pcDNA3.1-VEGF165转染BMSCs构建组织工程骨修复骨缺损的实验研究

目的：依据组织工程的原则，首先将pcDNA3.1-血管内皮生长因子（vascular endothelial growth factor, VEGF）165质粒转染至骨髓间充质干细胞（bone marrow stromal cells ，BMSCs），然后将其与纳米羟基磷灰（nano-hydroxyapatite，n-HA) /羧甲基壳聚糖（carboxymethyl chitosan，CMCS）支架材料复合构建组织工程骨，观察该组织工程骨在兔桡骨骨缺损模型处的成骨能力及降解速度。 方法：采用化学共沉淀法制备纳米羟基磷灰石，以京尼平（Genipin）为交联剂，通过粒子沥滤结合冷冻干燥工艺制备纳米羟基磷灰石/羧甲基壳聚糖复合支架材料。采用扫描电镜观察其微观结构；三点弯曲法测试其力学性能；通过细胞形态分析测试其体外细胞毒性；激光共聚焦显微镜观察其荧光性能。根据设计的引物反转录合成VEGF165,pcDNA片段，构建pcDNA3.1-VEGF165质粒。骨髓间充质干细胞取自兔骨髓，培养、传代，采用电转法将 pcDNA3.1- VEGF165转至 BMSCs 然后与 n-HA/CMCS支架材料复合构建组织工程骨；建立动物模型将其植入兔桡侧，通过大体观察，X射线，HE染色及三点弯曲强度评价其成骨能力及降解速度。 结果：京尼平交联的n-HA/CMCS支架材料微观结构、力学性能与天然松质骨相似，可满足支架材料的要求；与戊二醛做交联剂所得支架相比，本研究所得支架生物毒性更小，更利于细胞的吸附与增殖，并且使用京尼平交联是支架材料具有自发荧光特征；成功构建出 pcDNA3．1-VEGFl65质粒，将其转至 BMSCs，并与n-HA/CMCS 复合构建组织工程骨。大体标本观察表明复合材料植入骨缺损处可见骨缺损处愈合良好，植入材料大部分已转化为自体骨组织仅少许未降解。X射线观察显示实验侧可见明显骨痂生成，骨髓腔形成，骨髓腔基本贯通。HE染色结果表明实验组骨缺损愈合，皮质骨形成，皮质主要由编织骨组成，部分由新生的骨单位构成，髓腔再通。 结论： pcDNA3.1-VEGF165转染BMSCs复合n-HA/CMCS支架材料具有生物相容性好、无毒副作用、促进局部微血管形成、加快骨缺损修复的作用，其降解速度与骨生长速度基本匹配；使用京尼平交联的支架不同于其他骨组织工程支架材料，其具有自发荧光特征。通过激光共聚焦显微镜可以清楚地观察到支架和细胞的界面，细胞的粘附特征，以及支架的微结构和降解时微结构特征变化。综上可知本研究所得复合骨是一种潜在的性能优良的骨修复材料。

Transfect BMSCs with pcDNA3.1-VEGF to Construct Bone Tissue Engineering in Defect Repair

Background: We previously showed that nano- hydroxyapatite/carboxymethyl chitosan (n-Ha/CMCS) displayed excellent mechanical properties, good degradation rates and exceptional biocompatibility, with negligible toxicity. The aim of this study was to determine the effect of the same composite with vascular endothelial growth factor (VEGF)-transfected bone marrow stromal cells (BMSCs) in a rabbit radial defect model. Methods: The nano-hydroxyapatite was produced through co-precipitation. The n-HA/CMCS scaffold was produced by particle filtration and lyophilization followed by genipin crosslinking. Total RNA from rabbit bone was reverse-transcribed to synthesize VEGF165-pcDNA3.1 that was transfected into the BMSCs. The composite was implanted into a rabbit radial defect model, and the osteogenic activity examined by gross morphology, X-ray analysis and hematoxylin and eosin (HE) staining. Results: The microstructure and mechanical property of the n-HA/CMCS scaffold resembled natural cancellous bone. Compared with glutaric dialdehyde crosslinked scaffolds, the genipin crosslinked scaffold is less toxic, and displays a higher capacity to promote cell adhesion and proliferation. Spontaneous fluorescence of the composite permitted visualization the composite-bone interface and the adhesion behavior of cells on the scaffold under laser scanning confocal microscopy. The scaffold with VEGF-transfected BMSC bridged the bony defect and promoted healing, with most of the implanted material being replaced by natural bone over time with little residual implant. Using X-ray, we noted obvious callus formation and recanalization of the bone marrow cavity. Furthermore, HE stained sections showed new cortical bone formation. Conclusions: The n-HA/CMCS scaffold composite with VEGF-trasnfected BMSCs is biocompatible, nontoxic, promotes the infiltration and formation of the microcirculation, and stimulates bone defect repair. Furthermore, the degradation rate of the composite matched that of growing bone. Overall, this composite material is a potentially useful tool for bone defect repair.