3.2.3. Fibroblasts Biocompatibility and Adhesion

Assessment for the cytocompatibility (OD, optical density) towards fibroblasts of (a) the scaffold components after 3 and 6 days of growth, and (b) the scaffolds after 3, 6, and 10 days of growth is summarized and presented in Figure 9. All the scaffold components (solubilized or dispersed in growth medium, GM) were characterized by similar biocompatibility considering 3 or 6 days of interaction, attachment and exposure with the fibroblasts (Figure 9a). Both HNT and MMT showed good biocompatibility. Of note, the fibroblast cytocompatibility decreased with their concentrations: in these conditions, clay minerals as powders (not soluble in GM) could negatively influence cell viability due to their sedimentation.

Fibroblast cells showed to grow onto the scaffolds, and these were compared to those of the control (GM, cell growth in standard conditions) (Figure 9b). After 3 days of cell growth onto the scaffolds loaded with either 2% HNT or 2% MMT, these were able to proliferate similarly to that seen when in simple substrate exposed to growth medium (as reference control). Conversely, all the other compositions caused a significant decrease in cell viability, as shown in Figure 9a,b. After 6 days, the blank scaffold and the scaffolds loaded with MMT (at all the concentrations) and HNT (at 2% and 5%) enhanced cell proliferation similarly to control samples, and only the scaffolds loaded with HNT at 1% was not able to show any proliferation as the control. After 10 days, the GM was unable to progress into further cell growth due to the extensive cytotoxicity issues. Nonetheless, the scaffolds loaded with both the clay minerals at 2% and 5% showed increased cell proliferation, and HNT and MMT at 2% showed the best proliferative responses. A possible explanation could be due to

the specific properties of halloysite and montmorillonite which showed the enhancing fibroblast proliferation [9,10].

**Figure 9.** Cytocompatibility (OD, optical density) towards fibroblasts of (**a**) the scaffold components (GM: growth medium; P: pullulan; CS: chondroitin sulfate; CA: citric acid; CH: chitosan; Val: valinomycin) after 3 days (plain color) and 6 days (oblique line) of growth, and (**b**) of the scaffolds loaded with HNT or MMT after 3 days (plain colors), 6 days (oblique lines), and 10 days (spotted) of growth (**b**) (mean values ± SD; n = 8). Statistics: \* = Mann–Whitney (Wilcoxon) W test *p* < 0.05.

These results are in agreement with the analysis carried out on the observed CLSM and the SEM images (Figure 10). In particular, from the SEM and CLSM analysis it could be suggested that the loading of clay minerals in the scaffolds allowed homogeneous fibroblast attachment, spreading and growth all over the scaffolds. Interestingly, only the scaffold containing MMT at 5% caused cell growth in clusters and this could be associated with the irregular surface and morphology of the scaffold fibers, which prevented cell attachment and surface adhesion. However, the scaffolds loaded with HNT or MMT at 2% allowed the fibroblasts to maintain their fusiform structure and aligned and elongated the cytoskeleton filaments, enhancing cell confluency.

In a previous work [16], a blank scaffold was characterized for surface zeta potential by means of the measurements of a streaming current and streaming potential. Such a scaffold possessed 2.9 isoelectric point with a zeta potential plateau above pH 5, at about −13.8 mV, and this was related to the strong interaction between CS and the amino groups of CH. Moreover, the structural features at the mesoscale were characterized by means of SAXS analysis [16]. These evidenced that nanofibers in the scaffold were characterized by tubular structures and that the hydration caused polymer chains protruding and stretching out from the fibers surface. The scaffold swelling was due to the dilatation of the scaffold mesh rather than single fiber swelling [16]. In addition, the blank scaffold possessed a certain degree of antibacterial activity against *Staphylococcus aureus*, and this was attributable to chitosan that retained its antimicrobial properties although entangled in the scaffold structure. Furthermore, the blank scaffold demonstrated to be resorbed in vivo in a preclinical (burn excisional murine) model after the lesion healing [16]: the evaluation of the degradation pathway evidenced that lysozyme, continuously secreted by white cells (macrophages and neutrophils) during the inflammatory phase of wound healing, played a crucial role in scaffold degradation [30].

**Figure 10.** CLSM (scale bar: 50 μm) and SEM (scale bar: 20 μm) representative images of fibroblasts grown for 6 days onto the scaffolds loaded with HNT or MMT at 1%, 2%, and 5% (CLSM: in blue: nuclei; in red: cytoskeleton). Cell proliferation pattern showed by the nuclear staining increased frequencies in the HNT and MMT.
