In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing.

Electrospun (ES) fibrous nanomaterials have been widely investigated as novel biomaterials. These biomaterials have to be safe and biocompatible; hence, they need to be tested for cytotoxicity before being administered to patients. The aim of this study was to develop a suitable and biorelevant in vitro cytotoxicity assay for ES biomaterials (e.g. wound dressings). We compared different in vitro cytotoxicity assays, and our model wound dressing was made from polycaprolactone and polyethylene oxide and contained chloramphenicol as the active pharmaceutical ingredient. Baby Hamster Kidney cells (BHK-21), human primary fibroblasts and MTS assays together with real-time cell analysis were selected. The extract exposure and direct contact safety evaluation setups were tested together with microscopic techniques. We found that while extract exposure assays are suitable for the initial testing, the biocompatibility of the biomaterial is revealed in in vitro direct contact assays where cell interactions with the ES wound dressing are evaluated. We observed significant differences in the experimental outcome, caused by the experimental set up modification such as cell line choice, cell medium and controls used, conducting the phosphate buffer washing step or not. A more detailed technical protocol for the in vitro cytotoxicity assessment of ES wound dressings was developed.

Fluorescent reporters give new insights into antibiotics-induced nonsense and frameshift mistranslation.

We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.