构建大段组织工程骨的新型生物反应器的设计与制造

目的:设计、制造一种新的灌注式生物反应器,专门用于高效地构建大体积、β-磷酸三钙组织工程骨。方法:在普通模式灌注生物反应器的灌流室内生成间断性低压环境(-0.01 mpa,0.5 Hz),用材料色素颗粒洗脱实验进行验证后,将复合兔骨髓间充质干细胞的大段、管状β-磷酸三钙材料分别在静态、反应器内常压灌注和间断低压灌注三种环境下培养4周。期间收集培养液检测葡萄糖日耗量、细胞活力(MTT比色法)、碱性磷酸酶比活性、骨桥蛋白水平,并进行硬组织切片检查。结果:色素颗粒洗脱实验证明,间断性低压可以改善低流量液流在材料内的分布;在培养2周和4周时,负压灌注组日均葡萄糖消耗量和细胞活力均显著高于常压灌注组:(t=20.254 P<0.05,t=64.794 P<0.05)及(t=17.586 P<0.05,t=7.583 P<0.05);碱性磷酸酶(ALP)比活性测定和骨桥蛋白水平(OPN)反映间断低压灌注组中骨髓间充质细胞向成骨细胞分化效率更高,但高峰相晚于常压灌注组和静态培养组;在间断低压灌注组中材料深部的占孔率最高,并且分布更均匀。结论:此新型灌注式生物反应器适用于构建大体积、特殊构型组织工程骨;其高效的促进细胞增殖效应可减少初始复合的种子细胞数量,缩短构建周期。

Design and Building of A Novel Perfusion Bioreactor for Large Volume Engineering Bone

Objective: To elevate the effectiveness in fabricating a β-TCP tissue engineering bone with large volume,a novel custom-designed perfusion bioreactor was built.Methods: A normal perfusion bioreactor was fabricated first,then a system contain a mini-pump and digital controller was added to the chamber to produce intermit negative pressure(-0.01 mpa,0.5 Hz).The coloring particle flushing test under atmospheric pressure or negative pressure with either high or low perfusion rate was applied to testify the efficiency.Then the rabbit bone mesenchymal stem cell was seeded in the scaffold and cultured under static condition,atmospheric pressure or intermit negative pressure for 4 weekes.The cell proliferation and distribution were examined by daily glucose consumption,the cell viability and undecalcified histological study,the osteogenic differentiation was examined by alkaline phosphatase activity and osteopotin concentration.Results: In the negative pressure group the most homogenous layer composed of cells and mineralized matrix throughout the whole scaffold was observe by histological study,the cell/pore rate at 4-week was significant higher than the other two groups(P<0.05),the daily glucose consumption increased significantly during the culture(P<0.05),the cell viability was also the highest than the other two groups at the end of culture(P<0.05),alkaline phosphatase activity and osteopotin concentration indicated the cell’s osteogenic differentiation of the intermit negative pressure group is most effectively.Conclusion: These results manifested the feasibility and benefit of perfusion culture in conjunction with intermit negative pressure;the remarkable efficiency in cell proliferation can reduce the amount of cell originally seeded in the porous scaffold,so reduce the cost and time of fabricating engineering bone.