慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的实验研究

目的：探究慢病毒介导BMP-2过表达质粒转染骨髓间充质干细胞联合丝素蛋白支架向成骨细胞转化的作用效果。方法：构建慢病毒BMP-2过表达载体，培养骨髓间充质干细胞，构建细胞核支架的联合培养体系，体外实验利用茜素红染色和碱性磷酸酶染色检测骨髓间充质干细胞的成骨转化。选择10只新西兰大白兔，体重3.2～4.5 kg，平均3.9 kg；年龄（2.89±0.45）岁；使用口腔钻在兔子胫骨钻孔（长度5 mm、宽度2 mm、深度3 mm的锥形胫骨缺损）构建兔子胫骨骨缺损模型，HE染色观察动物模型内骨缺损的修复。实验组造模后植入丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物，阴性对照组造模后植入丝素蛋白支架+未转染骨髓间充质干细胞复合物。结果：实验组（丝素蛋白支架+转染BMP-2过表达载体骨髓间充质干细胞复合物）中支架表面黏附的细胞与对照组（丝素蛋白支架+未转染骨髓间充质干细胞）相比，细胞数明显增多。实验组细胞外基质分泌与对照组相比，支架间细胞外基质含量明显增多。对照组支架表面元素EDX分析显示钙离子含量为0.22%，实验组支架表面元素EDX分析显示钙离子含量为0.86%，可见实验组诱导钙离子形成的能力要比对照组强。钙结节茜素红染色结果显示，对照组肉眼观无明显变化，镜下观察可见少量钙结节点。实验组肉眼观可见明显红色区域染色，镜下观察可见大量钙结节点。碱性磷酸酶染色结果显示，对照组肉眼观无明显变化，镜下观察未见明显变化。实验组肉眼观可见紫色区域染色，镜下观察可见ALP染色呈强阳性。丝素蛋白支架与骨髓间充质干细胞联合培养体系可以对软骨缺损有较好的修复作用，转染BMP-2骨髓间充质干细胞后修复作用明显优于未转染组。HE染色结果显示，对照组炎性细胞减少，支架略有消失。实验组炎性细胞明显减少，支架消失，血管生成。结论：慢病毒介导BMP-2过表达质粒可以促进BMSC向骨细胞的分化作用，并且分泌更多的含Ca²⁺成分的细胞外基质，从而发挥其促进骨缺损修复的作用。

Lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells combined with silk fibroin scaffold for osteoblast transformation

Objective:To explore the effect of lentivirus-mediated BMP-2 overexpression plasmid transfection into bone marrow mesenchymal stem cells and silk fibroin scaffold on osteoblast transformation.Methods:The lentivirus BMP-2 overexpression vector was constructed,bone marrow mesenchymal stem cells were cultured,and the combined culture system of nuclear scaffolds was constructed. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic transformation of bone marrow mesenchymal stem cells in vitro. Ten New Zealand white rabbits,weighing 3.2 to 4.5 kg(averaging 3.9 kg),aged (2.89±0.45) years old,were selected to construct the rabbit tibial defect model by drilling a conical tibial defect (5 mm in length,2 mm in width and 3 mm in depth) with an oral drill. The repair of the tibial defect in the animal model was observed by HE staining. The experimental group was implanted with silk fibroin scaffold + BMP-2 overexpression vector bone marrow mesenchymal stem cell complex,while the negative control group was implanted with silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cell complex.Results:Compared with the control group(silk fibroin scaffold+non-transfected bone marrow mesenchymal stem cells),the number of adherent cells on the surface of the scaffold in the experimental group(silk fibroin scaffold+transfected BMP-2 overexpression vector BMP-2 complex) increased significantly. Compared with the control group,the ECM secretion in the experimental group increased significantly. EDX analysis showed that the content of calcium ion was 0.22% in the control group and 0.86% in the experimental group,which showed that the ability of inducing calcium ion formation in the experimental group was stronger than that in the control group. Alizarin red staining of calcium nodules showed that there was no obvious change in the naked eye of the control group,and a small amount of calcium nodules could be seen under the microscope. In the experimental group,obvious red area staining was observed by naked eye,and a large number of calcium nodules were observed by microscopy. The results of alkaline phosphatase staining showed that there was no obvious change in the naked eye of the control group,and no obvious change in the microscopic observation. In the experimental group,purple area staining was observed by naked eyes,and ALP staining was strongly positive by microscopy. The combined culture system of silk fibroin scaffold and bone marrow mesenchymal stem cells can repair cartilage defects. The repair effect of BMP-2 bone marrow mesenchymal stem cells after transfection is obviously better than that of non-transfection group. HE staining showed that inflammatory cells decreased and scaffolds disappeared slightly in the control group. In the experimental group,inflammatory cells were significantly reduced,scaffolds disappeared and angiogenesis was observed.Conclusion:Lentivirus-mediated BMP-2 overexpression plasmid can promote BMSC to differentiate into osteocytes and secrete more extracellular matrix containing Ca²⁺ to promote bone defect repair.