Electrical stimulation directs engineered cardiac tissue to an age-matched native phenotype.
Electrical stimulation directs engineered cardiac tissue to an age-matched native phenotype.
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DOI：
10.1177/2041731412455354
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发表时间：
2012
影响因子：
8.2
通讯作者：
Hitchcock RW
中科院分区：
文献类型：
--
作者：
Lasher RA;Pahnke AQ;Johnson JM;Sachse FB;Hitchcock RW
量化工程组织中天然心肌的结构特征对于创建功能性组织至关重要，该功能性组织可以用作体外测试或最终替换患病或受伤心肌的替代物。我们应用三维共聚焦成像和图像分析来定量描述天然和工程化心脏组织的特征。定量分析方法的开发和应用，以测试的假设，环境线索直接工程组织朝着一个表型相似的年龄匹配的天然心肌。该分析方法被应用于有和没有应用电刺激的工程心脏组织以及年龄匹配的成人天然组织。从共聚焦图像堆栈中分割单个肌细胞，并分配一个坐标系，从中计算细胞几何形状和连接蛋白-43空间分布的测量值。数据收集自9个非刺激和12个电刺激的工程组织构建体和5个出生后12天和7个成人心脏。与未刺激的工程组织相比，刺激的工程组织中的肌细胞体积分数几乎是两倍（0.34 ± 0.14 vs 0.18 ± 0.06），但不到出生后12天的天然心肌（0.90 ± 0.06）和成人心肌（0.91 ± 0.04）的一半。电刺激下的心肌细胞比未刺激的心肌细胞更长，并表现出相似的长度，宽度和高度，在年龄匹配的心肌。此外，连接蛋白-43阳性膜染色的百分比是相似的电刺激，出生后第12天，和成人的肌细胞，而它是显着较低的非刺激的肌细胞。缝隙连接蛋白-43被发现主要位于细胞末端的成年心肌细胞和不规则，但密集地聚集在膜上的nonstimulated，刺激，和出生后第12天的心肌细胞。这些发现支持了我们的假设，并揭示了环境线索的应用产生的组织结构特征更能代表年龄匹配的天然心肌比成人心肌。我们建议，所提出的方法可以应用于定量表征工程组织的发育过程和机制。
Quantifying structural features of native myocardium in engineered tissue is essential for creating functional tissue that can serve as a surrogate for in vitro testing or the eventual replacement of diseased or injured myocardium. We applied three-dimensional confocal imaging and image analysis to quantitatively describe the features of native and engineered cardiac tissue. Quantitative analysis methods were developed and applied to test the hypothesis that environmental cues direct engineered tissue toward a phenotype resembling that of age-matched native myocardium. The analytical approach was applied to engineered cardiac tissue with and without the application of electrical stimulation as well as to age-matched and adult native tissue. Individual myocytes were segmented from confocal image stacks and assigned a coordinate system from which measures of cell geometry and connexin-43 spatial distribution were calculated. The data were collected from 9 nonstimulated and 12 electrically stimulated engineered tissue constructs and 5 postnatal day 12 and 7 adult hearts. The myocyte volume fraction was nearly double in stimulated engineered tissue compared to nonstimulated engineered tissue (0.34 ± 0.14 vs 0.18 ± 0.06) but less than half of the native postnatal day 12 (0.90 ± 0.06) and adult (0.91 ± 0.04) myocardium. The myocytes under electrical stimulation were more elongated compared to nonstimulated myocytes and exhibited similar lengths, widths, and heights as in age-matched myocardium. Furthermore, the percentage of connexin-43-positive membrane staining was similar in the electrically stimulated, postnatal day 12, and adult myocytes, whereas it was significantly lower in the nonstimulated myocytes. Connexin-43 was found to be primarily located at cell ends for adult myocytes and irregularly but densely clustered over the membranes of nonstimulated, stimulated, and postnatal day 12 myocytes. These findings support our hypothesis and reveal that the application of environmental cues produces tissue with structural features more representative of age-matched native myocardium than adult myocardium. We suggest that the presented approach can be applied to quantitatively characterize developmental processes and mechanisms in engineered tissue.