Expansion Of Osteoblasts And Fabrication Of Engineered Bone In Bioreactor

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...