*2.5. Drug Release*

Samples from the different study groups (*n* = 3 each) were transferred to vials containing 5 mL of phosphate buffer saline (PBS, pH 7.4) at 37 ◦C. At different time intervals in a 14-day period, aliquots were taken for further analysis, and the vials were refilled with the same volume of fresh PBS. The collected aliquots were mixed with the mobile phase (1:1), consisting of a solution of acetonitrile: water (20:80), and filtered. The drug release was analyzed by means of high-performance liquid chromatography (HPLC) using a Shimadzu SL-200 apparatus (Shimadzu Scientific Instruments Inc., Columbia, SC, USA) with a Kromaphase C18 column (250 mm × 4.6 mm) and eluting rate of 1 mL/min, using a UV detector at 254 and 280 nm for CHX and RIF, respectively. Calibration curves using a free drug were previously obtained with good correlation values (r = 0.9887 for CHX and r = 0.9668 for RIF). Three measurements were taken for each sample. Results were plotted in a graph representing the cumulative percentage drug release over time.

### *2.6. Cell Viability*

A cell viability assay was conducted to determine the in vitro biocompatibility of the designed mesh coatings, by means of the alamarBlue test. The alamarBlue is a colorimetric reagent (active ingredient: resazurin) used to measure cell proliferation and cytotoxicity of eukaryotic cells or bacteria, following exposure to chemicals or drugs; cells exerting a proliferative status will reduce this reagent (converting resazurin to resorufin), which provokes a colorimetric change in the culture medium measurable by spectrophotometry. For this assay, rabbit skin fibroblasts were used, which were isolated by the explant method, as described elsewhere [17]. Biopsies were harvested from the dermis tissue of 3 male New Zealand White rabbits belonging to another study, (experimental protocol approved by the Committee on the Ethics of Animal Experiments of the University of Alcalá, reference PROEX <sup>045</sup>/18). Cells were cultured in a controlled humid atmosphere (37 ◦C, 5% CO2) in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) plus 1% antibiotic–antimycotic cocktail for cell culture (Life Technologies, Carlsbad, CA, USA), and visualized in a Zeiss Axiovert 40C phase-contrast inverted microscope (Carl Zeiss, Oberkochen, Germany). Cells were grown in 6-well plates (2.5 × 10<sup>5</sup> cells per well in 2 mL of DMEM) and incubated overnight under the conditions described above. Next, the medium was renewed, and a mesh fragment of the corresponding study group was transferred to each well (*n* = 9 per group). Following a 24 h incubation period under the same conditions, the medium was discarded, wells were washed in Hank's balanced salt solution (Life Technologies) and 2 mL of FBS-free DMEM supplemented with 10% alamarBlue viability reagent (Bio-Rad, Hercules, CA, USA) was added. After 5 h of incubation at 37 ◦C, several 100 µL aliquots were collected from each well to read absorbance (OD570 and OD600) in an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA). Data were analyzed using software provided by the manufacturer. Results were expressed as the mean percentage cell viability.
