van den Dolder Juliette, Bancroft Gregory N, Sikavitsas Vassilios I, Spauwen Paul H M, Jansen John A, Mikos Antonios G
Department of Biomaterials, College of Dental Science, University Medical Center Nijmegen, Nijmegen, The Netherlands.
J Biomed Mater Res A. 2003 Feb 1;64(2):235-41. doi: 10.1002/jbm.a.10365.
The objective of this study was to evaluate the effect of two cell culture techniques, static and flow perfusion, on the osteogenic expression of rat bone marrow cells seeded into titanium fiber mesh for a period up to 16 days. A cell suspension of rat bone marrow stromal osteoblasts (5 x 10(5) cells/300 microL) was seeded into the mesh material. Thereafter, the constructs were cultured under static conditions or in a flow perfusion system for 4, 8, and 16 days. To evaluate cellular proliferation and differentiation, constructs were examined for DNA, calcium content, and alkaline phosphatase activity. Samples were also examined with scanning electron microscopy (SEM) and plastic-embedded histological sections. Results showed an increase in DNA from day 4 to day 8 for the flow perfusion system. At day 8, a significant enhancement in DNA content was observed for flow perfusion culture compared with static culture conditions, but similar cell numbers were found for each culture system at 16 days. Calcium measurements showed a large increase in calcium content of the meshes subjected to flow perfusion at day 16. The SEM examination revealed that the 16-day samples subjected to flow perfusion culture were completely covered with layers of cells and mineralized matrix. In addition, this matrix extended deep into the scaffolds. In contrast, meshes cultured under static conditions had only a thin sheet of matrix present on the upper surface of the meshes. Evaluation of the light microscopy sections confirmed the SEM observations. On the basis of our results, we conclude that a flow perfusion system can enhance the early proliferation, differentiation, and mineralized matrix production of bone marrow stromal osteoblasts seeded in titanium fiber mesh.
本研究的目的是评估两种细胞培养技术，即静态培养和流动灌注培养，对接种于钛纤维网的大鼠骨髓细胞长达16天的成骨表达的影响。将大鼠骨髓基质成骨细胞的细胞悬液（5×10⁵个细胞/300微升）接种到网状材料中。此后，构建物在静态条件下或流动灌注系统中培养4天、8天和16天。为了评估细胞增殖和分化，检测构建物的DNA、钙含量和碱性磷酸酶活性。还通过扫描电子显微镜（SEM）和塑料包埋组织切片对样本进行检查。结果显示，流动灌注系统中DNA含量从第4天到第8天有所增加。在第8天，与静态培养条件相比，流动灌注培养的DNA含量显著增加，但在第16天，每个培养系统的细胞数量相似。钙含量测定显示，在第16天，进行流动灌注的网中钙含量大幅增加。SEM检查显示，经过16天流动灌注培养的样本完全被细胞层和矿化基质覆盖。此外，这种基质深入支架内部。相比之下，在静态条件下培养的网在其表面仅存在一层薄薄的基质。光学显微镜切片评估证实了SEM观察结果。基于我们的结果，我们得出结论，流动灌注系统可以增强接种于钛纤维网的骨髓基质成骨细胞的早期增殖、分化和矿化基质生成。
