*3.6. Effect of Piezoelectric Stimulation Induced by NG on MC3T3-E1 Cells*

We next verified the effect of the piezoelectric response of the P(VDF-TrFE) NG acted as electrical stimulus on the cell behavior under dynamic mechanical vibration. To further illustrate the effect of the accurate electrical stimulation modulated by NG on cell proliferation. Here, P100-NG and P80-NG were the experimental groups, and A-NFM was the control. The vibration frequency was set at 2 Hz for mimicking low-frequency biomechanics. The cell proliferation was quantitatively assessed by the CCK-8 assay to estimate the metabolic activity of the total number of MC3T3-E1 cells. Figure 8a shows that the MC3T3-E1 had an elongated morphology along the direction of the nanofibers for all the samples. There was no obvious difference in morphology between cells grown on the two membranes.

**Figure 8.** Proliferation of MC3T3 cells on P80-NG, P100-NG, and control A-NFM. (**a**) Fluorescence microscopy images of MC3T3 cells on A-NFM, P80-NG, and P100-NG. (**b**) MC3T3 cells proliferation analyzed by ImageJ software after 1, 3, and 5 days culture. All data represent the mean standard deviation (*n* = 3, \* *p* < 0.05). The scale bar is 100 μm.

The cell counts of all groups increased on days 1, 3, and 5. The CCK-8 assay showed that the cells grown on both the P100-NG and P80-NG had the higher proliferation rate than those grown on A-NFM illustrating that the piezoelectricity increased cell proliferation (Figure 8b). On day 3, the MC3T3-E1 cells proliferation on the P100-NG and P80-NG was enhanced by 1.24- and 1.10-fold versus that of A-NFM, respectively. No significant difference was observed between the P100-NG and P80-NG. There were significant statistical differences in proliferation rate between cells on P100-NG, P80-NG and A-NFM on day 3 and 5. On day 5, the cells proliferation on the P100-NG and P80-NG was 1.27- and 1.13-fold versus that of A-NFM, respectively; there were significant statistical differences in proliferation rate between cells on P100-NG and P80-NG. A preliminary in vitro assay suggested that piezoelectric stimulations induced by the P(VDF-TrFE) NGs are suitable for the biopotential of MC3T3-E1 cells and can significantly promote cell growth. These results indicate that the combination of customizable exact piezoelectric stimulation and aligned nanostructured NG show the potential application to meet the demand of electrical stimulation according to specific tissue repair.
