Data in support of Gallium (Ga3+) antibacterial activities to counteract E. coli and S. epidermidis biofilm formation onto pro-osteointegrative titanium surfaces

This paper contains original data supporting the antibacterial activities of Gallium (Ga3+)-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in “The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii” (Cochis et al. 2016) [1]. In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga3+ enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Finally biofilm morphology was analyzed by Scanning Electron Microscopy (SEM). Data regarding Ga3+ activity were compared to Silver.

E. coli S. epidermidis Biofilm Gallium Silver Titanium Anodic Spark Deposition a b s t r a c t This paper contains original data supporting the antibacterial activities of Gallium (Ga 3 þ )-doped pro-osteointegrative titanium alloys, obtained via Anodic Spark Deposition (ASD), as described in "The effect of silver or gallium doped titanium against the multidrug resistant Acinetobacter baumannii" (Cochis et al. 2016) [1].
In this article we included an indirect cytocompatibility evaluation towards Saos2 human osteoblasts and extended the microbial evaluation of the Ga 3 þ enriched titanium surfaces against the biofilm former Escherichia coli and Staphylococcus epidermidis strains. Cell viability was assayed by the Alamar Blue test, while bacterial viability was evaluated by the metabolic colorimetric 3- [4,5- The data presented here provide new insights on Gallium and silver as useful antibacterial biomimetic treatments for pro-osteointegrative titanium based surfaces.

Data
The data here presented are directly available in this article and related to our previous publication by Cochis et al. [1]. We provide new data regarding the cytocompatibility (Supplementary Fig. 1) of electrochemically modified gallium or silver doped titanium alloys by Anodic Spark deposition and their antibacterial ability in counteracting biofilm formation produced by Escherichia coli and Staphylococcus epidermidis (Supplementary Figs. 2 and 3).

Specimen preparation
A biomimetic treatment, named SiB-Na, was made by the Anodic Spark Deposition (ASD) technique, as previously described [1]. The resulting SiB-Na specimens were then further modified by the addition of the antibacterial agents Gallium and Silver; obtained samples were identified as GaCis, GaOss, AgCis, and AgNPs respectively. For specimens preparation and characteristics please refer to Table 1 described in the Ref. [1]. Specimens were sterilized for 2 h by 70% ethanol immersion.

Bacterial strains and culture conditions
The microorganisms used in this study, E. coli PHL628 [2] and S. epidermidis RP62A [3], are biofilmproducing strains. E. coli PHL628 was provided by Dr Roberta Migliavacca (University of Pavia, Italy) whereas S. epidermidis RP62A was a gift from Prof. Tim Foster (Department of Microbiology, Dublin University, Ireland). E. coli PHL628 and S. epidermidis RP62A were routinely grown overnight, respectively in Luria Bertani Broth (LB) and in tryptic soy broth (TSB) (Difco Laboratories Inc., Detroit, MI, USA), under aerobic conditions at 37°C using a shaker incubator (New Brunswick Scientific Co., Edison, NJ, USA).

Indirect evaluation of cytocompatibility
Titanium disks were sterilized by 100% ethanol immersion (1 mL/specimen) followed by UV irradiation (235 nm; 30 min/side). After washing with PBS, they were submerged in 2 mL of complete McCoy cell culture medium (Sigma-Aldrich, Milan, Italy); 1% (v/v) sodium pyruvate (Sigma), 1% (v/v) Penicillin-Streptomycin solution (Sigma), and 15% (v/v) FBS (Fetal Bovine Serum, Sigma) were added to each well [4] and incubated for 21 days at 37 7 2°C in a H 2 O saturated atmosphere under constant shaking (50 rpm, using a VDRL DIGITAL MOD. 711/D shaker). After 1, 4, 7, 14 and 21 days, conditioned medium from each specimen was harvest and stored at À80°C. Saos2 cells were seeded onto a 96well plate at a density of 25 Â 10 3 cells/cm 2 in 100 mL of complete McCoy cell culture medium. The cells were then incubated for 24 h at 37°C, 95% relative humidity, and 5% v/v CO 2 . After 24 h the medium was replaced with 100 mL of cell medium conditioned for different times (5 wells/treatment) and with fresh medium (for control wells); plates were then incubated for 24 h, after which the medium was removed from each well and the plates were incubated for 3 h with 100 mL of Alamar solution. Cell viability was then measured using a spectrophotometer plate reader (Tecan Genius Plus; Tecan Italia, Cernusco Sul Naviglio, Italy) at excitation wavelength 540 nm and emission wavelength 590 nm.

Antibacterial activity
For biofilm growth, overnight cultures of E. coli PHL628 and S. epidermidis RP62A were diluted in their own culture medium containing 0.25% glucose [5]. Aliquots (1 mL) of the diluted bacterial suspensions were applied to the surfaces of 1 cm diameter disks, placed in 24-well tissue culture plates (Costar, Corning, NY, USA) and incubated for 72 hours at 37°C. Biofilm that had formed on the disks was washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , pH 7.4) to remove planktonic cells and loosely adhering bacteria [6]. The quantitative 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT Sigma Aldrich, St Louis, MO, USA) test was used to assess dehydrogenase activity, as an indicator of the metabolic state of the biofilm cells on the different surfaces [7]. Moreover, 72 h after culturing on the disk surface, the bacterial morphology was analyzed by SEM. Briefly, the disks were washed several times and then fixed for SEM with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 1 h at 4°C. After additional washes, they were incubated with increasing concentrations of ethanol (25%, 50%, 75% and 96%) for 10 min each, dried to the critical point using an Emitech K-850 apparatus (Emitech, Ashford, Kent, UK), and placed on a mounting base. Finally, the disks were coated with gold and examined under a SEM (Zeiss EVO-MA10; Carl Zeiss, Oberkochen, Germany).

Statistical analysis of data
Data are expressed as means 7 standard deviation. Statistical analysis was performed using the SPSS v20 software (IBM, Chicago, USA) setting the significance level at p o0.05. Data were compared by one-way ANOVA followed by Sheffè post-hoc analysis.