*3.1. Morphology and Characterization of NFMs*

Figure 1 shows a schematic for the electrospinning process and post-treatment process. The P(VDF-TrFE) nanofibers were fabricated with the optimized electrospun parameters using the electrospinning setup. They had a strongly aligned and uniform morphology (Figure 1b). The mean diameter of electrospun nanofibers was 590 nm ± 26 nm with further size analysis in Figure S1a. After annealing (Figure 1c and Figure S1b), several microvoids appeared clearly on the surface of the nanofibers and the surface of single fiber became rough. Here, the A-NFM exhibited a porous structure and a higher surface-area-to-volume ratio than the U-NFM, which is beneficial for the migration of implanted cells. Although the P-NFM exhibited the flat surface owing to the mechanical pressing to make the nanofibers flattened, it still had nanofibrous surface. As shown in the insets of Figure 1b–d, the average contact angle of U-NFM was 129.13◦. Although the single nanofiber surface of A-NFM was rougher than that of U-NFM, the contact angle of A-NFM decreased to 113.38◦. This phenomenon can be explained as follows. In addition to the influence of surface roughness, the contact angle is also related to the surface free energy [45]. The annealing treatment resulted in the transformation of some nonpolar α phases into polar β phase. The presence of polar β phase increased the dipolar interaction between the NFM and water molecules, which increased surface energy and reduced the contact angle [46,47]. The influence of high surface energy on the contact angle of A-NFM is greater than that of surface roughness. The P-NFM had the smallest contact angle (91.14◦) due to the combined effects of nanostructure flattening and polar β phase, demonstrating the surface wettability of P(VDF-TrFE) NFM can be improved by poling treatment. In addition to the highly-aligned and porous properties, the fibers of the polarized P-NFM samples were arranged more closely, and the fibers were bonded to each other. This improves the material's mechanical properties (Figure 1d) [48]. Representative stress–strain plots are displayed in Figure S2. The mean elastic modulus of U-NFM, A-NFM, and P-NFM were 0.148 GPa, 0.426 GPa, and 0.876 GPa, respectively. This result proves that the poling treatment can significantly affect the mechanical robust. A well-aligned and uniform P-NFM with favorable mechanical properties shows its potential as a scaffold in tissue engineering.

**Figure 1.** The morphology and contact angle of electrospun nanofiber membranes (NFMs). (**a**) Schematic illustration of the preparation and treatment of different samples. Scanning electron microscope (SEM) micrographs of (**b**) U-NFM, (**c**) A-NFM, and (**d**) P-NFM. The insets represent the contact angles corresponding to U-NFM, A-NFM, and P-NFM, respectively. (U-NFM represented pristine poly(vinylidene fluoride-trifluoroethylene)(P(VDF-TrFE)) NFM without any postprocessing; A-NFM represented annealed P(VDF-TrFE) NFM; P-NFM represented the poled samples.)

The crystallinity of P(VDF-TrFE) NFMs treated with different postprocessing steps was determined via the XRD patterns (Figure 2a). There was a distinct reflection peak from 19 to 21 degrees for all NFMs corresponding to the diffraction of plane (200)/(110) of the β phase crystal [49,50]. The broad shoulder at ~18 degrees for U-NFM is associated with the amorphous phase. After annealing treatment, the shoulder completely disappeared for both A-NFM and P-NFM, but the diffraction intensity of the (200)/(110) plane increased. In addition, the increase in diffraction peak intensity of P-NFM versus A-NFM demonstrates that the β phase is enhanced after poling. By fitting the XRD patterns [51], the diffraction curve could be resolved into three regions: amorphous as well as α- and β-crystalline phases. Thus, α and β phases could be measured. The percentage of α phase in the U-NFM was 23.3% while that of the A-NFM was 26.5%. The P-NFM had less α phase—this might be due to the phase transformation from α-crystal phase to β-crystal phase after poling treatment. The percentage of β phase in the U-NFM and A-NFM was 43.1% and 46.6%, respectively; it was 69.2% in P-NFM.

The crystal phase structures could also be characterized by FTIR spectra (Figure 2b). The characteristic absorption bands [52,53] at 506, 840, 1285, and 1430 cm−<sup>1</sup> are recognized as β phase structures whereas the absorbance peaks of the α phase structure appears at 532, 614, 765, 870, and 976 cm−1. Versus U-NFM, the intensity of characteristic bands corresponding to β phase increased for the A-NFM, which suggests that the annealing treatment improves the β phase content. Furthermore, the highest peak intensity of β phase was seen with P-NFM. This phenomenon is mainly due to thermal poling that increases with the degree of dipole orientation and phase transition of the β phase. There are fewer crystalline defects and enhanced β-crystallinity.

The XRD patterns and FTIR spectra indicate that an annealing treatment can slightly increase the β phase content while the thermal poling treatment can significantly improve the β phase crystallinity.

**Figure 2.** Crystalline characterization of P(VDF-TrFE) NFMs treated with different postprocessing steps. (**a**) X-ray diffraction (XRD) patterns and (**b**) Fourier transform infrared spectroscopy (FTIR) spectra.

#### *3.2. Effect of Postprocessing on NFM Piezoelectric Properties*

The polarization-electric field hysteresis loops (P-E loops) of U-NFM, A-NFM, and P-NFM at various electric fields are presented in Figure 3 and illustrate the ferroelectric behavior of nanofiber membranes treated with different postprocessing steps. The remnant polarization (Pr) and the saturated polarization (Ps) of U-NFM was 17.1 mC/m2 and 37.9 mC/m2, respectively (Figure 3a). After annealing, the Pr and Ps of A-NFM could reach 26.9 mC/m<sup>2</sup> and 43.1 mC/m2, respectively (Table 1 and Figure 3b), indicating that the annealing process can increase the crystallinity. The Pr is mainly associated with a highly polar β-crystalline phase. Table 1 and Figure 3c show that a higher Pr of 32.6 mC/m<sup>2</sup> could be obtained by P-NFM under a polarizing electric field of 160 MV/m. In addition, Pr was closer to Ps in sample P-NFM, and the P-E loops tended to be saturated. This suggested that the ferroelectric domain trends toward a single-domain. In particular, the higher Pr mainly originated from a β phase crystal domain reflecting the better ferroelectric properties. This result suggests that thermal poling can increase the β-crystalline phase content. In addition, the coercive electric field (Ec) increased from 60.9 MV/m for U-NFM and 65.2 MV/m for A-NFM to 88.1 MV/m for P-NFM, suggesting that the ferroelectric domain of the β-crystal phase is not oriented easily. Consequently, the P-NFM had a strong ability to maintain polarization and possessed excellent piezoelectric property.

**Figure 3.** The P-E hysteresis loops of P(VDF-TrFE) NFMs treated with different postprocessing at a polarization electric field of 160 MV/m.

The relationship between charge density and applied stress is plotted in Figure 4 and can evaluate the piezoelectric coefficient d31. The slope of the line represents the piezoelectric coefficient d31 (Figure 4). The calculated d31 and the corresponding linear regression correlation coefficients (R2) are summarized in Table 1. The data illustrate that there is better agreement between the theoretical and experimental values of d31 for A-NFM and P-NFM than for U-NFM. The A-NFM and P-NFM showed good sensing performances. It means that P-NFM can provide accurate electrical stimulation under varying external stress and that P-NFM is more reliable in the application of NGs that provide electrical stimulation.

**Figure 4.** Experimental and theoretical studies of piezoelectric coefficient d31 of nanofiber membranes. (**a**) Experimental charge density–stress curve of U-NFM. Experimental (symbols) and linear fitting (lines) charge density–stress curves of (**b**) A-NFM and (**c**) P-NFM.

**Table 1.** Electric properties of P(VDF-TrFE) NFMs with different postprocessing steps at an electric field of 160 MV/m.


In addition, there was almost no piezoelectricity in A-NFM (d31 = 0.07 pC/N) and U-NFM (d31 = 0.03 pC/N); however, P-NFM showed the highest piezoelectric coefficient (d31 = 22.88 pC/N, d33 = −31 pC/N) among all samples (Table 1). This is due to the fact that U-NFM has many amorphous crystalline phases, some nonpolar α phases and a small amount of polar β phases. With an annealing treatment, the amorphous phase of the nanofibers decreases, the total crystallinity increases and some nonpolar α phases are transformed to polar β phase, which increases the content of β phase and thus the remnant polarization was increased compared to U-NFM, nevertheless, the dipole orientation is disorderly, so the total spontaneous polarization is zero and the piezoelectric property is weak. After poling treatment, the transformation of the α phase to the β phase is further promoted leading to high remnant polarization and most electric dipoles are oriented along the direction of the externally applied electric field, underscoring the high piezoelectric performance. With the favorable mechanical and ferroelectric properties of P-NFM confirmed, we next used this material as piezoelectric nanofiber NG and evaluated the electrical response as well as the arrangement and proliferation of cells cultured on this material.
