2.3.1. Biocompatibility Tests

Normal human dermal fibroblasts (NHDFs) from juvenile foreskin (PromoCell GmbH, Heidelberg, Germany) were used. All cells were between the 10th and 13th passages. NHDFs were grown in Dulbecco's modified Eagle medium (DMEM, Sigma Aldrich®-Merck, Milan, Italy), supplemented with 10% fetal bovine serum (FBS, Euroclone, Milan, Italy), 200 IU/mL penicillin and 0.2 mg/mL streptomycin (PBI International, I), kept at 37 ◦C in a 5% CO2 atmosphere with 95% relative humidity (RH). Fibroblasts were seeded in 96-well plates (area 0.34 cm2/well) at a density of 10<sup>5</sup> cells/cm2. Cells were grown 24 h to obtain sub-confluence. Then, cell substrates were washed with saline solution, and the cell substrates were put in contact with the samples. Biocompatibility of all samples was assessed after 24 h contact between samples and NHDF cultures. Powdered PS9 and G30 clay minerals

were used in concentrations of 1000, 500, 50 and 5 μg/mL. ALI and GR medicinal waters were used in concentrations 0.25, 2.5, 25 and 50% *v*/*v*. Regarding PS9ALI, PS9GR, G30ALI and G30GR, cell contact concentrations were prepared in order to have equal amounts of clay mineral with respect to powdery samples, bearing in mind that hydrogels were prepared with 10% *w*/*w* of the corresponding clay. Since the final amount of clay fibroblasts was put in contact with the same as in the experiments with pristine clays, the same concentration codes (1000, 500, 50 and 5 μg/mL) were used. Briefly, clay and hydrogel samples were dispersed in sterile Hanks' Balanced Salt Solution (Sigma-Aldrich) and mixed with Ultra-Turrax® (S 25N, -18G, IKA, Staufen, Germany) for 5 min, 150,000 rpm. These initial suspensions were subsequently diluted in order to obtain samples having a concentration in the range previously mentioned. Eight replicates were assessed for all samples and for the control (NHDF cultures in pure DMEM phenol red).

After the 24 h contact, growth medium and samples were withdrawn from each well, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was performed. This test is based on the activity of mitochondrial dehydrogenases of vital cells that convert MTT in formazan crystals. DMEM phenol red-free and 50 μL of MTT dissolution were added in each well, the final MTT concentration being 2.5 mg/mL. MTT-NHDF contact was maintained for 3 h before the whole supernatant was withdrawn and substituted by 100 μL of dimethyl sulfoxide solution (DMSO, Sigma-Aldrich®-Merck, Milan, Italy) to dissolve formazan purple salts. The absorbance was assayed at 570 nm by means of an ELISA plate reader (Imark Absorbance Reader, Bio-rad, Hercules, CA, USA), with a reference wavelength set at 655 nm. Cell viability was calculated as % ratio of the absorbance of each sample and the absorbance of the cells kept in contact with the growth medium (control).

#### 2.3.2. Cell Motility Assay for Wound Healing

The gap closure cell motility assay is based on the employment of a Petri μ-Dish35 mm, low (Ibidi, Giardini, Italy) in which a silicone insert is enclosed. The insert comprises two chambers with a growth area of 0.22 cm<sup>2</sup> divided by a septum with a width of cell-free gap of 500 <sup>±</sup> <sup>50</sup> <sup>μ</sup>m. NHDFs were seeded in each chamber at 105 cells/cm2 concentration and were grown until confluence in the same conditions described in Section 2.3.1. After 24 h, fibroblasts reached confluence, and the silicone inserts were subsequently removed with sterile tweezers, displaying two areas of cell substrates divided by the 500 μm (± 50) gap.

Cell substrates were washed with sterile phosphate buffer solution (PBS; 10% *v*/*v*) to eliminate debris. Then, they were put in contact with a final volume of 700 μL of phenol red DMEM in which samples were included at determined concentrations. These concentrations were selected according to MTT results. Particularly, PS9, G30, PS9ALI, PS9GR, G30ALI and G30GR were used in concentration B (50 μg/mL of clay mineral), and spring waters were used in 2.5% *v*/*v* concentrations. Cells kept in contact with pure growth medium were used as control.

Microphotographs were taken at prefixed time intervals (0, 24, 48 h) to evaluate cell growth inside the gap. Anoptical microscope (Leica, DMI3000-B model) equipped with LAS EZ software was used (Leica microsystems, Wetzlar, Germany). In order to analyze results in a more objective way, the full area of the wound healing space was photographed in all samples. Then, wound closure was monitored by measuring the remaining gap with ImageJ software. The percentage of wound closure was calculated according to Equation (2), where WS0 stands for "wound space at time 0" and WS24 is "wound space after 24 h", both of them measured as an area (μm2).

$$\% \text{ Wound closed after 24 h} = 100 - \frac{\text{WS}\_{24} \text{(\mu m}^2\text{)} \cdot 100}{\text{WSo} (\mu\text{m}^2)} \tag{2}$$

## *2.4. Confocal Laser Scanning Microscopy*

An additional sequence of wound healing experiments was stopped at 24 h of growth in order to study the morphology of fibroblasts during the wound closure procedure. NDHFs were washed three times with PBS (10% *v*/*v*) and fixed with glutaraldehyde solution in PBS (3% *v*/*v*, 800 μL; Sigma-Aldrich®-Merck, Milan, Italy). Contact with glutaraldehyde was maintained for 2 h (4–8 ◦C), and all samples were protected from light. Three PBS washes were once again performed prior to fibroblast permeabilization. Permeabilization was performed by adding Triton X-10 (0.1% *w*/*v*) for 10 min at room temperature. Triple PBS wash was again performed. Fluorescein isothiocyanate (FITC, λex = 495 nm; λem = 513 nm)-labeled phalloidin (Phalloidin-FITC, Sigma-Aldrich) was used to mark polymerized F-actin in the cytoplasm of NHDF (50 μg/mL, darkness, 40 min at room temperature). The procedure was defined according to fabricant indications. After several PBS washings aiming to eliminate unbound phalloidin-FITC, fibroblast nuclei staining was done. Blue fluorescence nucleic acid stain 4 ,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) was used. This molecule binds to double-stranded DNA, thus labelling nuclei (λex = 485 nm; λem = 552 nm). Contact between DAPI and cells was maintained for 10 min at room temperature in darkness. Finally, samples were washed and preserved in PBS (10% *v*/*v*) to avoid dryness. Confocal Laser Scanning Microscopy (CLSM) microphotographs were obtained by a Leica TCS SP2 (Leica Microsystems, Milan, Italy). Images were processed with ImageJ software.
