利用灌注型生物反应器促进干细胞在大段磷酸三钙载体内扩增

Using perfusion bioreactor for mesenchymal stem cell proliferation in large tricalcium phosphate scaffold

目的探讨灌注培养法体外构建大段细胞-载体复合物的可行性。方法利用灌注型生物反应器对复合骨髓基质干细胞的大段磷酸三钙柱状载体进行动态灌注培养(动态培养组)1、2、4周或传统静态培养(静态培养组)2、4周,通过葡萄糖日耗量、细胞活力(MTT比色法)检测以及硬组织切片观察、检测干细胞在载体内的扩增情况。结果葡萄糖的日消耗量随培养时间的延长而增加,培养前2周较后2周增加更迅速,动态培养组的葡萄糖日耗量明显大于静态培养组。细胞活力随培养时间的延长而增大,动态培养组的细胞活力明显高于静态培养组。动态培养4周时细胞活力高于2周(9·04±0·79vs7·98±0·67,P<0·05),而静态培养2周和4周时细胞活力未发生显著变化(2·55±0·23vs2·65±0·31,P>0·05)。在动态灌注培养下,骨髓基质干细胞能够在大段载体中心及周缘存活并增殖,而在静态培养下,骨髓基质干细胞仅能在载体周缘存活增殖,细胞数量明显少于动态培养组。动态培养4周组织占孔率明显大于2周(67·92%±12·28%vs50·04%±8·44%,P<0·05)。静态培养4周和2周时的组织占孔率无显著性变化(6·78%±1·85%vs6·63%±2·73%,P>0·05)。结论灌注型生物反应器使骨髓基质干细胞在大段三维载体内存活并增殖,提高细胞在载体内的复合效率。

Objective To investigate the feasibility of using perfusion culture bioreactor for bone mesenchymal stem cell proliferation in large scale β-tricalcium phosphate （β-TCP） scaffold. Methods In the dynamic perfusion culture group, the porous β-TCP cylindrical scaffolds combined with the sheep mesenchymal stem cells were continuously perfused with the complete α-MEM medium by a peristaltic pump for 1, 2 and 4 weeks. While in the static culture group, the hybrid constructs were immersed in the medium without perfusion for 2 and 4 weeks. The cell proliferation and distribution were examined by the daily glucose consumption, the cell viability and undecalcified histological study. Results The daily glucose consumption increased with time. The increase was much more evident in the first 2 weeks than in the last 2 weeks. The daily glucose consumption was higher in the dynamic culture group than in the static culture group. The cell viability also increased with time. It was higher in the dynamic culture group. In comparison to 2-week culture, the cell viability was significantly higher after 4-week culture in the dynamic culture group （P 〈 0. 05）, while it was not significantly different after 4-week culture in the static culture group （ P 〉 0. 05）. Under dynamic perfusion culture, the mesenchymal stem cells survived and proliferated through the scaffolds. However, the mesenchymal stem cells survived and proliferated only in the peripheral pores of the scaffolds under static culture. Histomorphometrical study indicated that there were much more cells in dynamic culture group than in the static group. The cell/pore rate was not significantly different between 2- week static culture and 4-week static culture （ P 〉 0. 05）. However, the cell/pore rate was significantly higher after 4-week dynamic culture than after 2-week dynamic culture （P 〈 0. 05）. Conclusion Perfusion culture permitted the persistent nutrition supply and gas exchange into the centre of large scaffold. This perfusion bioreactor makes the mesencymal stem cells survive and proliferate through a large threedimensional scaffold.