*2.6. Piezoelectric Stimulation and Cell Proliferation Assay*

The dynamic piezoelectric stimulus used the custom-made speakers that provided uniform mechanical vibration to cells in monolayer cultures. Specialized flexible-bottomed culture plates (BF-3001U, Flexcell Int. Co., Austin, TX, USA) made of silicone elastomer membrane were used to culture the cells. The strain applied to the silicone elastomer membrane was directly transmitted to the NGs to generate piezoelectricity. The synthesized function generator and power amplifier were used to control the frequency and amplitude of deformation applied to the culture plate (experimental vibration frequency: 2 Hz; amplified voltage: 4 V).

To clarify the feasibility of P(VDF-TrFE) nanofiber NGs as exact electrical stimulation and demonstrate the effects on the proliferation of MC3T3-E1 cells, P100-NG and P80-NG were selected as the experimental groups, and the nonpiezoelectric A-NFM served as the control group. The cells were seeded at a density of 2 × <sup>10</sup><sup>4</sup> cells per well on the various NGs. The piezoelectric stimulation was applied to MC3T3-E1 cells for 30 min per day for 1 day, 3 days, or 5 days. The proliferation of the cultured MC3T3-E1 cells was measured using the cell count kit-8 (CCK-8, Dojindo Molecular Technology). The culture medium was first replaced with 1.5 mL α-MEM medium plus 10% CCK-8 solution. After 4 h incubation at 37 ◦C, the production of water-soluble formazan dye was determined using a microplate reader (MULTISKA NMK3, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm. The culture medium was changed every 2 days. Three parallel replicates were examined each time for each group.

To observe the cell morphology on the NFMs, the MC3T3-E1 cells were fixed with 4% paraformaldehyde solution in PBS (Sigma) for 10 min and then washed three times with warm 1× PBS and blocked with 1% bovine serum albumin (BSA, Sigma) solution for 60 min. The cytoskeleton was stained with Phalloidin (Invitrogen) conjugated to Alexa Fluor 488 (1:200 diluted) for 2 h at 37 ◦C, and the nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI, 300 nM, Life Technology) for 10 min.
