灌注培养促进人胚成骨细胞在大体积可控微结构支架内的均匀扩增

flow perfusion culture enhances proliferation of human fetal osteoblasts in large scaffold with controlled architecture

目的 探讨采用灌注式生物反应器和可控微结构支架促进人胚成骨细胞在大体积支架内均匀扩增的可行性和优势.方法 设计灌注式生物反应器,制备大体积可控微结构磷酸三钙支架.将人胚成骨细胞与支架复合后分别行静态培养或灌注培养4、8、16 d.利用D-葡萄糖日消耗量、细胞活力（MTT法）检测,和组织学切片观察、扫描电子显微镜（SEM）观察及X射线能谱（EDX）分析,研究细胞在支架内的扩增、分布及功能情况.结果 葡萄糖日耗量随培养时间延长而升高（F=96.81,P＜0.05）,灌注培养组显著高于静态培养组（F=1848.91,P＜0.05）；细胞活力随培养时间延长而升高（F=125.67,P＜0.05）,灌注培养组显著高于静态培养组（F=1588.15,P＜0.05）.组织学切片及SEM观察均显示静态培养组细胞仅在支架边缘存活,而灌注培养组细胞在遍及支架内部的管道内均有良好增殖,并形成层状的新生组织.EDX分析证实细胞间的类球体基质为磷酸钙结节.结论 灌注培养结合可控微结构支架实现了人胚成骨细胞在大体积支架内的均匀扩增,可控微结构支架可作为深入研究载体内液流调配和细胞行为的有效工具,人胚成骨细胞可作为体外构建大体积骨移植物的种子细胞.

Objective To demonstrate the feasibility and benefits of custom designed perfusion bioreactor in conjunction with well-defined three-dimensional （3D） environment for enhanced proliferation and homogeneous distribution of human fetal osteoblasts in large scaffold in vitro.Methods Large-scale β-tricalcium phosphate （β-TCP） scaffolds with tightly controlled architectures were fabricated.And a custom designed perfusion bioreactor was developed.Human fetal osteoblasts were seeded onto the scaffolds,cultured for up to 16 days in static or flow perfusion conditions.At Days 4,8 and 16 post-incubation,the proliferation and distribution of osteoblasts were determined by daily D-glucose consumption,cell viability （methyl thiazolyl tetrazolium （MTT） assay）,histological evaluation and scanning electron microcopy （SEM）.Sphere like structures observed in the SEM images were assessed by energy dispersive X-ray （EDX） analysis.Results In both static and perfusion cultures,the daily D-glucose consumption increased with prolonged time.The daily D-glucose consumption was significantly higher in the perfusion culture than that in static culture （P 〈 0.05）.The increased cell viability with time during the culture was similar to the daily D-glucose consumption under both conditions.There was much greater cell viability under flow perfusion culture compared to static culture （P 〈 0.05）.Flow perfused constructs demonstrated improved cell proliferation and a homogeneous layer composed of cells and extracellular matrix in channels throughout the whole scaffold.However,the cells were biased to periphery in scaffolds culture statically.Sphere like structures present in the matrix were identified as calcium phosphate nodules via EDX analysis.Conclusion Flow perfusion culture plus well-defined 3D interconnected channel environments enhances the proliferation and improve the distribution of human fetal osteoblasts in large scaffolds.Scaffolds with controlled architecture may be a potential tool of studying the fluid flow configuration and cell behavior inside scaffold in details.And human fetal osteoblasts can be used as a cell source in large bone graft research.