基本形成骨桥，截骨面稍模糊。 8周时，A组骨缺损区域近远端新生骨痂继续向中间延伸，近远端新生骨痂未形成连接；B组骨痂量未见明显变化，截骨面较前模糊；C组骨痂已形成完整骨桥，截骨面已模糊不清；D组骨痂已完全长满骨缺损区域，截骨面模糊。 12周时，A组仅有少量骨痂增加，无明显变化，A组骨缺损最终未能完成修复；B组骨缺损区域骨痂较前明显增多，未形成完整骨桥；B组骨缺损最终未能完成修复；C组骨痂开始塑形，部分多余骨痂被吸收，髓腔基本再通；C组骨缺损修复完成；D组骨痂开始塑形，部分多余骨痂被吸收，髓腔再通；D组骨缺损修复完成。 A、B、C、D组4，8，12周的Lane-Sandhu X射线评分均有显著差异(P < 0.05)，D组2周的Lane-Sandhu X射线评分与A、B、C组相比有明显差异(P < 0.05)，见表1。提示在兔股骨大段缺损修复中慢病毒-hBMP-2/BMSCs/脱钙骨基质效果优于单纯脱钙骨基质和BMSCs/脱钙骨基质。随时间推移，各组Lane-Sandhu X射线评分依次增高，提示骨缺损的修复有一定的时间依赖性。 2.3 各组兔股骨生物力学测试结果骨缺损修复后兔股骨形状不规则，无法建立弹性模量计算模型，故实验只能通过三点弯曲试验测量出最大负载压力。修复兔股骨标本中无骨性连接的也无法完成力学测试。8、12周时C、D组组间及同正常股骨三点弯曲最大负荷比较，差异均有显著性意义(P < 0.05)，见表2。 2.4 兔股骨标本苏木精-伊红染色结果见图3。 A组造模后2周见机化血肿及纤维组织，无明显骨小梁形成；4周时见大量纤维组织增生，少量骨小梁形成；8周骨小梁较前有所增多，但仍然被大量的纤维组织填充；12周可见部分排列紊乱的骨小梁和大量的纤维组。 B组造模后2周大量脱钙骨基质骨架，少量机化血肿及纤维组织，无明显骨小梁形成；4周脱钙骨基质骨架已部分降解，周围纤维组织侵入其中，有少许不规则的骨小梁包绕；8周无形态典型脱钙骨基质骨架，纤维组织侵入较前增多，外层排列紊乱的骨小梁增多；12周无脱钙骨基质，可见较多排列紊乱的骨小梁和部分纤维组织。 C组造模后2周大量脱钙骨基质骨架，少量机化血肿及纤维组织，外层已有幼稚骨小梁形成；4周脱钙骨基质骨架已部分降解，骨小梁之间的连接增多；8周脱钙骨基质骨架基本降解，大量骨小梁形成，骨小梁之间连接较前增多；12周骨小梁已基本连接成线，开始塑形为皮质骨，髓腔再通不显著。 D组造模后2周大量脱钙骨基质骨架，少量机化血肿及纤维组织，外层已有大量幼稚骨小梁形成；4周脱钙骨基质骨架已部分降解，大量新生骨小梁出现，骨小梁之间的连接增多并连接成线；8周脱钙骨基质骨架基本降解，骨缺损区域大量骨小梁形成，骨小梁之间连接较前增多；12周已基本形成新的骨皮质，并已有髓腔再同。股骨正常形态可见皮质骨与髓腔分界清晰，骨细胞分布均匀，细胞基质平行排列有序，可见骨皮质中散在分布数个小血管腔隙。"

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