*2.1. Cellular Proliferation*

The preference of MC3T3-E1 cells for adhesion on surfaces with specific topographies has been previously reported [7] and was further assessed in the present study. Further, it is to notice that it is essential, from a molecular point of view, to obtain the materials in the electroactive phase, i.e., in the all-trans b-phase chain conformation, to provide electromechanical cues to the cells at the nano- [18] or microscale [15], depending on the poling state of the material [19]. It is confirmed that the scaffolds are obtained in the β-phase, identified by the vibration modes at ∼510, 840, 1287, and 1400 cm<sup>−</sup><sup>1</sup> [20], representing, respectively, the CF2 bending; CF2 symmetric stretching; CF2 and CC symmetric stretching and CCC bending; and CH2 wagging and CC antisymmetric stretching (see supplementary information Figure S1).

Proliferation assays were performed using control scaffolds (non-patterned) and patterned ones featuring topographies with 25, 75, and 150 μm wide lines or hexagons.

For both linear and hexagonal topographies, the smaller lines and hexagons (25 μm) show higher cell viability than the larger ones (Figure 1a). Checking the immunofluorescence images (Figure 1b), it is observed that cells show, contrary to the non-patterned control samples (see supplementary information Figure S4), the preferential orientation of the microstructure of the patterned P(VDF-TrFE) scaffolds, being slightly lower with smaller features (25 μm). Thus, the perception of more cellular viability is provided by the fact that cells have more contact area to proliferate, since they adhere to all the scaffolds' surfaces. Bone cells are quite resilient to different surroundings, since they are naturally present in different microenvironments. Therefore, MC3T3-E1 proliferate quite well in both topography types and dimensions, although significantly more on the isotropic hexagonal topography. Immunofluorescent images disclose the compromised cellular phenotype over linear topography, being elongated and not round as is common. Their elongated phenotype suggests that the normal fate of cells is being negatively influenced by this physical stimulus, contrary to the hexagonal topography. This may indicate that, in a differentiation phase, this stimulus may cause cells to be unable to differentiate, or to differentiate into an unwanted cell type.

**Figure 1.** (**a**) SEM images of patterned poly(vinylidene fluoride-co-trifluoroethylene) (P(VDF-TrFE)) scaffolds of hexagons and lines microstructures with 75 μm dimensional features; (**b**) cellular viability obtained after MTS assay of MC3T3-E1 cells in contact with control; (**c**) representative images of pre-osteoblast culture on linear topographies with 25, 75, and 150 μm (L25, L75, and L150, respectively) and hexagonal topographies with 25, 75, and 150 μm (H25, H75 and H150, respectively), at 24 h and 72 h timepoints (nucleus stained with DAPI-blue and cytoskeleton stained with tetramethylrhodamine (TRITC)-red). τ *p* < 0.0005 vs. control; \*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001 (two-way ANOVA); (**c**) MC3T3-E1 adhesion on patterned P(VDF-TrFE) scaffolds with 25, 75, and 150 μm dimensions, in 24 and 72 h of contact.
