Ionized Jet Deposition applied onto Calgary Biofilm Devices and titanium alloys

All coatings were obtained by Ionized Jet Deposition, starting from commercial metallic silver and zinc targets (Kurt J. Lesker, PA, USA and EVOCHEM Advanced Materials, respectively). In IJD, a solid target material, either a metal or a ceramic (here, metallic silver and zinc, respectively), is ablated by a pulsed electron beam. The interaction with the beam causes the ionization of the target, and the ionized material is accelerated towards the substrate where it grows in the form of a nanostructured thin film.

Here, for the deposition, the targets were mounted on a rotating holder inside the deposition chamber ablated by the electron gun (fast pulse—100 ns of high energy—10 J electrons with high-density power). The first 5 min of deposition were performed on a shutter to avoid deposition of surface impurities deriving from the target very surface. The chamber was initially evacuated down to a base pressure of 1.0 × 10–7 mbar by a turbomolecular pump (EXT255H, Edwards, Crawley, UK) and then pressure was raised by a controlled flow of oxygen (purity level = 99.999%) up to 3 × 10–4 mbar.

Deposition parameters were selected based on past results. More in detail, target-substrate distance, electron beam frequency, and working voltage were optimized to ensure uniformity of deposition and adjusted to 8 cm, 22 Hz, and 7 kV, respectively. Deposition time was fixed and set to 30 min, while different coatings characteristics and ion release were obtained by comparing different areas of the substrate. Indeed, the exact characteristics (thickness, surface roughness, aggregates geometry) depend on the distance from the plasma plume and its angle of incidence with the substrate.

Calgary Biofilm Devices were used as deposition substrates. As a results of the deposition on the CBD, three areas were selected in the Calgary, summarized in Fig. 1, having the same distance and angle with respect to the plasma plume and hence the same characteristics of the coating. In the CBD, only the wells showing complete coatings and high homogeneity were selected, while the others were discarded. Indeed, in IJD, the characteristics of the coatings depend on the distance and the angle of the plasma plume. As a consequence, the areas that are more distant from the plasma plume and have lower angles of incidence suffer relevant shadowing and incomplete coverage, both hampering a reliable evaluation of antimicrobial activity. All the compositional, morphological, and microbiological analyses performed in this work involved the three different areas selected on the CBD.

Then, to validate the approach, micro-rough medical grade titanium alloy disks (Grade 23 Titanium 6Al-4 V ELI alloy, 5 mm of thickness, 5/10 mm of diameter, Citieffe S.r.l.) were used as substrates for the deposition, after ultrasonic cleaning in isopropyl alcohol and water. A surface roughness (Ra) of 5 μm was specifically selected as representative of that of orthopaedic implants. Cylinders were placed in the deposition chamber, so as to correspond to zone “M” in the CBD.

Composition and morphological characterization of the coatings

The coatings morphological characteristics were analysed by evaluating their morphology and surface geometry by Field Emission Gun Scanning Electron Microscopy (FEG-SEM) and Atomic Force Microscope (AFM). To avoid cutting of the CBD, plastic coverslips of the same material and geometry of the wells were inserted in the different wells of the CBD and extracted for microscopy analyses. Coatings uniformity and surface texture associated with each CBD area were assessed by FEG-SEM (Tescan Mira3, CZ, working distance = 10 mm, voltage = 5 kV).

For samples topography and thickness a Multimode VIII AFM equipped with a Nanoscope V controller (Bruker, USA) was used. The AFM was operated in the ScanAsyst Imaging mode using a NT-MDT cantilever (NSG10, nominal spring constant 3.1 N/m). Based on the acquired images, minimum and maximum diameters of the metal aggregates and the clusters were determined by ImageJ. Surface roughness was calculated as root mean square (RMS) by Gwyddion after performing a plane subtraction 0 order line-flattening [24]. Thickness was measured by optical profilometer after scratching the samples.

Films composition was assessed by XRD, using a Malvern PANalytical Empyrean series III instrument (40 kV and 30 mA, 2θ range = 20–80, step size = 0.01, time per step = 30 s). XRD was performed on the thicker coatings (H), that were deposited on 2 × 2 mm2 glass slides, so that no bands deriv-420ing from the substrate could conceal those of the coating.

Silver and zinc ions concentration in the wells were measured by Inductively Coupled Plasma (ICP). In particular, the metal-coated CBD wells were filled with 150 µL Luria–Bertani (LB) medium (NaCl 1% w/v, tryptone 1% w/v, yeast extract 0.5% w/v) that was kept in the wells for 48 h, before being collected for ICP analysis. The media from all the wells belonging to the same CBD area (minimum 4 wells) were collected and pooled together. The data acquisition was carried out in triplicates, using the media from wells of three different CBD separately coated and analysed. To determine the concentration of Ag and Zn ions in the medium, the standards and samples were first diluted in a nitric acid solution with a final concentration of 4% (v/v) in ASTM type IV deionized water (Millipore MilliQ IQ7000). Calibration curves were obtained using standard solutions. To obtain the curves, a multi-element solution IV-ICPMS-71A (Inorganic Ventures) was used and progressively diluted. An Yttrium solution (100 ppb) was used as internal standard (isotope 89Y, obtained by dilution of a 1000 ppm solution, VWR). Data were collected by using an ICP-MS instrument (XSeriesII, ThermoFisher Scientific) with the isotopes 66Zn e 107Ag.

Preparation of the bacterial cultures for the antibacterial and antibiofilm assays

Four pathogenic bacteria were tested, i.e., the gram-negative strains Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, and the gram-positive strains Staphylococcus aureus ATCC 6538P, and Enterococcus faecalis DP1122. All cultures were conducted using LB medium, to which agar (1.5% w/v) was added to generate solid LB plates. The study was carried out by inoculating a single colony of each strain (grown on agar plate for 24 h) in 50 mL tubes with 5 mL of LB liquid medium. Cultures were grown overnight at 37 °C under agitation at 150 rpm and then diluted to reach specific optical density values measured at 600 nm (OD600).

Evaluation of bacterial planktonic and biofilm cell growth in the Calgary Biofilm Device

The cultures grown overnight (o/n) were diluted to reach an optical density of 0.03 measured at 600 nm (OD600). From these bacterial suspensions, a volume of 150 μL was transferred to each CBD well. The CBD plates were then incubated at 37 °C with gentle shaking at 100 rpm for 48 h in a humid environment to avoid desiccation. After incubation, the bacterial suspension was removed from each well and the OD600 was measured to evaluate the growth of planktonic cells. Biofilms formed on the wells and pegs were quantified through crystal violet (CV) staining.

After bacterial growth, the CBD wells and pegs were washed twice with saline solution (NaCl 0.85% w/v) and then fixed with 99% ethanol (v/v) for 10 min. Afterwards, wells and pegs were stained with an aqueous solution of CV (0.2% w/v) to stain the adhered cells and the biofilms by incubating the supports in the CV solution for 10 min at room temperature. The unbound dye was washed away with sterilized water. Lastly, 200 μL of an aqueous solution of acetic acid (33% v/v) was added to solubilize the stained biofilms and the optical density at 595 nm (OD595) was measured for biofilm quantification. Controls were performed on CBD under the same growth conditions using wells without metal deposition. The background staining was corrected by subtracting the mean value for CV bound to negative controls (CBD wells with the growth medium but without the bacterial inoculum).

Evaluation of bacterial biofilm formation on Ag- and Zn-coated titanium alloys

For the anti-adhesion and antibiofilm assays, the bacterial suspensions grown o/n were diluted to reach OD600 of 0.2 and 0.03, respectively. Then, 1 mL of the diluted suspensions was transferred into each well of 24-wells microplates. Sterile alloys coated with Ag, with Zn, and without coating (as control) were inserted with the coating facing up in the bacterial suspension. The microplates were incubated for 4 h (for anti-adhesion assay) or 48 h (for antibiofilm assay) at 37 °C with gentle shaking (90 rpm). The alloys were then removed from the cultures to quantify the adhered cells through CV staining and to visualize the biofilm through SEM.

Scanning Electron Microscopy of bacterial biofilms on Ag- and Zn-coated titanium alloys

The biofilms of the four bacterial strains were observed through SEM analysis on titanium alloys coated with silver, zinc, and without coating (as control experiment). Biofilms were produced by inoculating 700 μL of a bacterial suspension with OD600 = 0.03 in 48-multiwells plates with the titanium alloys and by incubating the plates at 37 °C for 48 h with gentle shaking. The titanium alloys were washed twice in NaCl 0.85% w/v, fixed in PBS 0.1 M pH 7.2 with glutaraldehyde 2.5% for 2 h, washed in PBS 0.1 M pH 7.2 for 10 min, and air-dried.

Ion release from Ag- and Zn-coated titanium alloys

Release of Ag and Zn from the coatings was measured in DMEM + 2 mM L-glut + 10%PBS + 1%es. The supernatant liquids were removed from the wells at increasing times up to 7 days and metal content was analyzed by means of Agilent 4210 (Agilent, Santa Clara, CA, USA) Molecular Plasma-Atomic Emission Spectroscopy (MP-AES). Silver lines at 338.289 nm and Zinc line at 213.857 nm were used. The calibration lines were made with 4 calibration standards (Ag: 1, 6, 10, 15 mg/L; Zn: 2, 40, 100, 140 mg/L), prepared by dilution of 1000 mg/L silver or zinc standard solutions in 0.5 M HNO3. Results from this analysis represent the mean value of three different determinations on different samples.

Cell cultures

Murine L929 fibroblasts cell line were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Life Technologies, UK) with 10% fetal bovine serum (FBS, Sigma-Aldrich, Milan, Italy), 1% penicillin/streptomycin, and 2 mM L-glutamine (Life Technologies). Cells were maintained at 37° C in a humidified 5% CO2 atmosphere.

Cell viability

The Ag and Zn cytotoxicity was tested using eluates from the two coatings, selecting the concentration corresponding to the highest ion release (i.e., 7 days). Zn/Ag coated plates were kept in medium (D-MEM, Gibco) at 37° C in a humidified 5% CO2 atmosphere on a plate-shaker (60 rpm). After 7 days the eluates were collected.

The cytotoxicity was evaluated by methylthiazolydiphenyl-tetrazolium bromide (MTT) assay. The assay is a colorimetric test based on the ability of viable cells to convert MTT solution in water-insoluble MTT formazan by mitochondrial dehydrogenases.

Briefly, 2x103 cells/well were plated in flat-bottomed 96 microplate wells (Costar, Cambridge, MA) in complete D-MEM. After cell adhesion, Ag and Zn eluates were added to culture medium and kept in contact with the cells for 24 h. Cells treated with medium conditioned with Ti alloy plates without Ag/Zn coatings were used as controls. After 24 h, cells were exposed to 5 mg/mL of MTT in complete medium for 4 h at 37 °C, washed with PBS, and 100 μL DMSO was added to each well to dissolve the MTT-formazan crystals. The absorbance was measured at 570 nm by a plate reader spectrophotometer (Tecan Infinite F200pro, Mannedorf, Switzerland). Results were recorded as optical density units (OD570) and averaged after blank subtraction. Data were expressed as percentage between OD measured in samples exposed to eluate and OD measured in negative control. The experiment was repeated two times in quadruplicate.

Data analysis

The growth inhibition (% inhibition in Fig. 5) was calculated as follows: % inhibition = (1 – T/C) × 100, where T and C are cell density (measured as OD600 for planktonic and OD595 for biofilm) in the target experimental samples (inoculated CBD wells with coatings) and control samples (inoculated CBD wells without coatings), respectively. All the microbiological and cytotoxicity results are expressed as average ± standard deviation (n > 3). Statistical significance was determined through a one-way Anova test in Graphpad Prism. Differences were considered significant when p < 0.05.