Juncosa-Melvin Natalia, Matlin Karl S, Holdcraft Robert W, Nirmalanandhan Victor S, Butler David L
Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio 45221-0048, USA.
Tissue Eng. 2007 Jun;13(6):1219-26. doi: 10.1089/ten.2006.0339.
Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
我们的研究小组已经表明，机械刺激可增加培养14天时干细胞 - 胶原海绵构建体的硬度，并在术后12周促进兔髌腱修复。目前尚不清楚的是哪些基因可能导致这种硬度增加。因此，本研究的目的是确定拉伸刺激如何影响用于修复兔中央髌腱缺损的干细胞 - 胶原海绵构建体的基因表达。通过将10只成年兔的间充质干细胞（MSCs）以0.14×10⁶个细胞/构建体的密度接种到I型胶原海绵中，创建组织工程构建体。一半的构建体每5分钟机械刺激一次，持续8小时/天，达到2.4%的峰值应变，持续2周。另一半在培养箱中不进行机械刺激，培养2周。培养14天后，将一半受刺激和未受刺激的构建体用于通过实时定量逆转录聚合酶链反应测定I型胶原、III型胶原、核心蛋白聚糖、纤连蛋白和甘油醛 - 3 - 磷酸脱氢酶基因的表达。其余构建体进行力学测试以确定其力学性能。两周的体外机械刺激显著增加了干细胞 - 胶原海绵构建体中I型胶原和III型胶原基因的表达。受刺激的构建体显示I型胶原（p = 0.0001）和III型胶原基因表达（p = 0.001）分别比未受刺激的对照高3倍和4倍。受刺激的构建体的线性刚度也是未受刺激构建体的2.5倍，线性模量是其4倍。然而，培养14天后，机械刺激并未显著增加核心蛋白聚糖或纤连蛋白基因的表达（p = 0.2）。本研究表明，对细胞 - 海绵构建体进行机械刺激会使2种结构基因的表达以及线性刚度和线性模量产生类似的增加。
