*3.5. Cell Morphology on NG without Piezoelectric Stimulaton*

For tissue engineering, the scaffold material should have excellent cytocompatibility to support cell growth and proliferation [58]. The cell viability data were validated by live/dead kit (Figure S3). The viability was similar between NG and control, suggesting that the P(VDF-TrFE) piezoelectric nanofiber NG has good cytocompatibility. Furthermore, the highly aligned micro-/nanostructure of the fiber-based scaffolds can provide morphological cues for cell attachment and behavioral modulation [59–61]. To study the effect of nanofiber morphology on the alignment of cells, the high aligned and random P(VDF-TrFE) nanofibers were used for cell culture. Figure 7a,c shows that

the MC3T3-E1 cells attached nicely on both the aligned and random P(VDF-TrFE) nanofibers. The cell cytoskeleton and nucleus showed an elongated morphology on the direction of nanofibers alignment of P(VDF-TrFE) NG, while the MC3T3-E1 cells seeded on random nanofibers displayed a random orientation. This was further verified in that the aligned P(VDF-TrFE) NGs not only have an excellent cytocompatibility but can also guide cell elongation and orientation.

**Figure 7.** Attachment and alignment of MC3T3-E1 cells on well-aligned and random P(VDF-TrFE) nanofibers after 3 days of culture. (**a**) The SEM image of well-aligned nanofiber substrate (left column) and the confocal fluorescence micrographs of MC3T3-E1 cells (right column). (**b**) 2-D FFT image analysis of cell nuclei (above) and cytoskeleton alignment (below) on well-aligned P(VDF-TrFE) nanofibers. (**c**) SEM image of random nanofiber substrate (left column) and the confocal fluorescence micrographs of MC3T3-E1 cells (right column). (**d**) 2-D FFT image analysis of nuclei (above) and cytoskeleton alignment (below) on random P(VDF-TrFE) nanofibers. F-actin was stained by Alexa Fluor 488-labeled phalloidin (green); cell nuclei were stained by DAPI (blue). The scale bar for confocal fluorescence micrographs is 100 μm.

The results of representative 2-D FFT image are shown in Figure 7b,d. There were two significant symmetrical peaks at 90◦ and 270◦ in the plot of aligned P(VDF-TrFE) NGs illustrating that the direction of actin filaments is the same as the nanofibers (Figure 7b below) [62]. The frequency distributions of nuclei were preferentially concentrated along 90◦ (Figure 7b above) suggesting a specific nuclei orientation. In comparison, the 2-D FFT plot of random samples showed no obvious peaks; the actin filaments were randomly arranged (Figure 7d blow). The arrangement angles of nuclei on the random nanofibers were disordered as the angular histogram exhibits. This confirms that the cell cytoskeleton orientation is caused by the well-aligned surface topography of P(VDF-TrFE) NGs. The results demonstrate that P(VDF-TrFE) NGs can provide not only electrical stimulation signals but also morphologic cues.
