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背景:干细胞的分化潜能与培养条件有密切关系,改变支架材料的表面特性,三维结构,增加生长因子均可实现对干细胞增殖分化的控制。目的:制备适合骨髓间充质干细胞附着生长的、具有最佳孔隙率和孔隙结构的药物缓释组织工程支架——三维大孔支架,提供能促进多能干细胞生长的微环境。设计:重复测量设计。单位:广州中医药大学基础医学院解剖教研室。材料:实验所用健康成年SD大鼠由广州中医药大学实验动物中心提供。壳聚糖、碱性成纤维细胞生长因子购自Sigma公司。方法:实验于2003-03/2006-12主要在广州中医药大学基础医学院解剖教研室完成。采用冷冻干燥的方法,用不同比例的壳聚糖-碱性成纤维细胞生长因子-明胶依次混匀,通过控制冷冻、复温和干燥时间处理使其具有最佳孔隙率和孔结构,制备具缓释碱性成纤维细胞生长因子功能的三维大孔支架。取SD大鼠股骨和胫骨骨髓,分离、培养骨髓间充质干细胞并移植于缓释碱性成纤维细胞生长因子的三维大孔支架上进行三维培养,与无碱性成纤维细胞生长因子的支架对照。实验过程中对动物的处置符合动物伦理学标准。主要观察指标:用ELISA和扫描电镜观察支架的三维结构和缓释性能,用苏木精-伊红染色、MTT、细胞计数及扫描电镜方法观察缓释碱性成纤维细胞生长因子的三维大孔支架对骨髓间充质干细胞生长状态和细胞活力的影响。结果:含有碱性成纤维细胞生长因子的三维大孔支架有碱性成纤维细胞生长因子缓释性能,孔隙尺寸与不含碱性成纤维细胞生长因子的支架三维结构相比,差异无显著性意义(P>0.05)。含有碱性成纤维细胞生长因子的三维大孔支架能提高在支架上立体培养的骨髓间充质干细胞存活率,促进骨髓间充质干细胞黏附、增殖和活力,与不含碱性成纤维细胞生长因子的支架相比,差异有显著性意义(P<0.05)。结论:含有碱性成纤维细胞生长因子的三维大孔支架能缓释碱性成纤维细胞生长因子,有利于在支架上立体培养的骨髓间充质干细胞存活,为其在组织工程中的应用打下基础。
BACKGROUND: The differentiation potential of stem cells is closely related to the culture conditions. Controlling the proliferation and differentiation of stem cells can be achieved by changing the surface characteristics of the scaffold material, three-dimensional structure and increasing growth factor. OBJECTIVE: To prepare three-dimensional macroporous scaffolds for drug-loaded tissue engineering scaffolds with the best porosity and pore structure for bone marrow mesenchymal stem cell attachment and growth, and to provide a microenvironment capable of promoting the growth of pluripotent stem cells. Design: repeat measurement design. Unit: Department of Anatomy, School of Basic Medicine, Guangzhou University of Chinese Medicine. MATERIALS: The healthy adult SD rats used in the experiment were provided by Experimental Animal Center of Guangzhou University of Chinese Medicine. Chitosan and basic fibroblast growth factor were purchased from Sigma Company. Methods: The experiment was performed at Department of Anatomy, School of Basic Medicine, Guangzhou University of Traditional Chinese Medicine from March 2003 to December 2006. Using freeze-drying method, with different proportions of chitosan - basic fibroblast growth factor - gelatin followed by mixing, by controlling the freezing, rewarming and drying time to make it has the best porosity and pore structure, preparation of slow Three-dimensional macroporous scaffolds for the release of basic fibroblast growth factor. Bone marrow of the femur and tibia of SD rats was taken, bone marrow mesenchymal stem cells were isolated and cultured, and then were transplanted into the three-dimensional macroporous scaffold with sustained release of basic fibroblast growth factor for three-dimensional culture. The scaffold with basic fibroblast growth factor Control. The handling of animals during the experiment is in line with animal ethics standards. MAIN OUTCOME MEASURES: The three-dimensional structure and sustained-release properties of scaffolds were observed by ELISA and scanning electron microscopy. The three-dimensional macropores of basic fibroblast growth factor were observed by hematoxylin-eosin staining, MTT, cell counting and scanning electron microscopy Effect of scaffold on the growth status and cell viability of bone marrow mesenchymal stem cells. RESULTS: The three-dimensional macroporous scaffolds with basic fibroblast growth factor had the sustained-release properties of basic fibroblast growth factor. The pore size was not significantly different from the three-dimensional scaffold with basic fibroblast growth factor Significance (P> 0.05). Three-dimensional macroporous scaffolds containing basic fibroblast growth factor can enhance the survival rate of stereospecifically cultured bone marrow mesenchymal stem cells, promote the adhesion, proliferation and viability of bone marrow mesenchymal stem cells, and do not contain basic fibroblast growth Compared with the scaffolds, the difference was significant (P <0.05). CONCLUSION: Three-dimensional macroporous scaffolds containing basic fibroblast growth factor can delay the release of basic fibroblast growth factor and promote the survival of bone marrow-derived mesenchymal stem cells on the scaffolds, laying the foundation for its application in tissue engineering basis.