Kim Hyeon Joo, Kim Ung-Jin, Leisk Gary G, Bayan Christopher, Georgakoudi Irene, Kaplan David L
Department of Biomedical Engineering, Biotechnology Center, Tufts University, Medford, MA 02155, USA.
Macromol Biosci. 2007 May 10;7(5):643-55. doi: 10.1002/mabi.200700030.
Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.
在成骨条件下使用旋转瓶培养法，研究接种于水相衍生多孔丝支架上的人骨髓间充质干细胞（hMSCs）的成骨结果。特别新颖的是使用了更大尺寸的支架（直径15毫米，厚5毫米）和大孔径（直径约900 - 1000微米）。在旋转瓶中培养84天，并与在其他条件相似的静态对照进行比较。与静态培养相比，旋转瓶培养显示出细胞增殖增强，并且改善的流体流动基于碱性磷酸酶（ALP）活性升高和矿化基质沉积，显著促进了与成骨相关的结果改善。基于实时PCR的成骨分化相关标志物的表达也显示在动态旋转瓶培养条件下反应增加。组织学分析显示，培养56天后，在旋转瓶中培养的构建体中有组织化的骨样结构。这些结构经冯·科萨染色后颜色很深。旋转瓶培养带来的改善的物质传输，以及使用具有大孔径的大孔三维水相衍生丝支架，即使在研究中使用了大尺寸支架，也导致了与骨组织工程相关的结果得到增强。这些结果表明，在动态培养过程中，丝生物材料基质的结构（水相制备与溶剂相制备）和大孔径对于改善骨样结果具有重要意义。
