转染碱性成纤维生长因子基因的骨髓间充质干细胞在珊瑚骨表面生长状况

背景:颌面骨缺损是临床上常遇到的问题,寻找理想的种子细胞同支架材料复合构建组织工程化人工骨成为该类疾病治疗的发展趋势.目的:将转染碱性成纤维生长因子基因的骨髓间充质干细胞与珊瑚骨的复合培养,观察转染碱性成纤维生长因子基凶的骨髓间充质干细胞在珊瑚支架材料上生长状况.设计、时间及单位:骨组织工程实验,于2006-0312008-06 在中山大学口腔医学研究所完成.材料:选用海南省浅海滩产石头状滨珊瑚为原料,将其制成8mm×8mm×2mm的珊瑚人工骨小块.方法:采用密度梯度离心法分离新西兰大白兔骨髓间充质干细胞,采用贴壁筛选法对分离出的骨髓间充质下细胞进行纯化,利用脂质体转染bFGF-pcDNA3到骨髓间充质干细胞.取生长良好的转染碱性成纤维生长因子基因骨髓间充质干细胞和未转染的骨髓间充质干细胞,分别接种于不同珊瑚表面.主要观察指标:MTT法观察细胞-支架联合培养骨髓间充质干细胞的增殖情况和利用扫描电镜观察珊瑚支架上细胞的生长状况.结果:MTT法检测显示细胞-支架联合培养转染组细胞与联合培养未转染组细胞增殖相比差异有显著性意义(P＜0.05),联合培养转染组细胞生长增殖强于未转染组细胞.而联合培养的转染组细胞同单纯培养转染组细胞增殖相比差异无显著性意义(P＞0.05).扫描电镜观察显示复合骨髓间充质干细胞细胞贴附在珊瑚上,并在材料上完全铺展,形态多样,细胞向孔内长入或跨越微孔表面,部分区域有细胞外基质形成.结论:转染碱性成纤维生长因子基因的骨髓间充质干细胞在珊瑚支架材料上生长状况较未转染组好,珊瑚人工骨不影响骨髓间充质干细胞的增殖,可以作为骨髓间充质干细胞支架材料构建组织工程骨.

Growth characteristics of basic fibroblast growth factor gene-transfected bone marrow mesenchymal stem cells seeded on coral skeleton in vitro

BACKGROUND: Jaw defects are common clinically. It is desirable to find ideal seed cells combined with scaffolds to construct tissue engineered jaws for curing these diseases. OBJECTIVE: To investigate the growth characteristics of bone marrow mesenchymal stem cells (BMSCs) transfected with basic fibroblast growth factor (bFGF) gene after seeded on coral scaffold in vitro. DESIGN, TIME AND SETTING: An experimental study of bone tissue engineering was performed in the Research Institute of Stomatology, Sun Yat-sen University between March 2006 and June 2008. MATERIALS: Natural coral from China Hainan bench was made into pieces of 8 mm×8 mm×2 mm. METHODS: BMSCs were isolated from New England rabbits by density gradient centrifugation and then purified by adherent separation. bFGF-pcDNA3 gene was transfected into BMSCs using Lipofectamine TM 2000. bFGF gene-transfected (transfected group) or untransfected (untransfected group)BMSCs were seeded on different coral scaffolds. In addition, bFGF gene-transfected BMSCs were simply cultured but not on the coral scaffold for control (simple culture group). MAIN OUTCOME MEASURES: BMSC proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay and BMSC growth on coral scaffold was observed under the scanning electron microscope. RESULTS: MTT assay showed that the BMSC proliferation rate was significantly higher in the transfected group than in the untransfected group (P < 0.05) and that there was no significant difference in BMSC proliferation between the transfected and simple culture groups (P > 0.05). Scanning electron microscope results displayed that BMSCs adhered to and spread over the coral scaffold, exhibiting various appearances, with some cells had grown into scaffold micropores or spanned micropore surface, and some extracellular matrix secreted by BMSCs were found. CONCLUSION: The transfected group exhibited better growth of BMSCs transfected by bFGF gene than the untransfected group. These findings indicate that coral skeleton does not influence BMSC proliferation and can be used as a scaffold of BMSCs to construct tissue-engineered bone.