灌注型生物反应器中流速对人骨髓间充质干细胞增殖及成骨分化的影响

Effect of perfusion flow rate on proliferation and osteoblastic differentiation of human mesenchymal stem cells

目的探讨在灌注型生物反应器中,大段磷酸三钙(β-TCP)载体内不同流速对人骨髓间充质干细胞(humanmesenchymal stem cells,hMSCs)增殖及成骨分化的影响。方法利用灌注型生物反应器对复合了hMSCs的大段β-TCP载体分别以3、6、9 mL/min的流速培养15 d,通过葡萄糖消耗量、细胞活力(MTT比色法)以及扫描电镜(SEM)观测细胞在载体内的增殖情况;以Real-time PCR检测成骨分化相关基因的表达。结果各组葡萄糖的日消耗量随培养时间的延长而增加,初期9 mL/min组细胞增殖明显快于3、6 mL/min组(P<0.001),但是灌注培养后期6 mL/min组的细胞增殖快于9、3 mL/min组(P<0.05)。灌注培养15 d后,6 mL/min组细胞活力显著高于3、9 mL/min组(P<0.001)。SEM观察发现3组β-TCP载体内均形成复合细胞层,3 mL/min组细胞层呈疏松的簇状,6 mL/min组复合细胞层部分呈膜片状,9 mL/min组复合细胞层多数呈膜片状。在进行成骨诱导灌注培养15 d后,6、9 mL/min组碱性磷酸酶(ALP)及骨桥蛋白(OP)的表达均显著高于3 mL/min组(P<0.01);3、6 mL/min组骨钙素(OC)的表达基本相同(P>0.05),而9 mL/min组OC的表达量则显著高于另外两组(P<0.001)。结论在利用灌注型生物反应器对hMSCs进行灌注培养的早期,9 mL/min的流速最有利于hMSCs的增殖,而晚期6 mL/min的流速最有利于hMSCs的增殖。β-TCP载体内流体剪切应力(flow shear stress,FSS)随灌注流速的增加而增加,适当的FSS可促进细胞外基质的合成和分布,并增加成骨相关基因的表达。

Objective To investigate the effect of different perfusion flow rates on proliferation and osteoblastic differentiation of human mesenchymal stem cells（hMSCs） in large scale β-TCP（tricalcium phosphate） scaffold at perfusion bioreactor.Methods hMSCs isolated from iliac bone marrow aspiration were loaded into large scale β-TCP scaffold and cultured in perfusion bioreactor at the perfusion flow rate of 3,6 or 9 mL/min for 15 days.The culture media were collected for D-glucose consumption assay every 3 days.After perfusion culture for 15 days,the cell-scaffold composites were harvested for assessment of cell viability by MTT colorimetric method,SEM observation and osteogenic gene expression by real-time PCR.Results The proliferation of hMSCs assayed by daily glucose consumption showed that at early stage of culture,cells proliferated faster at flow rate of 9 mL/min than at 3 or 6 mL/min（P0.001）;while at late stage of culture,cells proliferated faster at flow rate of 6 mL/min（P0.05）.The cell viability indicated that the cell-scaffold composites at flow rate of 6 mL/min exhibited the most viable cells（P0.001）.SEM indicated that all the macropores of the scaffold at different flow rates were filled with cellular layers.All cellular layers at flow rate of 3 mL/min were incompact,but that at 9 mL/min were compact;at flow rate of 6 mL/min,the cellular layers were either compact or incompact.Real-time PCR revealed that after perfusion culture for 15 days,the mRNA expression of osteobalstic genes including ALP and OP,were enhanced significantly at flow rate of 6 and 9 mL/min as compared to that at 3 mL/min（P0.01）;however,the 9 mL/min group presented the higher OC expression than 3 and 6 mL/min group（P0.001）.Conclusions At early stage of perfusion culture,the proliferation of hMSCs was promoted at flow rate of 9 mL/min,while at late stage,there was more viable cells in scaffolds at flow rate of 6 mL/min.The osteoblastic differentiation of hMSCs was facilitated with the increase of perfusion flow rate,which was attributed to the increased flow shear stress.