*3.4. Cytocompatibility: Fibroblast Adhesion and Proliferation*

Figure 10 reports the cytocompatibility (optical density (OD)) of the scaffolds towards fibroblasts after 3 or 6 days of growth. Fibroblast adhesion and proliferation onto the scaffolds were compared to those of the control (GM and cell growth in standard conditions).

**Figure 10.** Cytocompatibility (optical density (OD)) of fibroblasts grown for 3 days (**a**) and 6 days (**b**) onto CH (blue), CH/CS (red), CH/HA (green) loaded with norfloxacin at 1% (plain color) and 2% (oblique lines), and with norfloxacin–montmorillonite nanocomposite (N-VHS) at 1% (horizontal lines) and 2% (dots) in norfloxacin. N norfloxacin as a free drug and H (N-VHS nanocomposite) at the same concentrations of the scaffolds are evaluated (mean values ± SD; n = 8). Statistics: \* = Mann–Whitney (Wilcoxon) W test *p* < 0.05.

The cytocompatibility of the scaffolds loaded either with norfloxacin as a free drug or with the nanocomposite VHS-N at 1% or 2% were evaluated and N or H at the same concentrations as in the scaffolds were considered for comparison. After 6 days of growth, all the samples were characterized by higher OD than those after 3 days of growth, suggesting that the cells were in proliferation. Considering the scaffolds loaded with N as a free drug, the increase in drug concentration caused a significant decrease in cell viability with respect to the control. These results were similar to those obtained for N. This indicates that the decrease in cytocompatibility could be completely attributed to the drug and not to the scaffolds.

On the contrary, for the scaffolds loaded with the nanocomposite, the drug loading increased the cytocompatibility, suggesting that the nanocomposite in the scaffolds was able to prevent the negative effect of norfloxacin towards the fibroblasts (this was also evident considering the cytocompatibility of the nanocomposite, which was higher for the 1% solution than the 2% solution). Such an increase in cytocompatibility could be due to montmorillonite, which was able to control the drug release and could enhance fibroblast proliferation [39].

Figures 11 and 12 report CLSM and SEM microphotographs of fibroblasts grown for 6 days onto CH, CH/CS, CH/HA loaded with norfloxacin at 1% or 2%, either as a free drug or in the nanocomposite. The complementary information from the SEM and CLSM analyses suggests that in the scaffolds loaded with N as a free drug, the fibroblasts were not homogeneously distributed on the scaffolds and mainly formed aggregates as cell clusters. This behavior was dramatically influenced by drug concentration, in agreement with the cytocompatibility. However, both the scaffolds containing glycosaminoglycans allowed the fibroblasts to maintain their fusiform structure and cytoskeletons based on aligned and elongated filaments. However, norfloxacin concentration did not alter nuclei morphology. In the

hybrid scaffolds, loaded with norfloxacin nanocomposite, fibroblasts were spread out all over the scaffolds and, in some areas, confluence could be reached and, although all the scaffolds were effective to allow cell adhesion and proliferation, the scaffolds containing chondroitin sulfate were characterized by their better performance.

**Figure 11.** Confocal laser scanning microscopy (CLSM) microphotographs of fibroblasts grown for 6 days onto CH, CH/CS, CH/HA loaded with norfloxacin as a free drug (N) or norfloxacin–montmorillonite nanocomposite (H, N-VHS) at 1% or 2% (in blue: nuclei; in green: cytoskeleton) (scale bar: 50 μm).

**Figure 12.** SEM microphotographs of fibroblasts grown for 6 days onto CH, CH/CS, CH/HA loaded with norfloxacin as a free drug (N) or norfloxacin–montmorillonite nanocomposite (H, N-VHS) at 1% or 2% (scale bar: 50 μm).

These results are in agreement with others in the literature stating the biocompatibility and the proliferation enhancement properties of montmorillonite and halloysite, both phyllosilicates with a planar and rolled structure, respectively [40–42]. Moreover, the polymer matrix of the scaffolds had a synergic effect with montmorillonite, leading to effectiveness in enhancing cell growth in the presence of norfloxacin [43].

Moreover, the mechanical properties combined with norfloxacin release could better support fibroblast adhesion, proliferation and spreading all over the scaffold when the norfloxacin is loaded in the scaffolds as a nanocomposite, at 1% concentration, and chondroitin sulfate or hyaluronic acid are present in the composition.
