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Volume 9 - 2021 | https://doi.org/10.3389/fbioe.2021.686192 biofilm-associated infections have become a major problem in many medical fields leading to a high burden on patients and enormous costs for the healthcare system Microbial infestations are caused by opportunistic pathogens which often enter the incision already during implantation In the subsequently formed biofilm bacteria are protected from the hosts immune system and antibiotic action anti-microbial implant materials displays an indispensable task Thermoplastic polyurethane (TPU) represents the state-of-the-art material in implant manufacturing Due to the constantly growing areas of application and the associated necessary adjustments the optimization of these materials is essential modified liquid silicone rubber (LSR) surfaces were compared with two of the most commonly used TPUs in terms of bacterial colonization and biofilm formation The tests were conducted with the clinically relevant bacterial strains Staphylococcus aureus and Staphylococcus epidermidis Crystal violet staining and scanning electron microscopy showed reduced adhesion of bacteria and thus biofilm formation on these new materials suggesting that the investigated materials are promising candidates for implant manufacturing Despite their wide application as medical biomaterials and (iii) PPSP is not established for cardiovascular implants so far Considering the age of antibiotic resistance it is highly relevant to explore innovative strategies for bacterial defense were selected as target materials for the comparative analysis TPU samples were produced by extruding a film from the TPU granulate using a twin-screw extruder with a flat film nozzle Round samples with a diameter of 16 mm were cut out where 55 indicates a greater hardness than 80 the polymer was filled in a flat mold and vulcanized by a hot press round samples with a diameter of 16 mm were cut out LSR samples were swollen and treated with methyl methacrylate (MMA) and methacryloyloxyethyl phosphorylcholine (MPC) Afterward the samples were rinsed with VE water LSR samples were treated with PSU-DMAC (dimethy- lacetamid) solution LSR samples were treated with PPSP-DMAC solution. After coating, all samples were cured in a drying oven, washed in a surfactant solution, and underwent a sterilization process with ethylene oxide (ETO). The chemical structures of the modifications (i–iii) are depicted in Figure 1. All known material specifications and mechanical characterization are summarized in Table 1 abbreviations are used as follows: thermanox = tmx Chemical structures of the molecules for the modification of the liquid silicone rubber (LSR) The asterisk marks the point at which the monomers combine (A) polymethylmethacrylat+ 2-methacryloyloxyethyl phosphorylcholine (PMMA + MPC) (B) polysulfone (PSU) (C) poly(1,4-phenylene ether-ether-sulfone) (PPSP) Material specification and mechanical properties Contact angles were assessed in duplicates for each sample and calculated by averaging the values of both drop sides Surface free energy (SFE) as well as polar and dispersive components were calculated using Krüss Advance software v Snap frozen Staphylococcus (S.) aureus (ATCC35556) and Staphylococcus (S.) epidermidis (ATCC35984) were purchased from the American Type Culture Collection (ATCC). S. aureus was cultivated in Luria broth (LB) according to the supplier’s instructions and S. epidermidis was cultivated in tryptic soy broth (TSB). To ensure robust biofilm formation, media were supplemented with 1 or 0.25% glucose, respectively (Kwasny and Opperman, 2010) For all experiments the inoculum of each strain was prepared by adjusting the concentration of an overnight bacterial broth culture to 1 × 108 CFU/ml (colony forming unit/ml) in LB or TSB medium corresponding to an OD600 of 0.2 All experiments were carried out aerobically at 37°C with 5% CO2 in 24-well polystyrene plates with a culture volume of 2 ml unless stated (Nunc Germany) and were carried out in three independent replicates Hydrophobicity was assessed by microbial adherence to n-hexadecane in the MATH/BATH (Microbial Adhesion to Hydrocarbons/Bacterial Adherence to Hydrocarbons) test according to Lather et al. (2016) and Di Ciccio et al. (2015) cells were harvested by centrifugation at 3,000 g for 5 min washed three times in ice-cold phosphate buffer and finally resuspended in phosphate buffer to achieve an OD500 of 0.5 The bacterial suspension was overlayed with 0.5 mL of n-hexadecane (Sigma Aldrich) the phases were separated for 15 min at room temperature The results were expressed as the percentage of the cells excluded from the aqueous phase determined by the equation as follows: % adherence = [(1 -A/A0)] × 100 with A0 and A as initial and final optical densities of the aqueous phase The strains were classified as: highly hydrophobic for values >50%; moderately hydrophobic for values ranging from 20 to 50% and hydrophilic The measurement was performed twice with two independent and three technical replicates Bacterial viability was assessed using water-soluble tetrazolium (WST Germany) according to the manufacturer’s instructions 1 × 108 CFU were added to a 24-well plate containing the different rounded material disks with a diameter of 16 mm which were kindly provided from Biotronik (Berlin) and incubated for 1 h The coloring reagent was added and after 2 h of incubation the absorbance was measured at 450 nm using a microplate reader (FLUOstar Omega Tmx was used as reference and normalized to 100% The ability of bacteria to adhere to the different material surfaces was analyzed using scanning electron microscopy (SEM) 2 × 108 CFU were added to a 24-well plate containing the different material disks and incubated for 3 each well was gently washed in 0.1 M phosphate buffer (PBS) to remove planktonic germs The adherent bacteria were fixed with 2.5% buffered glutaraldehyde at 4°C over night The glutaraldehyde was removed and the disks were washed two times for 10 min with PBS and dehydrated in a series of ethanol (50–100%) Samples were dried with hexamethyldisilazane and subsequently gold-coated using a gold sputtering unit Biofilm formation on the materials was analyzed using crystal violet staining as described before (Kwasny and Opperman, 2010) 2 ml of the bacterial suspension containing 2 × 108 CFU were incubated on the materials for 24 h supernatants were removed and the disks were washed three times with double-distilled water to remove non- or loosely adherent bacteria the plates were incubated at 60°C for 1 h 200 μl of 0.06% crystal violet were added into each well and incubated for 5 min The crystal violet was removed and the disks were washed three times with double-distilled water Generated biofilms on the disks were eluted with 200 μl of 30% acetic acid Photometric measurement of supernatants was performed at 600 nm in a multilabel microtiter plate (FLUOstar Omega A material eluate test was performed to analyze the impact of diffusing substances Four disks of each material were incubated in 6 ml TSB or LB broth for 24 h 2 × 108 CFU were resuspended either in undiluted or diluted (1:2) eluate broth of each material 200 μl of the suspensions were added in a 96-well plate as triplicates and incubated for 24 and 48 h the measurement of biofilm formation was carried out with crystal violet staining as described before Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software Normal distribution was tested using the D’Agostino and Pearson Omnibus Normality Test Non-normally distributed samples were compared using the Kruskal–Wallis test followed by a Dunn’s post-hoc test p < 0.05 were considered significant ∗∗∗p < 0.001; # to PMMA-MPC: #p < 0.05 ###p < 0.001; † to PSU: †p < 0.05 †⁣†⁣†p < 0.001; + to PPSP: +p < 0.05 Surface attraction properties of liquid silicon rubbers (LSRs) is decreased compared to thermoplastic polyurethane materials (A) water contact angles (B) CH2I2 contact angles (C) calculated SFE values (D) dispersive component of SFE and (E) polar component of SFE Material surface characterization were carried out with n = 4 The cell surface hydrophobicity was determined by the MATH/BATH (Microbial Adhesion to Hydrocarbons/Bacterial Adherence to Hydrocarbons) assay Comparison of the percent hydrophobicity of the cell surface of S Both strains are classified as highly hydrophobic with cell surface hydrophobicity of S LSR coated with polymethylmethacrylate-2-methacryloyloxyethyl phosphorylcholine (PMMA-MPC) suppresses S Materials were incubated with 1 × 108 CFUs of (A) S epidermidis and bacterial viability was quantified after 1 h by WST assay Values were normalized to tmx control (100%) Data are represented as mean + SEMean of three independent experiments carried out in duplicates Materials were incubated with 2 × 108 CFUs of S aureus and colonialization was analyzed after 3 Investigated materials have no influence on S epidermidis and colonialization was analyzed after 3 LSR coated with polymethylmethacrylate-2-methacryloyloxyethyl phosphorylcholine (PMMA-MPC) significantly reduces S Materials were incubated with 2 × 108 CFUs of (A) S epidermidis and biofilm formation was quantified after 24 h and 48 h by crystal violet staining Representative pictures of biofilm formation stained with crystal violet after 24 h are shown Soluble material components do not inhibit biofilm formation of S epidermidis 48 h were incubated with undiluted and diluted eluates from the materials and biofilm formation was quantified after 24 and 48 h by crystal violet staining unmodified LSR and three modified LSRs (PMMA-MPC PPSP) were evaluated for their anti-bacterial potential We were able to demonstrate that the modified LSR with PMMA-MPC has a high resistance to Staphylococcus aureus adherence and biofilm formation we provide strong evidence that the inhibition of S aureus biofilm formation was due to an anti-adhesive effect of the material surface coli to polymer substrates despite of a low polar component of the surface energy in the same study an air plasma treated material with the highest polar component (31.3 mN m–1) showed low bacterial adhesion This suggests that the underlying mechanisms are very complex and can vary greatly in individual cases there is a need for further research on the relationship between bacterial adhesion and the components of SFE The datasets generated for this study are available on request to the corresponding author MK performed the analysis and wrote the manuscript KS and AB designed the materials and contributed data and NG contributed data and analysis tools All authors contributed to the article and approved the submitted version This study was financially supported by the Federal Ministry of Education and Research (BMBF) with RESPONSE “Partnership for Innovation in Implant Technology.” The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We gratefully thank Babette Hummel for her excellent technical support processing SEM specimen we would like to thank Nicole Deinet for her excellent technical support Presence 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