双基因转染犬骨髓间充质干细胞复合HA/ZrO2生物材料新型组织工程骨的构建及其体外成骨能力的研究

The construction of double gene-modified dog bone marrow mesenchymal stem cells tissue engineered bone with Zirconia-Hydroxyapatite biological materials and its osteogenesis ability in vitro

目的：构建骨形态发生蛋白2（BMP-2）、血管内皮细胞生长因子165（VEGF165）双基因修饰的骨髓间充质干细胞（BMSCs）复合羟基磷灰石复合二氧化锆（HA/ ZrO2）生物材料的新型组织工程骨,并观察该组织工程骨在体外的成骨能力。方法采用有机泡沫作为模版,干铺烧制法制备新型的蜂窝状 HA/ ZrO2梯度生物材料,电镜观察新型生物材料的表面特性,生物力学试验机检测其力学性能。采取1岁龄健康 beagle 犬骨髓分离原代 BMSCs 进行培养,建立双基因修饰的 BMSCs 复合蜂窝状 HA/ ZrO2梯度生物材料的共培养体,构建新型组织工程骨。实验分为4组：未转染组,只转染BMP-2（BMP-2组）和 VEGF165（VEGF165组）单一目的基因的 BMSCs,以及转染 BMP-2、VEGF165共基因慢病毒的 BMSCs 组（BMP-2＋ VEGF165组）。显微镜下观察细胞在支架材料上的生长情况,用碱性磷酸酶染色检测各组细胞成骨分化能力,免疫组织化学染色检测其成骨细胞特异性蛋白骨Ⅰ型胶原及骨钙素的分泌。结果新型材料电镜下其表面整体呈多孔状,孔径125~550μm,各孔之间存在缝隙联结；其平均抗弯强度为812.25 MPa,最高可达987.12 MPa；共培养体建立后扫描电镜观察转染后的 BMSCs 在支架材料上黏附生长状况良好,双基因联合转染组细胞分泌基质旺盛；BMP-2＋VEGF165组细胞碱性磷酸酶活性检测明显高于其他各组（F =1029.398,P 〈0.01）,免疫组织化学染色在不同阶段发现成骨细胞早晚期分泌的骨Ⅰ型胶原及骨钙素特异性蛋白。结论新型的蜂窝状HA/ ZrO2梯度生物材料是一种合适种子细胞生长的支架材料,并且其力学满足人体四肢承重骨的需要；VEGF165、BMP-2双基因转染 BMSCs 后具有协同作用,能够促进其在体外的成骨分化。

Objective To establish a new tissue engineering bone with Zirconia-Hydroxyapatite （HA/ ZrO2 ） composite bioceramic as scaffold, combined with dog bone marrow mesenchymal stem cells （BMSCs） coexpressed with vascular endothelial growth factor 165 （ VEGF165） and bone morphogenetic protein-2（ BMP-2） by lentiviral transfection and observe its osteogenesis ability in vitro. Methods The HA/ ZrO2 graded composite was synthesized by dry-laying sintering method with polymer foam template. Then made the honeycomb HA/ ZrO2 graded composite as scaffold with VEGF165 / BMP-2 double gene-modified BMSCs construction a new tissue engineering bone. BMSCs of each group were collected and seeded on the HA/ ZrO2 graded composite, the scaffolds combining BMSCs were scanning electron microscopy observation after cell seeding, and Alkaline phosphatase activity （ALP） staining as well as ALP activity and immunohistochemistry assay were performed after cell seeding. Results The surface of the new tissue engineering bone was porous structure and the pore size was from 125 - 550 μm. The averagenbsp;compressive strength was 812. 25 MPa and the maximum strength was 987. 12 MPa. After seeding BMSCs onto the scaffolds, SEM observation showed that MSCs grew and proliferated well. The ALP activity observed in the co-transfection group was higher than that of groups（F = 1 029. 398, P 〈 0. 01）, collagenaseⅠand osteocalcin which were osteoblast secrete proteins could be observed in co-transfection group by immunohistochemistry assay. Conclusions The honeycomb HA/ ZrO2 graded composite was a good scaffold which was suitable for seed cells proliferation, and the combined use of VEGF165 and BMP-2 gene can produce better osteogenesis for BMSCs in vitro than each single gene alone.