Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes.

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).