Cytocompatibility of Macrophages and Pro-Inflammatory Immune Response

Human monocytic cell line THP-1 (American Type Culture Collection, Manassas, VA, USA) was cultured in RPMI-1640 medium (Gibco, Thermo Fisher, Ireland) supplemented with 10% fetal bovine serum (FBS, Sigma, Ireland), and with 200 IU/mL penicillin/0.2 mg/mL streptomycin kept at 37 ◦C in a 5% CO2 atmosphere with 95% relative humidity (RH). A total of 1 <sup>×</sup> 105 cells/mL were treated with 100 nM phorbol-12-myristate-13-acetate (PMA, Sigma Aldrich, Germany) for 48 h. After 48 h the cells were differentiated into macrophages and let to rest for 24 h before being treated.

Preliminarily, the cytocompatibility of the pure components was assessed considering sample concentrations as previously described in Section "Fibroblasts Biocompatibility and Adhesion" paragraph. Valinomycin (Val, Fisher Scientific, Ireland) or cysplatinum (Cys, Sigma Aldrich, Ireland) (final concentration 120 μM) was used as the cytotoxic control and GM (growth medium) as the biocompatible control.

THP-1 was differentiated using PMA and 20 <sup>×</sup> 103 cells were seeded in each well of the 96-well plates. After 24 h rest, the components were added to each well and the biocompatibility was assessed after 24 or 72 h of contact time, using MTT test, as previously described.

Subsequently, the scaffold cytocompatibility was assessed. Scaffolds were cut to have an area of 7.65 cm<sup>2</sup> (diameters of 1.5 cm) and placed on a cell crown (Sigma, Italy). THP-1 cells were differentiated by seeding 200 <sup>×</sup> 103 cells on the bottom of each well of the 24-well plates. After 24 h rest, the scaffold placed on the cell crown was inserted in the well. The cytocompatibility was assessed using an MTT assay (Sigma Aldrich, Ireland) after 24 or 72 h of contact time, as previously described.

TNF-α, pro-inflammatory cytokine, was assayed to evaluate the pro-inflammatory immune response using the commercially available ELISA kit (BioLegend, Medical Supply Co. Ltd., Ireland). Supernatants were collected from the cultures at 24 or 72 h after the treatment with the components or the scaffolds.

The cytokine secretion by macrophages was assayed at 450 nm with 570 nm as the reference wavelength (Epoch microplate reader, Biotek, Mason Technologies, Ireland). The method was linear in the concentration range from 7.8 to 500 pg/mL with the R2 always higher than 0.995. Lipopolysaccharide (LPS, 100 ng/mL for 24 h) was used as the positive control.
