CLSM Analysis

The substrates were then washed three times with PBS. Then cell actin cytoskeleton was stained with phalloidin FITC Atto 488 (50 μL at 20 μg/mL in PBS in each well, contact time 30 min) (Sigma-Aldrich, Milan, Italy). Subsequently, after three PBS washes, the cell nuclei were stained with Hoechst 33258 (100 μL of solution at 1:10,000 dilution in PBS per each well, contact time 10 min in the dark) (Sigma-Aldrich, Milan, Italy), for 10 min. After three further PBS washes, the scaffolds were mounted on glass slides, covered using coverslips and analyzed using CLSM (Leica TCS SP2, Leica Microsystems, Milan, Italy) at λex = 346 nm and λem = 460 nm for Hoechst 33258 and λex = 501 nm and λem = 523 nm for phalloidin FITC. The acquired images were processed by means of Leica software (Leica Microsystem, Milan, Italy).

#### 2.2.7. In Vitro Antimicrobial Assay

The antimicrobial activity of norfloxacin-loaded scaffolds, either as free drug, N, or in nanocomposite, H, was evaluated against two bacteria strains—*Staphylococcus aureus* ATCC 6538 and *Pseudomonas aeruginosa* ATCC 15442. In particular, killing time was determined as the exposure time required to kill a standardized microbial inoculum [8,10,23]. Bacteria used for killing time evaluation were grown overnight in Tryptone Soya Broth (Oxoid, Basingstoke, Hampshire, UK) at 37 ◦C. The bacteria cultures were centrifuged at 2000 rpm for 20 min to separate the cells from the broth and then suspended in phosphate buffer saline (PBS, pH 7.3). The suspension was diluted to adjust the number of cells to 107–108 CFU/mL (CFU = colony forming unit).

For each microorganism strain, a suspension was prepared in PBS without scaffolds and used as the control. Unloaded scaffolds were also tested for comparison. Bacterial suspensions were incubated at 37 ◦C. Viable microbial counts were evaluated after contact for 0, 5, and 24 h with scaffolds and in control suspensions; bacterial colonies were enumerated in Tryptone Soya Agar (Oxoid, Basingstoke, Hampshire, UK) after incubation at 37 ◦C for 24 h. The microbiocidal effect (ME value) was calculated for each test organism and contact times were calculated according to the following equation [10,23]:

$$\text{ME} = \log \text{Nc} - \log \text{Nd}\_{\prime} \tag{2}$$

where Nc is the number of CFUs in the control microbial suspension and Nd is the number of CFUs in the microbial suspension in the presence of the scaffold.

#### 2.2.8. Statistical Analysis

Statistical differences were evaluated by means of a one-way ANOVA post-hoc Fisher's Least Significant Difference (LSD) or Mann–Whitney (Wilcoxon) W test (Statgraphics Centurion XV, Statistical Graphics Corporation, Statgraphics Technologies, Inc., The Plains, Virginia, USA). Differences were considered significant at *p* < 0.05.

#### **3. Results and Discussion**
