新型旋转壁式生物反应器内三维组织工程骨的构建

利用微载体悬浮培养法将成骨细胞在旋转壁式生物反应器内进行大规模扩增，并检测细胞的组织形态和生物功能.然后以此作为种子细胞，分别以2×10⁶个/ml和1×10⁶个/ml两种密度接种到支架材料上，于旋转壁式生物反应器(RWV)内进行三维组织工程骨的构建.并将所构建的骨组织分别进行倒置显微镜(inverted microscope)、扫描电镜(SEM)、碱性磷酸酶(ALP)、矿化结构和AO/EB双重荧光染色等生物学性能检测，以及对培养过程的营养物质代谢情况进行监控和分析.结果表明，在RWV中培养的骨组织生长良好，分泌大量胶原纤维，并有矿化基质和新骨样组织形成. 由上述结果可断定，通过RWV内部流体对流所产生的应力刺激，可提高成骨细胞碱性磷酸酶的活性表达，并加速矿化结节的形成，从而完成成骨细胞的快速增殖与分化以及工程化组织的三维构建.

Three-dimensional Fabrication of Engineered Bone in Rotating Wall Vessel Bioreactor

The osteoblasts were expanded in large-scale by microcarrier suspension culture in rotating wall vessel bioreactor(RWV), then the biological functions of the cells were detected by histomorphometry and the cells were seeded at 2×10⁶ cells/ml and 1×10⁶ cells/ml onto scaffolds respectively to culture for 2 weeks. The biological properties of the fabricated bone tissue were detected by inverted microscope, scanning electron microscope (SEM), alkaline phosphatase (ALP), von-Kossa staining on mineralized nodules and AO/EB fluorescence staining. Meanwhile the cells' metabolism of nutrients was monitored and analyzed during the whole culture process. The bone tissue fabricated in RWV with two different seeded cell densities grew well. Osteoblasts in the scaffolds secreted much collagen fibers and formed mineralized nodules and new osteoid tissue. It can be conclueled that: With the stress stimulation inside the fluid in the RWV, the active expression of ALP can be increased, the formation of mineralized nodules can be accelerated. The rapid proliferation and differentiation of osteoblasts are possible and the three-dimensional fabrication of engineered bone could be realized.