Tobita Kimimasa, Liu Li J, Janczewski Andrzej M, Tinney Joseph P, Nonemaker Jill M, Augustine Serena, Stolz Donna B, Shroff Sanjeev G, Keller Bradley B
Rangos Research Center, Rm. 3320E, 3460 Fifth Ave., Pittsburgh, PA 15213, USA.
Am J Physiol Heart Circ Physiol. 2006 Oct;291(4):H1829-37. doi: 10.1152/ajpheart.00205.2006. Epub 2006 Apr 14.
Embryonic myocardium has a high rate of cell proliferation and regulates cellular proliferation, contractile function, and myocardial architecture in response to changes in external mechanical loads. However, the small and complex three-dimensional (3D) structure of the embryonic myocardium limits our ability to directly investigate detailed relationships between mechanical load, contractile function, and cardiomyocyte proliferation. We developed a novel 3D engineered early embryonic cardiac tissue (EEECT) from early embryonic ventricular cells to test the hypothesis that EEECT retains the proliferative and contractile properties of embryonic myocardium. We combined freshly isolated White Leghorn chicken embryonic ventricular cells at Hamburger-Hamilton (HH) stage 31 (day 7 of a 46-stage, 21-day incubation period), collagen type I, and matrix factors to construct cylindrical-shaped EEECTs. We studied tissue architecture, cell proliferation patterns, and contractile function. We then generated engineered fetal cardiac tissue (EFCT) from HH stage 40 (day 14) fetal ventricular cells for direct comparison with EEECT. Tissue architecture was similar in EEECT and EFCT. EEECT maintained high cell proliferation patterns by culture day 12, whereas EFCT decreased cell proliferation rate by culture day 9 (P < 0.05). EEECT increased active contractile force from culture day 7 to day 12. The culture day 12 EEECT contractile response to the beta-adrenergic stimulation was less than culture day 9 EFCT (P < 0.05). Cyclic mechanical stretch stimulation induced myocardial hyperplasia in EEECT. Results indicate that EEECT retains the proliferative and contractile properties of developing embryonic myocardium and shows potential as a robust in vitro model of developing embryonic myocardium.
胚胎心肌具有较高的细胞增殖率，并能根据外部机械负荷的变化调节细胞增殖、收缩功能和心肌结构。然而，胚胎心肌小而复杂的三维（3D）结构限制了我们直接研究机械负荷、收缩功能和心肌细胞增殖之间详细关系的能力。我们从早期胚胎心室细胞开发了一种新型的3D工程化早期胚胎心脏组织（EEECT），以检验EEECT保留胚胎心肌增殖和收缩特性的假设。我们将汉堡-汉密尔顿（HH）第31阶段（46阶段、21天孵化期的第7天）新鲜分离的白来航鸡胚胎心室细胞、I型胶原蛋白和基质因子结合起来，构建圆柱形的EEECT。我们研究了组织结构、细胞增殖模式和收缩功能。然后，我们从HH第40阶段（第14天）的胎儿心室细胞生成了工程化胎儿心脏组织（EFCT），以便与EEECT进行直接比较。EEECT和EFCT的组织结构相似。到培养第12天，EEECT保持了较高的细胞增殖模式，而到培养第9天，EFCT的细胞增殖率下降（P<0.05）。从培养第7天到第12天，EEECT的主动收缩力增加。培养第12天的EEECT对β-肾上腺素能刺激的收缩反应小于培养第9天的EFCT（P<0.05）。周期性机械拉伸刺激诱导EEECT心肌增生。结果表明，EEECT保留了发育中的胚胎心肌的增殖和收缩特性，并显示出作为发育中的胚胎心肌强大体外模型的潜力。
