*3.2. Cell Culture Study*

The SEM micrographs of fibroblast on electrospun fibers and composites shown in Figure 3 indicate a clear difference in cell behavior on the three types of tested meshes. After one day of incubation, cells started to attach to the fibers, but still kept spherical shapes (Figure 3A,D,G). The cells on the PS mats did not flatten even after 7 days of culturing (Figure 3C), in contrast to PA6 nanofibers and PS-PA6 composite meshes, where cells clearly started already spreading on the fibers after the third day. The cells' attachment and spreading prove their integration with the mesh [15,46]. Importantly, by incorporating PA6 nanofibers to PS fibers, we decreased the surface roughness of meshes significantly, as the PA6 fibers were 100 nm in diameter [44]. Fibroblasts prefer a lower surface roughness [43,47] for spreading and migration [48]. Additionally, the hydrophilic character of PA6 fibers leads to evident cell spreading and attachment, causing further enhanced cell development [30,31]. The SEM observations, shown in Figure 3 prove that PS-PA6 composite meshes were enhancing cell flattening and proliferation.

**Figure 3.** SEM micrographs showing fibroblasts growth on electrospun fiber after the 1st, 3rd, and 7th day in cell culture on (**A**–**C**) PS meshes, (**D**–**F**) PA6 meshes and (**G**–**I**) PS-PA6 composite meshes, respectively.

The surface properties of meshes are essential for cell attachment. In the first few hours, the process of cell anchoring already begins, leading to further cell development and proliferation [49–51]. The adhesion test performed during the first 4 h of cell culture indicates no significant difference in cell attachment between all fibrous meshes and TCPS, see Figure 4A. Interestingly, cell geometry and attachment to PS, PA6, and PS-PA6 composite varied. In Figure 5, we show the SEM images focused on cell filopodia anchoring to fibers. Additionally, the shape of the created filopodia and the cell flattening were different between the three types of samples. The cells kept the round shape on PS fibers (Figure 5A) and PA6 nanofibers, but PS-PA6 composites were flattened (Figure 5B,C). Moreover, the changes in filopodia's morphology defined totally different cells spreading, which is crucial for tissue regeneration and biomaterials integration with the living system. The further proliferation assay in Figure 4B shows no differences between materials after 1 day of cell culture. However, after the third day, the absorbance

values started to increase for PA6, especially for the PS-PA6 composite, which continued up to the seventh day. The MTS test for PS meshes was close to constant over one-week of cell culture, indicating that the cell proliferation was only kept at a minimal level. Importantly, after 7 days, the number of cells on PS-PA6 composite meshes was greater than on any other tested samples and TCPS (Figure 4B). Even though the statistical analysis showed differences between materials after the same period, SEM images of cells also have to be taken into consideration to conclude which material has the most suitable properties for cell proliferation. It was previously shown that decreasing the surface roughness and hydrophobicity enhances cell proliferation [52]. In our study, the incorporation of PA6 nano-sized fibers into PS fibers decreased the roughness by half Ra, and only slightly decreased the water contact angle values; see Figure 2. However, cell proliferation was visibly higher, as presented in Figure 4B. Indeed, the decreased Ra and hydrophilic character of PA6 fibers enhanced cell proliferation. As Ansleme et al. described, the short-term adhesion and proliferation were more influenced by surface chemistry, while surface roughness affects long-term behavior [31]. We noticed a better adhesion for more hydrophilic materials, such as PA6 meshes or TCPS. The PS-PA6 composite meshes were characterized by a water contact angle above 130◦, thus showing that hydrophobic behavior does not have a straightening effect. Noticeably, by adding PA6 to meshes, we also changed the surface chemistry by including the oxygen groups that were eventually detected by XPS [38]. This type of surface with increased oxygen content is preferable for cell adhesion [31]. Indeed, after one week of cell culture, the Ra decreased by half for PS-PA6 composite meshes in comparison to PS showed the highest value of absorbance for cell proliferation. Interestingly, the size of PS fibers in order of magnitude was higher than for PA6, with increased cell penetration into meshes and growth inside them, whereas in just PA6 meshes, the small distances between nanofibers were limiting cells to the top surface of the samples. The increase of PA6 fraction decrees the spacing between fibers as the smaller fiber diameter in electrospun random meshes cause the smaller distance between fibers [53]. The fiber diameters also control the roughness of meshes [44]. Therefore, the content of PA6 nanofibers was selected not to limit the cell's integration with electrospun meshes. We showed previously on PMMA nanofibers, microfibers, ribbons, and films how the cell morphology is changing according to the surface topography. A diameter of fiber exceeding 3.5 μm is required to provide enough spacing for cell migration into the 3D meshes and enhance the filopodia attachments to fibers underneath [53]. The increased fraction of nanofibers facilitates more cell spreading on the top of the surface in comparison to the microfibers; therefore, in our PS-PA6 hierarchical scaffolds, the layering electrospinning providing a higher number of PA6 fibers were not investigated in vitro in this study.

**Figure 4.** Cell culture study on electrospun PS, PA6 and PS-PA6 composite meshes showing (**A**) the adhesion test: 1.5 h, 2 h, and 4 h and (**B**) proliferation assay after 1, 3 and 7 days after cell seeding. \*\* statistical significance calculated with ANOVA, followed by Tukey's post-hoc test, p < 0.02, error bars are based on standard deviation.

**Figure 5.** SEM micrographs focused on cell–fiber attachment after the 3rd day of cell culture on (**A**) PS microfibers; (**B**) PA6 nanofibers and (**C**) hierarchical PS-PA6 composite meshes.
