Fibroblasts Biocompatibility and Adhesion

NHDFs (normal human dermal fibroblasts from juvenile foreskin, Promocell WVR, Italy) were grown with Dulbecco's Modified Eagle Medium (Sigma, I) supplemented with 10% fetal bovine serum (FBS, Sigma, Italy) and with 200 IU/mL penicillin/0.2 mg/mL streptomycin (Sigma-Aldrich, Italy), kept at 37 ◦C in a 5% CO2 atmosphere with 95% relative humidity (RH).

Preliminarily, the cytocompatibility and proliferation in the presence of pure components were assessed. Fibroblasts were seeded with a seeding density of 25 <sup>×</sup> 103 cells/well in 96-well plates. After 24 h of growth (at sub-confluence), the following samples (in growth medium, GM) were considered: CS (0.08 mg/mL), P (1.5 mg/mL), CA (0.4 mg/mL), CH (0.4 mg/mL), MMT (MMT1: 0.2 mg/mL; MMT2: 0.8 mg/mL; MMT5: 1.2 mg/mL), and HNT (HNT1: 0.2 mg/mL; HNT 2: 0.8 mg/mL; HNT5: 1.2 mg/mL). Valinomycin (Val, Fisher Scientific, Ireland) (final concentration 120 μM) was used as the cytotoxic control and GM (growth medium) as the biocompatible control.

After 24 or 72 h of contact, the MTT test was performed. Briefly, the MTT test evaluates the activity of mitochondrial dehydrogenase of vital cells that convert MTT into formazan salts. The MTT was solubilized in PBS at a concentration of 5 mg/mL per well. A total of 50 μL of the MTT solution and 100 μL of the DMEM (DMEM w/o phenol red, Sigma, Italy) were dispensed into each well and subsequently the plates were placed in an incubator at 37 ◦C for 3 h. The reagent was then removed from each well and the cells were washed with 150 μL of PBS to remove the samples and the un-reacted MTT solution. After PBS removal, 100 μL of DMSO were added to each well and the absorbance was detected with an ELISA plate reader (ELISA plate reader, Biorad, Italy; Epoch, Microplate Spectrophotometer, BioTek, Ireland) at a wavelength of 570 nm with a wavelength of reference of 690 nm.

Subsequently the scaffold cytocompatibility was assessed. For this purpose, scaffolds were cut to have an area of 0.36 cm2 to cover the bottom of a well in a 96 well-plate and fibroblasts were seeded onto each scaffold with 35 <sup>×</sup> 103 cells/well and grown for 3, 6, and 10 days. An MTT assay was performed, as previously described. In addition, SEM and CLSM analysis were performed to visualize the fibroblasts adhered and proliferated to each scaffold.

Fibroblasts grown onto the scaffolds were fixed with a 3% glutaraldehyde solution for 1 h at 4 ◦C (glutaraldehyde 50%—Sigma Aldrich, Italy), and washed twice with PBS. As for SEM, scaffolds were dehydrated in increasing concentrations of ethanol, placed onto stub and sputtered with graphite. The images were acquired at a high voltage of 8 kV, in high vacuum, at room temperature and different magnifications (5.00 kX; 10.00 kX; 20.00 kX) (SEM: Tescan, Mira3XMU, CISRIC, University of Pavia).

As for CLSM the fixed scaffolds were stained by dipping the scaffolds in contact with 50 μL of phalloidin Atto 488 (50 μg/mL in PBS) (Sigma Aldrich, Italy) for 40 min. Cell nuclei were subsequently stained by dipping the scaffolds in 100 μL of Hoechst 33258 solution (0.5 μg/mL in PBS) (Sigma Aldrich, Italy) for 10 min. Subsequently, the samples were washed twice for 10 min with PBS and placed on microscope slide and analyzed by using a CLSM (Leica TCS SP2, Leica Microsystems, Italy) using λex = 346 nm and λem = 460 nm for Hoechst 33342 (Sigma, Italy) and λex = 501 nm and λem = 523 nm for phalloidin Atto 488 (Sigma, Italy).
