*3.4. Cell Proliferation*

The in vitro bioactivity of the PVDF composite nanofibers was evaluated to assess the potential of the nanofibers as GBR membranes. The cell proliferation on the PVDF composite nanofibers was studied by the MTT assay, resulting that the relative cell viability of the MC3T3-E1 cells cultured on the composite nanofibers indicated good cytocompatibility of the nanofibers (Figure 7). For all the tested nanofibers, time-dependent actions were observed in cell proliferation. Changes in the content of the POSS–EGCG conjugate in the composite nanofibers affected the cellular activities. All the composite nanofibers accelerated more rapid cell proliferation as compared with the pure PVDF nanofiber. Among the composite nanofibers, cell proliferation was significantly enhanced on PE06 in comparison with on the other nanofibers. These results were associated with the contents of piezoelectric β-phase and POSS–EGCG conjugate in the PVDF composite nanofibers.

**Figure 7.** Proliferation behavior of MC3T3-E1 cells cultured on the PVDF composite nanofibers (*n* = 6). Significant difference from the pure PVDF nanofiber at each time point was denoted as *p*\* < 0.05.

During tissue regeneration, piezoelectricity can induce an increase in the surface charge density of ECM materials for the delivery of an electrical stimulus without external energy sources and consequently causes higher levels of cell proliferation and differentiation [14,16]. In addition, EGCG causes a significant elevation of osteoblastic survival as well as a decrease of osteoblastic apoptosis caused by reactive oxygen species, resulting that the proliferation and differentiation of osteoblasts can be stimulated because of antioxidant and free-radical scavenging activities of EGCG [26,40]. As shown in Figure 8, the POSS–EGCG conjugate exhibited excellent superoxide anion radical scavenging activity similar to that of pure EGCG, which could have a positive effect on cell viability. Therefore, the piezoelectric property and antioxidant activity of PVDF composite nanofibers may play an important role in osteoblast cell proliferation.

**Figure 8.** Superoxide anion scavenging activities of EGCG and the POSS–EGCG conjugate (*n* = 4).

To confirm cell growth on the PVDF composite nanofibers, the interactions between the cells and nanofibers was observed using SEM on periods of 3 and 7 days after cell seeding. SEM images exhibited that the cells adhered and spread on the surface of the composite nanofibers, and cell proliferation increased time-dependently in all the tested nanofibers (Figure 9). This indicates excellent biocompatibility of the composite nanofibers. Moreover, almost the whole surface of the PE06 was covered by the cells at day 7 of cell culture.

**Figure 9.** Morphologies of MC3T3-E1 cells on (**a**) PVDF, (**b**) PE02, (**c**) PE04, (**d**) PE06, and (**e**) PO06 composite nanofibers after culturing for 3 and 7 days.
