**1. Introduction**

Electrical stimulation is widely used to compensate for the altered electrical communication in diseased tissue and thus improve tissue regeneration [1–3]. External electric fields can improve physiological strength and can guide cell-orientated growth and influence cell proliferation and differentiation including for nerves, cardiac cells, and osteoblasts [4–10]. However, traditional electrical stimulator requires invasive microelectrodes, an external power supply, and electrical wires. This is

very uncomfortable, inconvenient, and unreliable. Thus noninvasive, wireless, portable, self-powered, and wearable electronic devices are urgently needed for electrical stimulation system. Recent work in nanogenerators (NGs) has exhibited significant progress in noninvasive and self-powered electrical stimulation [8,11]. In the framework, piezoelectric nanogenerators (PENGs) based on piezoelectric polymers that can generate electric surface charges under external mechanical vibration and thus achieve cordless electrical stimulation, have attracted a lot of attentions [12–15]. The electrical output produced by the PENG acts as an electrical stimulation signal whose value is related to the piezoelectric property of the material [16,17]. One such material is poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) with outstanding piezoelectric properties due to steric hindrance from the extra fluorine atoms in the TrFE inducing an all-trans stereochemical configuration [18,19]. It is well-known that the β phase is the most highly polar crystalline phase of PVDF and its copolymers [20]. Accordingly, P(VDF-TrFE) with a high content of piezoelectric β-crystalline offers excellent piezoelectric properties.

The electrospinning process can produce piezoelectric fibers by stretching as well as in situ poling during the fabrication process, paving the way for piezoelectric nanofiber NGs [21–24]. These electrospun aligned fibers have a higher content of β-crystalline phase versus random fibers [25]. In addition, various post-treatments, such as annealing and poling treatments, can produce piezoelectricity via dipole orientations; however, the influence of poling treatments on the β-crystalline phase content is rarely studied [26]. Therefore, to further improve the piezoelectricity of electrospun fibers and thus enhance the performance of PENG, it is critical to understand the specific significance of post-treatments on the piezoelectric performance [27,28].

In addition to its good piezoelectric properties, P(VDF-TrFE) also offers easy processing and nanomaterial formability. Therein, electrospinning has been widely employed to fabricate the micro-/nanofibers in ordered, random, and specific patterns. The electrospun fibrous films have a high surface-area-to-volume ratio similar to the structure and characteristics of extracellular matrix (ECM) [29]. In this framework, research has demonstrated that the alignment of nanofibers can structurally mimic the parallel orientation of the tissues and modulate cell adhesion, migration, proliferation, and differentiation [30–33].

Kai et al. prepared electrospun aligned and randomly oriented poly(e-caprolactone)/gelatin (PG) scaffolds. They found that the aligned PG scaffold could enhance the cells attachment and alignment [34]. Hitscherich et al. reported that mouse embryonic stem cell-derived cardiomyocytes (mES-CM), cultured on the aligned P(VDF-TrFE), were aligned along the fibers and expressed classic cardiac-specific markers [35]. Therefore, based on its piezoelectric property and processability, P(VDF-TrFE) has been widely used for biomedical scaffolds for tissue engineering and electrical stimulation. Genchi et al. fabricated P(VDF-TrFE)/BaTiO3 composite films as a substrate for piezoelectric stimulation to enhance the differentiation of neuroblastoma cells [36]. Later Deng's group used the BaTiO3/P(VDF-TrFE) nanocomposite membrane and leveraged the piezoelectric properties to promote bone regeneration [37]. However, the exact value of electrical stimulation induced by the piezoelectric substrate is not clear when external mechanical vibration is also applied to the cells in vitro. Thus, to better understand the effect of piezoelectric regulation on cell behavior, there is a need to measure this electrical stimulation.

In this work, we explored the effects of the exact electrical signals generated by the P(VDF-TrFE) piezoelectric nanofibers NGs on the proliferation fate of preosteoblasts. Here, the fabrication of self-powered piezoelectric nanofiber NG used as cell scaffold was based on electrospun well-aligned P(VDF-TrFE) nanofiber membranes (NFMs). The effects of annealing and poling post-treatments on the surface wettability, piezoelectric β phase, piezoelectricity, and sensing performance of P(VDF-TrFE) NFMs were investigated. In order to study the dependence of the electrical outputs of NGs on the degree of polarization, two kinds of NGs processed by different poling electrical fields were prepared. In particular, they were fixed to the flexible bottom of the culture plate, and the accurate electrical response was measured in real time under dynamic mechanical stimulation, thereby restoring the real scene of the electrical stimulation of the cells in vitro. In addition, we simulated the interaction between

piezoelectric nanofiber NG and cells through an equivalent circuit model. In order to study the role of NFMs as a scaffold, the effects of well-aligned and random interfaces of NFMs on the morphology of preosteoblasts were investigated. The well-aligned nanofibrous platforms could guide and elongate the cells. Finally, we compared the effects of different outputs stimulation of P(VDF-TrFE) nanofiber NGs on cell proliferation in vitro by applying a dynamic piezoelectric stimulus. This work demonstrates a significant potential of P(VDF-TrFE) piezoelectric nanofiber NG as self-powered electrical stimulation system for assisting tissue repair and regeneration.
