Tensile Stimulation of Murine Stem Cell-Collagen Sponge Constructs Increases Collagen Type I Gene Expression and Linear Stiffness
Tensile Stimulation of Murine Stem Cell-Collagen Sponge Constructs Increases Collagen Type I Gene Expression and Linear Stiffness
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DOI：
10.1089/ten.tea.2008.0451
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发表时间：
2009-09-01
影响因子：
4.1
通讯作者：
Butler, David L.
中科院分区：
文献类型：
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作者：
Chokalingam, Kumar;Juncosa-Melvin, Natalia;Butler, David L.
本研究的目的是确定培养中长达 14 天的拉伸刺激如何影响胶原海绵中培养的干细胞中 I 型胶原基因表达，并确定使用荧光方法测量的基因表达是否与既定方法实时定量逆转录酶聚合酶链反应 (qRT-PCR) 相关。使用新型模型系统，从六只双转基因小鼠中收获间充质干细胞，其中 I 型和 II 型胶原蛋白启动子分别与绿色荧光蛋白黄玉和增强的青色荧光蛋白连接。通过将 0.5 x 10(6) 间充质干细胞接种到硅胶培养皿中的 I 型胶原海绵支架上来创建组织工程构建体。然后将构建体转移到安装在标准培养箱中的定制气动机械刺激系统中，并使用重复曲线（2.4％峰值应变，1 Hz，20秒，然后以0％应变休息100秒），每天在张力下刺激5小时，持续7或14天。对照样本暴露于相同的培养条件但没有机械刺激。在三个时间点（0、7 和 14 天），制备构建体以使用荧光分析和 qRT-PCR 评估基因表达，其余构建体在张力下失效。两种分析方法均表明，刺激 7 天和 14 天的构建体显示出比相同时间间隔未刺激的对照显着更高的 I 型胶原蛋白基因表达。使用 qRT-PCR 和荧光分析测量的基因表达呈正相关 (r = 0.9)。在第 7 天和第 14 天，受刺激的结构的线性刚度均显着高于相同时间间隔内未受刺激的对照。第 14 天刺激结构的线性刚度与第 7 天显着不同。未来的研究将改变机械信号以优化 I 型胶原蛋白基因表达，从而改善结构生物力学和体内肌腱修复。
The objectives of this study were to determine how tensile stimulation delivered up to 14 days in culture influenced type I collagen gene expression in stem cells cultured in collagen sponges, and to establish if gene expression, measured using a fluorescence method, correlates with an established method, real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Using a novel model system, mesenchymal stem cells were harvested from six double transgenic mice in which the type I and type II collagen promoters were linked to green fluorescent protein-topaz and enhanced cyan fluorescent protein, respectively. Tissue-engineered constructs were created by seeding 0.5 x 10(6) mesenchymal stem cells onto type I collagen sponge scaffolds in a silicone dish. Constructs were then transferred to a custom pneumatic mechanical stimulation system housed in a standard incubator and stimulated for 5 h/day in tension for either 7 or 14 days using a repeated profile (2.4% peak strain for 20 s at 1 Hz followed by a rest period at 0% strain for 100 s). Control specimens were exposed to identical culture conditions but without mechanical stimulation. At three time points (0, 7, and 14 days), constructs were then prepared for evaluation of gene expression using fluorescence analysis and qRT-PCR, and the remaining constructs were failed in tension. Both analytical methods showed that constructs stimulated for 7 and 14 days showed significantly higher collagen type I gene expression than nonstimulated controls at the same time interval. Gene expression measured using qRT-PCR and fluorescence analysis was positively correlated (r = 0.9). Linear stiffness of stimulated constructs was significantly higher at both 7 and 14 days than that of nonstimulated controls at the same time intervals. Linear stiffness of the stimulated constructs at day 14 was significantly different from that of day 7. Future studies will vary themechanical signal to optimize type I collagen gene expression to improve construct biomechanics and in vivo tendon repair.