Bone marrow mesenchymal stem cells co-transfected by lentivirus-mediated bone morphogenetic protein 2 and vascular endothelial growth factor 165 in combination with demineralized bone matrix for treating rabbit femoral head necrosis

doi:10.3969/j.issn.2095-4344.1828

Abstract

Abstract:

BACKGROUND: Bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor 165 (VEGF-165) play important roles in bone repair. It is of great significance to treat bone injury by lentivirus-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix.
OBJECTIVE: To observe the therapeutic effects of bone marrow mesenchymal stem cells with lentivirus-mediated BMP-2 and VEGF-165 co-transfection and demineralized bone matrix in a rabbit model of osteonecrosis of the femoral head, so as to explore the feasibility of dual genes in the bone repair.
METHODS: Femoral head necrosis model was prepared by liquid nitrogen freezing combined with core decompression. Rabbit models successfully prepared were divided into three groups (n=12/group): non-transfection group, treated with bone marrow mesenchymal stem cells combined with demineralized bone matrix; single gene transfection group, treated with BMP-2-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix; dual-gene transfection group, treated with BMP-2 and VEGF-165 co-transfected bone marrow mesenchymal stem cells combined with demineralized bone matrix. Four rabbits from each group were sacrificed at 3, 6, and 9 weeks postoperatively. Western blot was used to detect the expression of BMP-2 protein in the femoral head of each group. Hematoxylin-eosin staining was used to observe the repair of femoral head necrosis in each group. The study protocol was approved by the Animal Ethics Committee of Guilin Medical University.
RESULTS AND CONCLUSION: BMP-2 positively expressed in all groups at 3, 6, and 9 weeks postoperatively, and the positive expression of target protein was significantly higher in the two transfection groups than the non-transfection group (P < 0.05). The positive expression of BMP-2 in the two transfection groups showed an increasing trend at three time periods. At 3 weeks postoperatively, there was no difference in the BMP-2 expression between the two transfection groups, while the BMP-2 expression was significantly increased in the dual-gene transfection group compared with the single gene transfection group at 6 and 9 weeks postoperatively (P > 0.05). In the dual gene transfection group, new osteophytes could fill in the defect of the femoral head at 9 weeks postoperatively, while in the other two groups, new osteophytes formed were not enough to fill in the defect. Hematoxylin-eosin staining indicated that primary trabeculae were formed in the non-transfection group, but there were more necrotic cells and less bone trabeculae. In the single gene transfection group, a large amount of trabeculae formed with good shape, some of which however were inferior to normal bone tissues because of fractures. In the dual gene transfection group, a mass of mature and dense trabeculae were formed and had normal shape. To conclude, lentivirus-mediated BMP-2 and VEGF-165 co-transfection is superior to single BMP-2 transfection in the combination of bone marrow mesenchymal stem cells and demineralized bone matrix for treating femoral head necrosis. Moreover, in vivo expression of target protein persists for a long time.

Key words: lentivirus, bone marrow mesenchymal stem cells, bone morphogenetic protein 2, vascular endothelial growth factor 165, demineralized bone matrix, double gene transfection, femoral head necrosis, National Natural Science Foundation of China

CLC Number:

Ma Yuegang, Li Qiang, Tao Xuan, Zhou Zhenjie, Zhu Lunjing, Duan Jiangtao. Bone marrow mesenchymal stem cells co-transfected by lentivirus-mediated bone morphogenetic protein 2 and vascular endothelial growth factor 165 in combination with demineralized bone matrix for treating rabbit femoral head necrosis[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(33): 5275-5280.

Figures/Tables 1

2.1 复苏后骨髓间充质干细胞的形态复苏后7 d即长至80%-90%培养皿体积，呈漩涡、鱼群状分布，生长状态良好，见图1。 2.2 病毒转染骨髓间充质干细胞的效率转染后7 d在普通倒置显微镜下观察可见细胞生长状态良好，倒置荧光显微镜下显示细胞转染效率在90%以上，见图2，证明BMP-2及VEGF-165基因已成功转入细胞中。 2.3 转染骨髓间充质干细胞复合脱钙松质骨支架观察结果在细胞和支架复合48 h后，倒置显微镜下见脱钙松质骨具有良好的三维结构与孔隙，见图3A；置于荧光显微镜下观察，双基因BMP-2/VEGF-165转染组可见支架发出明亮红绿亮光，伴有红绿点状亮点，见图3B；单基因BMP-2转染组可见支架发出明亮绿色亮光，伴有绿色点状亮点，见图3C。 2.4 Western blot 检测目的蛋白BMP-2表达术后3，6，9周，3组BMP-2蛋白均呈阳性表达，但单基因转染组、双基因转染组表达量明显多于未转染组，差异有显著性意义(P < 0.05)。随着时间延长，单基因转染组、双基因转染组BMP-2表达量逐渐增加。术后3周，单基因转染组、双基因转染组BMP-2表达量差异无显著性意义(P > 0.05)，术后6，9周时，双基因转染组BMP-2表达量较单基因转染组明显升高(P < 0.05)，见图4。灰度分析显示：双基因转染组在术后3，6，9周时目的蛋白BMP-2/内参GAPDH表达量比值分别为0.481±0.025，0. 612±0. 012，0. 837±0.018，两两比较差异均有显著性意义(P < 0.05)，上述结果证明慢病毒介导BMP-2和VEGF-165双基因转染骨髓间充质干细胞的目的蛋白BMP-2能在体内稳定表达，且随着时间的推移，此蛋白表达能力逐渐增强，效果好于单基因BMP-2转染形式。 2.5 苏木精-伊红染色结果股骨头正常结构：骨小梁排列连续、完整，与周边纤维组织分辨清晰，伊红染色后呈红色网状结构，成骨细胞形态正常，胞核清晰完整，苏木精染色后呈蓝紫色，见图5A。坏死的骨小梁结构不规则，连续性不完整，网格状结构消失，边缘不整齐，可被苏木精染成蓝紫色。未转染组：术后3周骨小梁较少，呈点状分布，未见连续性，成骨细胞坏死较多，见图5B；术后6周骨小梁仍较少，结构不规则，少许连续性，为初级骨小梁，点状分布，可见成骨反应，见图5C；术后9周时可见纤维组织填满钻孔处，孔隙附近存在成骨反应，向心性生长，坏死组织含量高，存在初级骨小梁，见图5D。单基因转染组：术后3周见钻孔处有少许新生骨痂填充，可见少许初级骨小梁，骨小梁边缘粗糙，形态不规则，连续性较差，骨组织坏死较未转染组少，见呈向心性生长的成骨反应趋势，见图5E；术后6周可见钻孔处新生骨痂增多，但仍不及钻孔范围一半；骨小梁含量增多，形态较前好，呈网状，有部分连续性，可见明显成骨反应，见图5F；术后9周见钻孔处骨痂超过孔深一半，但仍有部分孔隙未被填满；骨小梁明显，形态较好，呈网状分布，较前致密，连续性好，仅有部分中断，见图5G，但仍不及正常骨组织。双基因转染组：术后3周变化与单基因转染组相似，见图5H；术后6周即可见新生骨痂超过孔深一半，孔隙内可见毛细血管长入，未见炎症反应，可见大量初级骨小梁，形态规则，网状，连续性较好，少许中断，见图5I；术后9周可见骨痂填满孔隙，填充区充满成熟骨小梁，骨小梁致密饱满，形态较好，丝瓜络样，连续性好，无中断，骨小梁形态及分布与正常骨组织无明显差异；表面成骨细胞生长状态好，数量多且清晰可见，见图5J。"

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