Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials
Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials
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DOI：
10.1089/10763270360728116
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发表时间：
2003-12-01
影响因子：
--
通讯作者：
Tateishi, T
中科院分区：
文献类型：
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作者：
Wang, YC;Uemura, T;Tateishi, T
骨髓源性成骨细胞与多孔陶瓷复合材料作为骨组织工程的骨移植模型已被广泛应用。灌注培养在三维（3D）培养中对许多细胞类型具有潜在的实用性。我们的假设是，灌注介质将增加细胞的活力和生物合成活性的BMO在多孔陶瓷材料，这将被揭示的碱性磷酸酶（ALP）的活性和骨钙素（OCN）的水平增加，并增强骨形成在体内。为了体外测试，在灌注容器（Minucells和Minutissue，Bad Abbach，德国）中培养BMO/β-磷酸三钙复合物，其中通过蠕动泵以2 mL/h的速率输送新鲜培养基。分别于传代1、2、3、4周后测定ALP活性和OCN含量。对于体内测试，在传代培养2周后，将复合物皮下植入同系大鼠中。4或8周后收获这些植入物。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。灌注组ALP活性和OCN含量均显著高于对照组（p < 0.01）。苏木精和伊红染色切片的组织形态学分析显示，植入后灌注组的BMO/β-TCP复合材料中骨与孔的平均比率较高：4周时灌注组为47.64 +/- 6.16，对照组为26.22 +/- 4.84（n = 6，p < 0.01）;在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，在多孔陶瓷支架上传代培养BMO期间，灌注培养系统的应用有利于它们的成骨。经灌流培养系统体外分化培养后，成骨细胞的活性和体内成骨能力明显增强。这些结果表明，灌注培养系统是一种有价值的和方便的工具，用于组织工程，特别是在人工骨组织的产生。
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/β-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.