Wang Yichao, Uemura Toshimasa, Dong Jian, Kojima Hiroko, Tanaka Junzo, Tateishi Tetsuya
Age Dimension Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Tissue Eng. 2003 Dec;9(6):1205-14. doi: 10.1089/10763270360728116.
Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue.
骨髓来源的成骨细胞（BMOs）与多孔陶瓷的复合材料已被广泛用作骨组织工程的骨移植模型。灌注培养对于三维（3D）培养中的许多细胞类型具有潜在的应用价值。我们的假设是，培养基的灌注会增加多孔陶瓷材料中BMOs的细胞活力和生物合成活性，这将通过碱性磷酸酶（ALP）活性和骨钙素（OCN）水平的升高以及体内骨形成的增强来体现。为了进行体外测试，将BMO/β-磷酸三钙复合材料置于灌注容器（Minucells和Minutissue，德国巴特阿巴赫）中培养，通过蠕动泵以2 mL/h的速率输送新鲜培养基。在传代培养1、2、3和4周结束时测量复合材料的ALP活性和OCN含量。为了进行体内测试，传代培养2周后，将复合材料皮下植入同基因大鼠体内。4周或8周后取出这些植入物。然后对样品进行ALP活性和OCN含量的生化分析，并通过光学显微镜观察。无论是在体外还是体内，灌注组复合材料中的ALP活性和OCN水平均显著高于对照组（p < 0.01）。对苏木精和伊红染色切片的组织形态计量分析显示，灌注组植入后BMO/β-TCP复合材料中骨与孔隙的平均比例更高：4周时，灌注组为47.64 +/- 6.16，对照组为26.22 +/- 4.84（n = 6，p < 0.01）；8周时，灌注组为67.97 +/- 3.58，对照组为47.39 +/- 4.10（n = 6，p < 0.05）。这些结果表明，在多孔陶瓷支架中对BMOs进行传代培养期间应用灌注培养系统有利于其成骨作用。在体外使用灌注培养系统进行分化培养后，成骨细胞的活性以及随之而来的体内骨形成均显著增强。这些结果表明，灌注培养系统是组织工程应用中一种有价值且便捷的工具，尤其是在人工骨组织的生成方面。
