【作者】 宋克东；

【导师】 刘天庆； 崔占峰；

【作者基本信息】 大连理工大学 ， 化学工程， 2006， 博士

【摘要】 骨组织工程(Bone tissue engineering)概念的提出为解决由于创伤、肿瘤等各种因素引起的骨缺损等疾病提供了一种崭新的方法和思路，已成为治疗骨科疾病的新突破点。生物反应器系统既可以揭示在三维培养环境中细胞功能的基本机制，又有提高工程化组织质量的功能。旋转壁式生物反应器(Rotating wall vessel bioreactor，RWVB)具有剪切力低、培养的细胞之间有三维联系的机会、极高的溶氧效率、营养物质浓度梯度低等特点，是一种非常理想的骨组织工程用三维培养系统。 本研究使用Sprague-Dawley(SD)大鼠和新西兰兔的成骨细胞(Osteoblasts，OBs)作为骨组织工程的种子细胞，对其原代、传代培养的方法和其特异性和形态学等生物学性能进行了检测，确定所培养的细胞是成骨细胞，并通过纯化确保没有其他细胞的污染，可为后续的实验提供数量充足的骨组织工程种子细胞，也为实验的准确性和可重复性提供了保证。 在相同条件下分别在培养瓶、转瓶(Spinner flask)和RWVB中使用微载体悬浮培养法进行了SD大鼠成骨细胞扩增培养的研究。培养7天后，经倒置相差显微镜、扫描电镜(Scanning electron microscope，SEM)、苏木精-伊红染色(Hematoxylin-eosin，HE)、碱性磷酸酶(Alkaline phosphatase，ALP)、MTT、von-Kossa染色和茜素红染色等生物学性能检测，结果显示RWVB中扩增的细胞能最好地保持成骨细胞的各种生物学特征，可以作为骨组织工程的种子细胞。且当微载体的浓度为10mg／ml、成骨细胞的接种密度为5×10~4 cells／ml时，细胞在RWVB中可以获得最佳的扩增效果，每代可以扩增十倍以上，明显高于其它培养方式。 使用FLUENT软件模拟计算了旋转壁式生物反应器内的二维流场，计算了在反应器内进行细胞扩增和工程化组织构建时培养室内的动压、总压、剪切力和流体速度的分布，具体分析了当反应器的转速和旋转方向、中空纤维膜的径向位置和直径等发生改变时膜壁面各点的受力情况和流场分布。此外，还详细分析了当反应器的转速和旋转方向以及细胞-支架材料构建物的径向位置发生改变时构建物壁面各点的受力和流场分布情况。模拟结果显示在旋转壁式生物反应器内不同位置的中空纤维膜壁面和细胞-支架材料构建物壁面各点的动压、流体速度和剪切力随着反应器的旋转而发生周期性的变化，培养物可以获得周期性的应力刺激；反应器内流体的剪切力较低。 在上述理论分析的基础上，设计了旋转壁式中空纤维膜生物反应器(Rotating wall hollow-fiber embrane bioreactor，RWHMB)，利用中空纤维膜作为成骨细胞的载体，以1×10~5 cells／ml接种密度在该反应器内对其进行大规模扩增，并在静态环境中以相同条件对照培养。每隔12 hr对细胞进行取样，培养5天后对细胞进行收获。对所扩增的细胞分别进行扫描电镜(SEM)观察、碱性磷酸酶(ALP)染色、钙化结节染色等生物学更多还原

【Abstract】 The concept of bone tissue engineering provides a fire-new method and idea to repair bone defects caused by some accidents and diseases, such as trauma and tumour, and has become a novel breakthrough to treat these orthopedic diseases. Bioreactor systems provide both the technological means to reveal fundamental mechanisms of cell function in a three-dimensional (3D) environment, and the function to improve the quality of engineered tissues. Rotating wall vessel bioreactor (RWVB) has many virtues, such as uniform velocity and concentration fields, low shear stress acting on cells, great deals of 3D communications among cells. It is thus an ideal 3D culture system for engineered bone tissue.This research used the osteoblasts from Sprague-Dawley (SD) rats and Zelanian rabbit as the seed cells of the bone tissue engineering. The methods of primary and passaged cultures were tested and their biological properties and morphology were assayed. The cultured cells were purified and identified to be osteoblasts. The expanded osteoblasts can provide sufficient seed cells of bone tissue engineering for later researches and ensure the experimental veracity.Large-scale expansion of osteoblasts was studied by using microcarrier in suspension culture in a RWVB, spinner flasks and T-flasks under the same conditions. After 7 days, the cells were evaluated with inverted microscope, scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodules formation. The results show that the expanded cells in RWVB can retain the best biological properties of osteoblasts and be regarded as seed cells of bone tissue engineering. The cells can attain the best expanded results in RWVB when the microcarrier density is 10 mg/ml and the cell inoculated density is 5 × 10~4 cells/ml. And the cellular expansion multiple in the RWVB was more than ten, which was considerably higher than those in the other culture vessels.The two-dimensional flow field of rotating wall vessel bioreactor was simulated and calculated with Fluent software. The distributions of dynamic and total pressure, shear stress and velocity within the chamber were calculated when hollow fiber membrane assembly or the engineered bone tissues with different size were installed in different positions in the vessel. The results showed that the dynamic pressure, velocity and shear stress around the surface of a hollow-fiber membrane and cell-scaffold construct at different locations in RWVB were changed periodically, which could result in the periodical stress stimulation for the cultured objects. Additionally, the shear stress in the bioreactor is relatively low and the更多还原