动态培养对骨髓间充质干细胞在三维多孔支架中成骨分化的影响

目的  研究动态培养条件下人骨髓来源的间充质干细胞在三维多孔支架中的成骨状况。方法  从人骨髓中分离间充质干细胞进行体外培养扩增，将第3代细胞与多孔β-磷酸三钙支架复合。对细胞/支架复合体进行动态灌注培养（动态培养组）以及静态培养（静态培养组），28d后采用MTT法检测三维支架中细胞的增殖，通过p-磷酸硝基苯法检测碱性磷酸酶活性，以及应用ELISA方法检测培养过程中骨桥蛋白及骨钙素的分泌。同时对培养后的细胞/支架复合体进行电镜观察及组织学检测，观察人骨髓间充质干细胞形成矿化细胞外基质的能力。结果  培养28d后，动态培养组细胞增殖及ALP活性显著高于静态培养组（P<0.05）。整个培养过程中，动态培养组骨桥蛋白及骨钙素的分泌高于静态培养组。静态培养组中细胞仅在多孔支架周缘增殖，而动态培养组中细胞则在整个支架内增殖。另外，动态培养组支架中形成的组织及新生骨明显多于静态培养组（P<0.05）。结论  动态培养有利于人骨髓来源的间充质干细胞在三维多孔支架中增殖并向成骨方向分化。

Effects of dynamic culture on the osteogenic differentiation of BMSCs in the 3D porous scaffold

Objective  To study the osteogenic differentiation of hBMSCs seeded in the three dimensional porous scaffold and cultured in a dynamic environment. Methods   Human bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from human bone marrow and cultured. After passage and proliferation,  human BMSCs were seeded on the porous β-tricalcium phosphate (β-TCP) scaffold and cultured in a dynamic perfusion environment. After 28 days of culture,  cell proliferation, alkaline phosphatase activity, and the secretion of osteopontin and osteocalcin were measured. The calcification of the extracellular matrix was observed through SEM, histological assay and histomorphometry. A static culture was used as a control. Results  After 28 days of culture, the proliferation of cells and expression of alkaline phosphatase activity were higher in the dynamic culture group than that in the static culture group. In the culture period, the secretion of osteopontin and osteocalcin were higher in the dynamic culture group than that in the static culture group. In the static culture group, human BMSCs only survived and proliferated on the periphery of the β-TCP scaffold and the new bone volume was lower than in the dynamic culture group. However, in the dynamic culture  condition,cells survived and proliferated on the whole scaffold, and the new bone volume was more than that in the static culture group. Conclusion  The dynamic culture environment benefits the proliferation and osteogenic differentiation of human BMSCs in the three dimensional porous scaffold.