Au Hoi Ting H, Cheng Irene, Chowdhury Mohammad F, Radisic Milica
Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St., Toronto, Ont., Canada M5S 3E5.
Biomaterials. 2007 Oct;28(29):4277-93. doi: 10.1016/j.biomaterials.2007.06.001. Epub 2007 Jul 2.
In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1-80 microm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13 microm, and the average depth in the range from 140 to 700 nm (AFM). The surfaces with abrasions 13 microm wide and 700 nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1 ms duration, 1 Hz, 2.3 or 4.6 V/cm) was initiated 24 h after cell seeding and maintained for additional 72 h. The cover slips were positioned between the carbon rod electrodes such that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability. The presence of a well-developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that (i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation, (ii) electrical field stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular to the field lines and (iii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. The orientation and elongation response of cardiomyocytes was completely abolished by inhibition of actin polymerization (Cytochalasin D) and only partially by inhibition of phosphatidyl-inositol 3 kinase (PI3K) pathway (LY294002).
在诸如心肌等收缩性组织中，功能特性与细胞取向和伸长直接相关。因此，功能性心脏补片的组织工程关键取决于我们对多种引导线索（如拓扑、粘附或电线索）之间相互作用的理解。本研究的主要目的是确定接触引导和电场刺激对心肌主要细胞群体成纤维细胞和心肌细胞伸长和取向的交互作用。使用粒度为1 - 80微米的研磨纸对聚乙烯表面进行研磨，产生V形磨损，平均磨损峰峰宽度在3至13微米范围内，平均深度在140至700纳米范围内（原子力显微镜测量）。宽度为13微米且深度为700纳米的磨损表面对新生大鼠心肌细胞的伸长和取向表现出最强的影响，对成纤维细胞的取向也有统计学上的显著影响，因此将它们用于电场刺激。电场刺激采用与体内心脏组织以及心脏组织工程相关的方案进行。在细胞接种24小时后开始刺激（方波脉冲，持续时间1毫秒，频率1赫兹，2.3或4.6伏/厘米），并持续另外72小时。盖玻片放置在碳棒电极之间，使得磨损要么平行于要么垂直于电场线。未磨损的表面用作对照。电场刺激不影响细胞活力。在存在电场刺激的磨损表面上培养的组中，鉴定出新生大鼠心肌细胞中存在发育良好的收缩装置（心肌肌钙蛋白I和肌动蛋白丝染色）。总体而言，我们观察到：（i）电场刺激显著增强了未磨损表面上成纤维细胞和心肌细胞的伸长；（ii）当磨损也垂直于电场线放置时，电场刺激促进成纤维细胞在垂直于电场线的方向上取向；（iii）拓扑线索对心肌细胞取向的决定作用比电场刺激显著更强。通过抑制肌动蛋白聚合（细胞松弛素D），心肌细胞的取向和伸长反应完全被消除，而通过抑制磷脂酰肌醇3激酶（PI3K）途径（LY294002）仅部分被消除。
