Decreased bacterial colonization of additively manufactured Ti6Al4V metallic scaffolds with immobilized silver and calcium phosphate nanoparticles

Applied Surface Science 480 (2019) 822–829

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Decreased bacterial colonization of additively manufactured Ti6Al4V metallic scaffolds with immobilized silver and calcium phosphate nanoparticles

T

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Maria Surmenevaa, , Ales Lapanjeb, Ekaterina Chudinovaa, Anna Ivanovaa, Andrey Koptyugc, Kateryna Lozad, Oleg Prymakd, Matthias Eppled, Franka Ennen-Rothe, Mathias Ulbrichte, ⁎ Tomaž Rijavecb, Roman Surmeneva, a

Physical Materials Science and Composite Materials Centre, National Research Tomsk Polytechnic University, Tomsk, Russia Jozef Stefan Institute, Ljubljana, Slovenia Mid Sweden University, Östersund, Sweden d University of Duisburg-Essen, Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), Essen, Germany e University of Duisburg-Essen, Technical Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), Essen, Germany b c

A R T I C LE I N FO

A B S T R A C T

Keywords: Additive manufacturing Electron beam melting Electrophoretic deposition Nanoparticles Antimicrobial assay Bacteriostatic activity

The design of an ideal bone graft substitute has been a long-standing effort, and a number of strategies have been developed to improve bone regeneration. Electron beam melting (EBM) is an additive manufacturing method allowing for the production of porous implants with highly defined external dimensions and internal architectures. The increasing surface area of the implant may also increase the abilities of pathogenic microorganisms to adhere to the surfaces and form a biofilm, which may result in serious complications. The aim of this study was to explore the modifications of Ti6Al4V alloy scaffolds to reduce the abilities of bacteria to attach to the EBM-manufactured implant surface. The layers composed of silver (Ag), calcium phosphate (CaP) nanoparticles (NPs) and combinations of both were formed on the EBM-fabricated metallic scaffolds by electrophoretic deposition in order to provide them with antimicrobial properties. The assay of bacterial colonization on the surface was performed with the exposure of scaffold surfaces to Staphylococcus aureus cells for up to 17 h. Principal component analysis (PCA) was used to assess the relationships between different surface features of the studied samples and bacterial adhesion. The results indicate that by modifying the implant surface with appropriate nanostructures that change the hydrophobicity and the surface roughness at the nano scale, physical cues are provided that disrupt bacterial adhesion. Our results clearly show that AgNPs at a concentration of approximately 0.02 mg/сm2 that were deposited together with CaPNPs covered by positively charge polyethylenimine (PEI) on the surface of EBM-sintered Ti6Al4V scaffolds hindered bacterial growth, as the total number of attached cells (NAC) of S. aureus remained at the same level during the 17 h of exposure, which indicates bacteriostatic activity.

1. Introduction Despite the progress achieved in bone tissue engineering, the treatment of segmental large bone defects caused by tumour resection, osteoporosis, and open fractures or infections remains a major challenge in orthopaedic and trauma surgery. The development of porous materials with complex structures for filling segmental bone defects has been gaining ground in recent years. The novel and promising additive manufacturing (AM) technologies have the potential to overcome some currently known disadvantages and problems associated with the ⁎

treatment of large bone defects [1,2]. The scientific community in the field of tissue engineering and regenerative medicine has focused on the development of biocompatible composite materials that possess interconnected gradient cellular structures in combination with an appropriate biomechanical compatibility with native bone tissue. Since bones are rigid organs, some of the important factors that have to be taken into account are the following: the individual configuration of the skeleton, the anatomic location, the loading conditions, the age, the gender, and the character of the defect. It is important to take into account all these factors and create a composite scaffold with a

Corresponding authors. E-mail addresses: [email protected] (M. Surmeneva), [email protected] (R. Surmenev).

https://doi.org/10.1016/j.apsusc.2019.03.003 Received 21 December 2018; Received in revised form 16 February 2019; Accepted 1 March 2019 Available online 01 March 2019 0169-4332/ © 2019 Elsevier B.V. All rights reserved.

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2.2. Synthesis and deposition of silver and calcium phosphate NPs

personalized design for the damaged area following the requirements of a particular clinical situation [3]. Additively manufactured implants from titanium alloys can replicate the complex microstructure of bones by integrating the required porosity with the desired pore size, shape, and curvature into monolithic implants, thus improving the implant integration process and its long term stability [3,4]. 3D scaffolds with different morphologies and geometries (including lattice or mesh structures of different dimensions) have been successfully fabricated from metallic powders using electron beam melting (EBM) [5–7]. The rationally designed biomaterials with complex porous structures possess several orders of magnitude larger surface areas than those of solid biomaterials. The biofunctionalization of such scaffolds for improving the bone tissue regeneration performance is one of the directions that has received significant attention [8–10]. In our recent study [11], we demonstrated that covering the entire surface of 3D printed titanium scaffold with calcium phosphate nanoparticles (CaPNPs) using the electrophoretic deposition technique could lead to enhanced adhesion, proliferation and osteogenic differentiation of human mesenchymal stem cells in vitro. The advantage of electrophoretic deposition is that this method allows one to easily cover the inner parts of complex structures, which can be hardly achieved with other techniques [12]. However, the increasing surface area of the implant additionally promotes the adherence of pathogenic microorganisms and consequently formation of pathogenic biofilms, which are extremely challenging to treat, due to its resilience against antibiotics [13] and is frequently leading to life threatening complications, such as sepsis. Bacterial biofilms are formed in several steps: (i) preparation of cells for sessile life during the stage of reversible attachment, (ii) irreversible attachment, (iii) propagation and colony formation, (iv) biofilm maturation and (v) detachment of cells. Since bacterial cells explore the surfaces during the first stage, by using appropriate types or surface modifications, the prolongation of the phase of the reversible attachment can be expected and therefore slower progression towards the formation of the fully matured biofilm. Hence, in this study we have investigated the effect of various surface modifications of titanium on the initial phases of the biofilm formation. Since the initial phases can be observed only by quantification of the attached cells, we have developed an discontinuous time lapse fluorescent microscopy (DTLFM) approach focusing on S. aureus bacterium, which is the most common pathogen associated with infections of surgical implants and other prosthetic devices [14]. Silver nanoparticles (AgNPs) have been chosen as the antibacterial agent since it is known that they are bactericidal and act against different gram-positive and gram-negative bacteria [15–18]. The mixture of AgNPs and CaPNPs was deposited on the highly developed surface area of EBM-manufactured titanium alloy substrates in order to combine the advantages of both methods and achieve a multifunctional surface. Following the NPs' synthesis and deposition and the characterization of the formed layers, their activity against S. aureus formation of the biofilm was assessed.

For the synthesis of silver NPs, silver nitrate (Roth, p.a), polyvinylpyrrolidone (PVP K30, Povidon 30; Fluka analytical, molecular weight 40,000 g·mol−1) and D-(+)glucose (Sigma life Science, p.a.) were used as a metal salt precursor, stabilizing agent and reductant, respectively [19]. The PVP solution was prepared by dissolving 6 g of PVP and 12 g of Glucose in 240 ml distilled water together and heating it to 90 °C. A total of 3 g of AgNO3 was dissolved in 1 mL of distilled water and was quickly added to the PVP solution under magnetic stirring. After the mixed dispersion was stirred at 90 °C for 1 h, it was then allowed to cool down to room temperature. The synthesized particles were separated using centrifugation with a Sorvall WX Floor Ultra Centrifuge at 20,000 rpm for 30 min, redispersed in distilled water for 30 s and collected again using ultracentrifugation. The polyethylenimine (PEI)-stabilized CaPNPs were prepared as previously described in [20]. Briefly, aqueous solutions of 6 mM of calcium-L-lactate (Merck chemicals Darmstadt, Germany) and 3.6 mM of di-ammonium hydrogeNPshosphate (Aldrich, Steinheim, Germany) with pH 9 were mixed in a tubular reactor with a flow rate of 25 mL min−1 each. After that, the PEI solution (2 g L−1, Aldrich, Steinheim, Germany) was added with a flow rate of 12.5 mL min−1. Next, the particles were separated by centrifugation (4000 rpm, 30 min) and redispersed in ethanol. Ti6Al4V scaffolds were functionalized with NPs via electrophoretic deposition. We have demonstrated before a schematic setup for the electrophoretic deposition of CaPNPs on titanium alloy scaffolds [20]. Briefly, 3 mL stainless-steel beaker, forceps, tripod and voltage source were used for all the depositions. The sample was fixed using forceps above the beaker. Thus, Ti6Al4V scaffolds and the beaker were used as the electrodes. They were separated 3 mm apart. Positively charged CaPNPs and negatively charged AgNPs were deposited on the negatively and positively charged cathodes, respectively. The deposition process of both types of NPs in ethanol was carried out at a DC voltage of 50 V for 30 min at room temperature. After that, the scaffolds were dried at 50 °C. 2.3. Characterization The morphologies of the obtained biocomposites were investigated by scanning electron microscopy (Au/Pd [80:20]-sputtered samples; ESEM Quanta 400, Thermo Scientific). A microscope equipped with an energy-dispersive X-ray spectroscope (EDX; detector: S-UTW-Si(Li)) was also used to analyse the elemental compositions of the coated EBMfabricated metallic scaffolds. The hydrodynamic diameters of the NPs were measured by dynamic light scattering (DLS) with a Malvern Zetasizer Nano ZS instrument and NP tracking analysis (NTA) with a NanoSight LN 10 instrument. The zeta (ζ) potentials of the NPs were determined by DLS. The concentrations of Ca and Ag were determined by atomic absorption spectroscopy (AAS; M-Series, Thermo Electron Corporation) with a detection limit of 1 μg L−1. The scaffolds with CaP- and AgNPs were dissolved in HCl and HNO3, respectively. The Ultraviolet spectroscopy (UV/Vis Spectrometer Cary 300) was used for determining the PO43− contents on the scaffolds with CaPNPs dissolved in HCl. The contact angle measurements were made by the sessile drop method in air at room temperature with an optical contact angle apparatus (OCA 15 Plus Data Physics Instruments GmbH, Germany) using the SCA20 software (Data Physics Instruments GmbH, Germany). At least ten drops (2 μL) of water, ethylene glycol and diiodomethane were formed on the surfaces of the titanium alloy scaffolds, and the resulting mean contact angle values were then used for the calculations. To determine the total surface energy of the modified scaffolds and their polar and dispersive components, the Owens-Wendt-Rabel-Kaelble (ORWK) method was used. Three different media (water,

2. Materials and methods 2.1. Manufacturing of EBM scaffolds The titanium alloy scaffolds with a diameter of 7 mm and a height of 1 mm were produced on an electron beam melting machine (EBM® A2, ARCAM EBM, Mölndal, Sweden). Ti6Al4V powder supplied by Arcam AB with a powder particle size of 75–125 μm was used. The EBM process was maintained in a vacuum chamber with the working area kept at 730 °C. After manufacturing, all samples were carefully blasted to remove the residual titanium powder within the porous structure using a standard ARCAM powder recovery system with a high-pressure air stream with working powder suspended in it. 823

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approach enabled us to focus our observation only to the process of attachment of cells and then the further progress of their formation of microcolonies. Chamber was incubated at 37 °C for 3 days and the biofilm formation was monitored at 0 h, 3 h, 20–24 h, 46–48 h and 70–72 h. The imaging was performed at 100× and 400× magnification using Pln 10×/0.25 Ph1 (Zeiss) and LD Pln 40×/0.6 Ph2 DICII (Zeiss) objective lenses, respectively, and the 43 HE DsRed (Zeiss) filter set for the excitation and detection of the Syto82 dye. The imaging of the surfaces was obtained using an Orca camera (Hammamatsu, Japan) and the Micromanager software [21]. The figures were treated and the amount of bacteria were measured using the ImageJ software [22]. All experiments were performed in three biological replications.

diiodomethane and ethylene glycol) were used for these calculations. The surface roughness was measured at room temperature using a three-dimensional profilometer Micro Measure 3D Station by STIL (France). The 3D roughness parameters (average roughness, Sa; root mean square roughness, Sq; skewness, Ssk; kurtosis, Sku; maximum hole depth, DptH; maximum peak depth, DptP) were calculated. The three measurements and average values were obtained for each of the scaffolds. The average roughness (Sa) is the average of the individual heights (asperities) and depths from the arithmetic mean elevation of the profile. The root mean square roughness (Sq) is the square root of the sum of the squares of the individual heights and depths from the mean line. Skewness (Ssk) is a measure of the average of the first derivative of the surface profile (the departure of the surface from symmetry). A negative Ssk indicates that the surface is dominated by valleys, whereas a surface with a positive skewness is said to contain mainly peaks and asperities. Kurtosis (Sku) is a measure of the sharpness of the profile peaks.

2.6. Statistical analysis The significance of the observed differences between the means of different samples were analysed and compared using one-way ANOVA. Probabilities below p < 0.05 were considered as being statistically significant. The obtained results of the attached bacteria were analysed using the Wilcoxon rank signed test to assess the differences in the degrees of biofilm formation among the samples. All statistical comparison tests were performed using the R software [23]. The relation of the physicochemical surface properties of samples with the cell numbers present on the surfaces were determined using the multivariate principal component analysis (PCA) allowing us to identify correlating factors. In the PCA approach we used all values obtained from the measurements of the properties of the sample and the measurements of the number of bacterial cells obtained at particular time point. PCA was performed using R software [23]. The PCA values were calculated using FactoMineR [24] and graphs were visualized within ggplot2 environment [25] using factoextra packages [26]. Using the confidence intervals (p = 0.1) we visualized ellipses. The characteristics and variables are represented as vectors and the measured characteristics of different samples, the individuals, as points.

2.4. Bacterial cell culture In all experiments, we used S. aureus strain NCTC 6571 bacterial cells. The culture was grown at 37 °C on nutrient agar plates (SigmaAldrich). Prior to the experiments, we prepared an overnight liquid culture in a nutrient broth (NB) medium from a single colony to obtain a stationary phase culture (OD600 = 1.0–1.3), which was used for cell staining. The liquid cultures were always incubated at 37 °C in a rotary incubator at 200 rpm (Multitron, Infors). 2.5. Assessment of bacterial attachment in the early stages of biofilm formation To assess the development of the biofilm on the sample surfaces, we performed a DTLFM observation of the attachment of cells and biofilm formation under the Axio Observer Z1 inverted fluorescence microscope (Zeiss, Jena, Germany). The method enabled the observation of individual cells and microcolonies attached on the surface. We assessed the formation of the biofilms within small incubation chambers (width: 3 cm, height: 1 cm, and depth: 1–3 mm) where we placed the studied sample. The liquid NB medium containing 0.2 nM of Syto82 (Thermo Scientific) and the Syto82 stained S. aureus cells together were placed in the chamber where cells were exposed to the sample surface and were fluorescently labelled. The added Syto82 to the medium was added to stain new cells after division and to keep intensity of the fluorescent signal from bacterial cells high. The cells were stained in a 5 μM of Syto82 in 0.9% NaCl for 30 min. The stained cells were washed 3 times through centrifugation at 3000 g for 5 min. The chamber was made on the surface of a clean microscope slide (76 × 26 mm) by attaching a rectangular frame made from doublesided adhesive tape that was 5 cm long and 1 mm thick by cutting out the rectangular shaped aperture 1 × 2.5 cm in size. Single, double or triple layer frame was used, depending on the tested sample thickness. The spacer between the sample and the surface cover slip was made of a low-density polyethylene mesh with 400 μm pore sizes and 100 μm thickness. The chamber was assembled as follows: (i) the frame was attached to the surface of the microscope slide, (ii) the sample was placed into the chamber, (iii) the spacer was applied to the surface of the sample, (iv) the chamber was filled with stained cells suspended in NB containing Syto82 and (v) the chamber was sealed with the cover slip by attaching it on the exposed side of the double-sided adhesive tape. Prior to incubation, the chamber was turned upside down so that the cover slip and the spacer were facing down. Since S. aureus cells are non-motile only the cells that can get attached on the surface of the sample were quantified. Detached cells were deposited on the surface of the bottom glass plate of the chamber. The fluorescent signal of the cells from the bottom glass plate did not interfere with fluorescent signal from cells present on the surface of the sample. Accordingly, this

3. Experimental results 3.1. Morphological, chemical and phase compositions of samples Prior to electrophoretic deposition, the size distribution and zeta potential of the synthesized AgNPs and CaPNPs were analysed. According to the DLS measurements, the PVP-functionalized AgNPs were negatively charged (ζ = −6 ± 12 mV) particles with a hydrodynamic diameter of 70 ± 12 nm. The PEI-stabilized CaPNPs were positively charged (ζ = +22 ± 9 mV) with a hydrodynamic diameter of 90 ± 20 nm. In both cases, the polydispersity index (PDI) was below 0.3, indicating the absence of larger agglomerates. The surface of the as-printed EBM-scaffold has a complex microtopography consisting of micron-size globules (Fig. 1A). Partly fused and loosely attached powder grains are quite common for EBM-manufactured products, as was described in great detail in refs. [27, 28]. The scaffold's micro-topography remains unchanged after the electrodeposition of assemblies of NPs. The distribution of the deposited AgNPs, CaPNPs and AgNPs+CaPNPs assemblies can be clearly distinguished in the magnified SEM images (Fig. 1C, D, E). The discrete and uniform AgNPs are distributed evenly on the scaffold surface whereas the CaPNPs and AgNPs+CaPNPs assemblies form multiple layers of NPs. Although only Ti peaks can be found from the XRD patterns (Fig. S1), obvious Ag, Ca and P peaks emerge from the full EDX spectra of the studied samples (Fig. 2), thus suggesting that there are limited amounts of Ag and amorphous CaPNPs present on the surface of the studied scaffolds. The Ca/P ratio of the deposited layer of CaPNPs that was calculated based on the at.% of the elements that were retrieved from the EDX spectra was approximately 1.4, which is characteristic of amorphous CaP. In [20] the authors also revealed amorphous structure 824

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Fig. 1. SEM images of the Ti6Al4V alloy (A) covered with AgNPs (B), CaPNPs (C) and (CaP+Ag)NPs (D).

However, it can be observed that all the specimens have positive Ssk values, indicating that the surface has many peaks, the NPs are uniformly distributed on the whole substrate and the substrate valleys are covered. All the measured surfaces exhibited positive kurtoses. It is known that the surfaces with few high peaks and low valleys presented kurtoses < 3, and the surfaces with higher peaks and low valleys had kurtoses > 3 [31]. The samples of Ti6Al4V+(Ag + CaP)NPs have a high positive kurtosis and skewness, thus indicating that the surface has high peaks and deep valleys, which probably appeared due to presence of two kinds of NPs with different characteristic sizes. To determine the wettability properties of the surfaces, we measured parameters such as the contact angle for all liquids, the surface free energy (γ) and its dispersive (γd) and polar (γp) components (Table 3). Since the values of the contact angles of water are 95.9 ± 0.9° and 54.7 ± 0.7° for the as-manufactured and the AgNPscoated scaffolds, respectively, the hydrophilicity of the scaffolds with modified surfaces is significantly increased. Moreover, the titaniumbased scaffolds covered with CaPNPs and (CaP+Ag)NPs assembles exhibited super-hydrophilic surfaces with an observed water contact angle < 5° with the instant spreading of either water, diiodomethane or ethylene glycol drops. In view of this, the surface free energy and its

of synthesized CaPNPs. According to ref. [29], Banerjee et al. showed that the reduction of silver with glucose in the presence of PVP leads mostly to spherical NPs with pentagonally twinned crystals. The Ag and Ca concentrations were determined by atomic absorption spectroscopy. The phosphate (PO43−) content was estimated by means of UV-spectroscopy. The obtained results are summarized in Table 1. The CaPNPs layer has some cracks in the top of the micron-size globules of the metal scaffold surface. These cracks probably result from the shrinkage of the scaffold coatings during drying, which was previously reported by [30]. The surface roughness and contact angle of the studied samples are characteristic features that can limit bacterial colonization. The 3D roughness parameters (average roughness – Sa, root mean square roughness – Sq, skewness – Ssk and kurtosis – Sku) are listed in Table 2. The deposition of the NPs onto the EBM-manufactured Ti6Al4V surface did not result in significantly different surface roughness values (p > 0.05) except for the Ti6Al4V + AgNPs specimens (p < 0.01). It may be concluded that the roughness characteristics of all studied samples possess commensurate values that are dominated by the features of the as manufactured surface of the EBM-manufactured samples.

Fig. 2. EDX spectra of the Ti6Al4V alloy covered with AgNPs (A), CaPNPs (B) and (CaP+Ag)NPs (C). 825

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Table 1 Composition (AAS analysis) of the deposited Ag-, CaP- and (Ag + CaP)NPs. Sample

Concentration of Ca, mg/сm2

Concentration of PO4, mg/cm2

Concentration of Ag, mg/сm2

Ti6Al4V alloy covered with AgNPs Ti6Al4V alloy covered with CaPNPs Ti6Al4V alloy covered (Ag + CaP)NPs

Not applicable 0.77 ± 0.25 0.93 ± 0.14

Not applicable 0.48 ± 0.18 0.58 ± 0.08

0.025 ± 0.001 Not applicable 0.021 ± 0.001

with some selected physical properties, especially for the samples that do not contain Ag (CaPNPs) or the Ag is not exposed on the surface (AgNPs+CaPNPs). Large contact angles affect only the initial attachment but have the least effects after 3 h (see the distribution of vectors). Contrary, low contact angles contribute to the decreased growth of bacteria in CaPNPs and AgNPs+CaPNPs samples. The chemical variables describing the amount and ratio of Ag content are in the opposite direction of most of the physical variables describing surface roughness. The CaPNPS and AgNPs+CaPNPs samples act similar against the bacterial attachment and the most prominent effect was observed at 3 h of exposure time. According to the graphs (see Fig. 1A), the CaPNPs showed the biggest initial attachment of cells among all samples. For the CaPNPS and AgNPs+CaPNPs samples, the highest antimicrobial effect had a higher zeta potential; larger surface free energy, Ssk, and Sku; a bigger particle size; and low contact angles. In the case of the samples covered with AgNPs, the contact angle seems to be less important for bacterial attachment than the surface roughness parameters that are the lowest among all the samples. Although three coatings, AgNPs, CaPNPs and AgNPs+CaPNPs, have strong effects on the bacteria growth on their surfaces, the CaPNPs based coating outperformed the other ones since the overall distances of the whole group of replicates between the vectors of variables reflecting the amount of bacteria is the biggest when considering all time points. Based on our analysis, the AgNPs+CaPNPs coating is less consistent between replicates since it is formed over a much larger spread of observations than CaPNPs. The Ag inside the coating is well covered by the CaPNPs and resembles the properties of CaPNPs since these two groups of observations overlap in the space of the first two components.

components for these types of surfaces were calculated for 1°. Gennes [32] reported a high hydrophilicity of a similar surface, which was due to the contributions of strong polar chemical bonds towards the free surface energy in compounds with PO43− and OH– groups. This fact is also confirmed by the results reported elsewhere [33,34]. Free surface energy for titanium consistent with the results reported in this study, similarly to that known for hydroxyapatite [35]. 3.2. Assay for bacterial colonization of surfaces An assay for bacterial colonization was performed with the shortterm exposure of S. aureus to the scaffold surfaces (Fig. 3). In addition, the calculations of the raw data in relative units were also presented (Fig. S2). The amount of adhered S. aureus bacteria was calculated for each test group at three experimental points and compared to the mean value for the uncoated controls (equated to 100% bacterial adhesion) to give a relative amount of adhered bacteria. Contact with the Ti6Al4V + AgNPs (p < 0.05) and Ti6Al4V + CaPNPs (p < 0.001) samples reduced the number of attached S. aureus cells by 40% compared to the as-manufactured Ti6Al4V sample at the beginning of the experiments. After 3 h, there was an increase in the NAC on the Ti6Al4V + AgNPs implants relative to those on the Ti6Al4V + CaPNPs and Ti6Al4V +(Ag + CaP)NPs samples; however, after 17 h, the NAC was reduced and reached 55% of the NAC on the uncoated sample. In case of the Ti6Al4V + CaPNPs samples, the NAC increased within the exposure time, thereby indicating an absence of long-term antimicrobial activity. The Ti6Al4V +(Ag + CaP)NPs sample possessed a reduced NAC after 3 h of exposure, which remained at the same level for 17 h (70% of the NAC detected on the uncoated sample, p < 0.05). All the surface coatings had lower amounts of bacteria observed at all time points in comparison with the uncoated samples (KruskalWallis, p < 0.05). The comparisons between the different treatments showed similar effects on bacteria growth except at 3 h, where we observed more bacteria on the AgNPs surface than on the other coatings (Kruskal-Wallis, p = 0.05). In the principal component analysis (PCA), the first two principle components (Dim1 and Dim2) explain > 83% of the variance. The chemical variables (yellow vectors) are grouped together with those physical variables that determine the charge of the surface of the attached particles, the surface energy, the skewness (Ssk) and the kurtosis (Sku) of the surface. According to the results obtained, these parameters together with the contact angle correlate well with the first principal component (dim1) and the second principal component (dim2) correlates well with the content of Ag and surface roughness parameters. According to the PCA, the most important components are the chemical variables such as the amounts of Ca, P and Ag (which are opposite to the “bacteria3h” vector), but they must be in the right combination

4. Discussion Implant-associated infections remain a serious complication in orthopaedic surgery and lead to implant failure. A number of studies have been carried out over the past few decades in order to modify the bacterial adhesion and growth on the surfaces of implanted materials [36]. Early implant-associated infections usually arise within several months after surgery [36–38]. The main focus of the researchers and clinicians is on the prevention of bacterial biofilm formation on the implant surface since terminating the bacteria protected by a biofilm is extremely hard [36]. The most effective approach to minimize the risk of bacterial infections may be to prevent the formation of biofilms within the first few hours to the first days after implantation [36]. It is widely accepted that one of the predominant features determining bacterial adhesion is surface roughness [39]. Teughels et al. demonstrated that the higher the surface roughness is, the better it is for biofilm formation and maturation [40]. Moreover, biofilm formation was not suppressed significantly further when the roughness was reduced to below 0.2 μm (Ra), as shown in ref. [41]. The authors of this

Table 2 Surface roughness parameters of the Ti6Al4V, Ti6Al4V with Ag-, CaP- and (Ag + CaP)NPs. Parameter

Sa, μm

Ti6Al4V Ti6Al4V + AgNPs Ti6Al4V + CaPNPs Ti6Al4V+(Ag + CaP)NPs

50.0 34.0 39.0 42.5

± ± ± ±

Sq, μm 11.0 8.0 2.0 15.0

61.0 40.0 49.0 53.0

826

± ± ± ±

Ssk 12.0 10.0 4.0 18.0

1.1 1.2 1.4 1.5

Sku ± ± ± ±

0.2 0.3 0.2 0.1

3.2 3.3 3.7 4.1

± ± ± ±

0.3 0.9 0.4 0.4

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Table 3 The contact angle values of and free surface energy calculated for Ti6Al4V, Ti6Al4V with Ag-, CaP- and (Ag + CaP)NPs. Parameters Contact angle, °

Ti6Al4V substrate

Ti6Al4V + AgNPs

Ti6Al4V + CaPNPs

Water

95.9 ± 0.9

54.7 ± 0.7

< 5° Drop spreads

Diiodomethane

41.6 ± 0.3

22.4 ± 0.2

Ethylene glycol

61.7 ± 0.4

15.0 ± 0.3

38.40 ± 4.84 0.39 ± 0.03 38.01 ± 4.05

45.16 ± 5.24 16.85 ± 2.03 28.31 ± 3.16

Ti6Al4V + (Ag + CaP)NPs

Superhydrophilic state

Free surface energy, mN/m Polar, mN/m Dispersive, mN/M

study highlighted that with Ra > 0.2 μm, there is a positive correlation between Ra and biofilm formation. The as-manufactured EBM samples possess a naturally rough surface because of the layer-by-layer melting process and the presence of attached precursor powder grains. The measured roughness parameters of the studied samples were much higher than the threshold Ra established by Bollen et al. Interestingly, we also observed the reduction of the adherence of S. aureus on the untreated samples with the increase in the exposure time. It is assumed that the antimicrobial activity of the as manufactured by EBM Ti6Al4V surface may be attributed either to the presence of a few nanometres thick native titanium oxide passivation layer that is typically observed on the surface of titanium alloys or the surface physical properties which allow cell attachment and proliferation on the surface. Although the TiO2 layer was shown to reduce bacterial adhesion due to the presence of reactive ion species [42,43], however, in our case the materials were not photo activated. Perhaps, high values of contact angle of the native surface, chemically inert material and the setup of the experiment, where cells can get sedimented at the bottom glass plate of the chamber if they are not irreversibly attached, result in decreased number of the cells on the native material. In contrast, in the case of materials with modified surfaces, which are chemically active, small contact angle values enable good interaction of the antibacterial surface with bacteria and thus the decrease of the number of bacteria even further compared with the case of the unmodified surfaces. In addition, the nanoscale features of the samples coated with NP had a considerable impact on inhibiting bacterial adhesion. This is also a known phenomenon as a number of studies proved that the nanoscale roughness of the surface exhibited significant antimicrobial effects against gram-positive and gram-negative bacteria [39,44]. For instance, a Ti–6Al–4 V substrate coated with hydroxyapatite NPs (110–170 nm) using electrophoretic deposition alone decreased the bacterial attachment of different bacteria strains, such as P. aeruginosa, S. aureus and ampicillin resistant E. coli [45]. The bactericidal activity of nanostructured hydroxyapatite and CaP coatings or dispersed NPs were also recently demonstrated [46]. These studies indicated that the nanostructured surface along with the chemical features can increase antibacterial effect. Moreover, the advantage of the CaP deposits is that they have a positive effect on osteogenic cell regulation and bone regeneration [45,47,48], and thus the deposition of the AgNPs and CaPNPs together can be beneficial for bone implants. As a result, the decreased number of microbial cells on the surface of the samples after the electrophoretic deposition of NPs can be besides the chemical composition also due to the higher surface nanoroughness caused by the embedded NPs. It is also noticeable from PCA that the hydrophilicity of the samples also contributes to the reduction of bacterial adhesion. This can be explained either by (i) physical repelling or decreased efficiency of attachment of bacterial cells, (ii) increased chemical antimicrobial effects due to the closer contacts contributed by the hydrophilicity or (iii) mixed effect where both the repulsion and

72.39 48.09 24.30

72.39 48.09 24.30

chemical antimicrobial activity act on cells. In the case of S. aureus the initial attachment is governed by hydrophobic interactions [49]. S. aureus cells exhibit hydrophobic properties (water contact angle of 72°) due to the presence of highly negatively charged and hydrophobic polymers composing S. aureus cell walls [50,51]. This intrinsic property is common to all gram positive bacteria and especially pronounced in Actinobacteria and Mycobacteria. Thus, S. aureus has a lower propensity to adhere to hydrophilic surfaces, which we observed at the initial exposure (see native material vs. modified at 0 h – Fig. 3a). Moreover, the chemical effects of CaPNPs has been reported [46] and we can assume that in our observations the mixed effect attributed to the decreased number of cells during the consecutive time points after the initial exposure. In the case of hydrophilic CaPNPs containing samples, we assume that the measured contact angle is affected by the positive zeta potential and low surface energy induced by the positively charged PEI covering the particles. This is in line with the findings of Hua et al. that show the enhanced hydrophilicity of a silicone surface covered by nanometre thick layers of PEI [52]. Therefore, in case of the Ti6Al4V + CaPNPs and Ti6Al4V +(Ag + CaP)NPs samples, the presence of PEI on the surface of CaPNPs may cause decreasing NAC on the sample surfaces at the beginning of the experiment. It is well known that PVP has no antibacterial and toxic effects on cells. However, PEI can have these effects because of its positive charge, which affects the cell membranes [53]. The thickness of the polymer layer is quite difficult to determine, and it should be at the order of the molecule size of about a few nanometres according to a study by Wallat K. [53]. In the case of CaPNPs, the thermogravimetric analysis could show the amount of polymer adsorbed on CaPNPs with the synthesis performed in this study. We assume that up to 3–10 wt% of PEI can be expected. According to the colonization assay, the bactericidal effect of PEI on the surface of CaPNPs is limited. The observed colonization rate is considered to be a physical effect, which makes these surfaces free from drug resistance and may lead to long-term effects. The impact of AgNPs on the cell adhesion seems to appear after 17 h of exposure, which manifested itself in the reduction of the amount of bacteria on the Ti6Al4V + AgNPs samples. The latter observation is consistent with the fact that the biological effects of silver are size and dose-dependent [54–56]. The antibacterial effect of Ag is mainly based on the Ag+ ions and is therefore dependent on the dissolution rate of AgNPs [57]. According to Stebounova et al. [58], AgNPs have very low dissolution rates in bodily fluids (< 0.6%) and the antibacterial potential of AgNPs is greatly reduced as their size increases above 20 nm. As the size of AgNPs used in this work was 70 ± 12 nm, we assume that at 17 h of exposure, the concentration of silver ions in the media became enough to hinder the bacteria growth. In the case of the assembly of particles covering the surface of the scaffold, the synergistic effect from both components is considered to prevent biofilm formation. Thus, the obtained results indicated that the electrophoretic 827

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the cytocompatibility of the developed coatings. 5. Conclusions Antibiotic resistance in the context of surgical site infections is one of the most crucial problems in surgery. This problem is especially acute when a foreign object (implant) is placed into human body. To address the problem of implant-associated infections, Ag and CaPNPs and their assemblies were incorporated on the surface of the metallic scaffolds manufactured by electron beam melting and electrophoretic deposition, and the bacterial colonization abilities on these coatings were investigated. The synthesis conditions of the NPs applied in this study resulted in PVP-stabilized AgNPs with an average size of 70 ± 12 nm and PEI-stabilized CaPNPs with diameters of 90 ± 20 nm. The electrophoretic deposition of NPs allowed us to achieve tailored morphology without changing the microscale structure of the EBM-manufactured scaffolds. The assay for surface colonization by bacteria was performed with the exposure of S. aureus to the scaffold surfaces occurring for up to 17 h. The results indicate that modifying the implant surface with appropriate nanostructures that change the hydrophobicity and the surface roughness at the nano scale disrupt the bacterial adhesion. Our results clearly show that AgNPs at the concentration of approximately 0.02 mg/сm2 that are deposited together with the CaPNPs covered by a positively charged PEI on the surface of the EBMmanufactured Ti6Al4V scaffolds hindered bacteria growth since the total amount of NAC of S. aureus remained at the same level during the 17 h of exposure, which is the indication of bacteriostatic activity.

B

Acknowledgements This research was supported by the Russian Science Foundation (Grant 15-13-00043) and the Slovenian Research Agency projects J16746, J4-7640, J1-9194 and BI-RU/16-18-039. We acknowledge the support from the Deutscher Akademischer Austauschdienst (DAAD) within the framework of the Leonhard-Euler programme. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.apsusc.2019.03.003. References [1] S. Giannitelli, D. Accoto, M. Trombetta, A. Rainer, Current trends in the design of scaffolds for computer-aided tissue engineering, Acta Biomater. 10 (2014) 580–594. [2] S.M. Peltola, F.P. Melchels, D.W. Grijpma, M. Kellomäki, A review of rapid prototyping techniques for tissue engineering purposes, Ann. Med. 40 (2008) 268–280. [3] S.M. Ahmadi, S.A. Yavari, R. Wauthle, B. Pouran, J. Schrooten, H. Weinans, A.A. Zadpoor, Additively manufactured open-cell porous biomaterials made from six different space-filling unit cells: the mechanical and morphological properties, Materials 8 (2015) 1871–1896. [4] S. Van Bael, Y.C. Chai, S. Truscello, M. Moesen, G. Kerckhofs, H. Van Oosterwyck, J.-P. Kruth, J. Schrooten, The effect of pore geometry on the in vitro biological behavior of human periosteum-derived cells seeded on selective laser-melted Ti6Al4V bone scaffolds, Acta Biomater. 8 (2012) 2824–2834. [5] L.E. Murr, Frontiers of 3D printing/additive manufacturing: from human organs to aircraft fabrication, Journal of Materials Science & Technology 32 (2016) 987–995. [6] M.A. Surmeneva, R.A. Surmenev, E.A. Chudinova, A. Koptioug, M.S. Tkachev, S.N. Gorodzha, L.-E. Rännar, Fabrication of multiple-layered gradient cellular metal scaffold via electron beam melting for segmental bone reconstruction, Mater. Des. 133 (2017) 195–204. [7] X. Wang, S. Xu, S. Zhou, W. Xu, M. Leary, P. Choong, M. Qian, M. Brandt, Y.M. Xie, Topological design and additive manufacturing of porous metals for bone scaffolds and orthopaedic implants: a review, Biomaterials 83 (2016) 127–141. [8] E.A. Lewallen, S.M. Riester, C.A. Bonin, H.M. Kremers, A. Dudakovic, S. Kakar, R.C. Cohen, J.J. Westendorf, D.G. Lewallen, A.J. Van Wijnen, Biological strategies for improved osseointegration and osteoinduction of porous metal orthopedic implants, Tissue Eng. B Rev. 21 (2014) 218–230. [9] W. Tang, D. Lin, Y. Yu, H. Niu, H. Guo, Y. Yuan, C. Liu, Bioinspired trimodal macro/ micro/nano-porous scaffolds loading rhBMP-2 for complete regeneration of critical size bone defect, Acta Biomater. 32 (2016) 309–323. [10] S.A. Yavari, J. van der Stok, Y.C. Chai, R. Wauthle, Z.T. Birgani, P. Habibovic,

Fig. 3. Amount of bacteria on the Ti6Al4V, Ti6Al4V with CaPNPs, AgNPs, and (Ag + CaP)NPs scaffolds, respectively. A) Amount of bacteria on the Ti6Al4V, Ti6Al4V with CaPNPs, AgNPs, and (Ag + CaP)NPs scaffolds. *indicates the mean difference is significant at p < 0.001, and ** indicates the mean difference is significant at p < 0.05. B) Principle component analysis (PCA) of the properties of different material coatings. The vectors and dots represent the variables and observations (individuals), respectively. Yellow, red and blue vectors are the variables representing the chemical, antimicrobial and physical properties of the material, respectively. The replicates for each of the material coatings are encircled with ellipses. The larger symbol in the middle of each of the ellipses is a centroid.

deposition of nanosized features of AgNPs and the assembly of AgNPs and CaPNPs on the EBM-manufactured scaffold surface induce a bacteriostatic effect against S. aureus. Therefore, further dedicated studies on the obtained scaffolds would be required to assess whether such surface treatments can have the serious potential to prevent/combat infections on time scales of several weeks. It is also important to assess 828

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[11]

[12] [13]

[14]

[15] [16]

[17]

[18]

[19]

[20]

[21]

[22]

[23] [24] [25] [26] [27]

[28]

[29]

[30]

[31]

[32] [33]

[34]

Charact. 49 (2002) 129–137. [35] E. Dos Santos, M. Farina, G. Soares, K. Anselme, Surface energy of hydroxyapatite and β-tricalcium phosphate ceramics driving serum protein adsorption and osteoblast adhesion, J. Mater. Sci. Mater. Med. 19 (2008) 2307–2316. [36] R.O. Darouiche, Treatment of infections associated with surgical implants, N. Engl. J. Med. 350 (2004) 1422–1429. [37] A. Han, J.K. Tsoi, F.P. Rodrigues, J.G. Leprince, W.M. Palin, Bacterial adhesion mechanisms on dental implant surfaces and the influencing factors, Int. J. Adhes. Adhes. 69 (2016) 58–71. [38] A.W. Smith, Biofilms and antibiotic therapy: is there a role for combating bacterial resistance by the use of novel drug delivery systems? Adv. Drug Deliv. Rev. 57 (2005) 1539–1550. [39] W. Teughels, N. Van Assche, I. Sliepen, M. Quirynen, Effect of material characteristics and/or surface topography on biofilm development, Clin. Oral Implants Res. 17 (2006) 68–81. [40] C.M. Bollen, W. Papaioanno, J. Van Eldere, E. Schepers, M. Quirynen, D. Van Steenberghe, The influence of abutment surface roughness on plaque accumulation and peri-implant mucositis, Clin. Oral Implants Res. 7 (1996) 201–211. [41] A.K. Suresh, D.A. Pelletier, M.J. Doktycz, Relating nanomaterial properties and microbial toxicity, Nanoscale 5 (2013) 463–474. [42] D. Campoccia, L. Montanaro, C.R. Arciola, A review of the biomaterials technologies for infection-resistant surfaces, Biomaterials 34 (2013) 8533–8554. [43] I.A. van Hengel, M. Riool, L.E. Fratila-Apachitei, J. Witte-Bouma, E. Farrell, A.A. Zadpoor, S.A. Zaat, I. Apachitei, Selective laser melting porous metallic implants with immobilized silver nanoparticles kill and prevent biofilm formation by methicillin-resistant Staphylococcus aureus, Biomaterials 140 (2017) 1–15. [44] G. Bhardwaj, H. Yazici, T.J. Webster, Reducing bacteria and macrophage density on nanophase hydroxyapatite coated onto titanium surfaces without releasing pharmaceutical agents, Nanoscale 7 (2015) 8416–8427. [45] S.M.G. El-Rab, S.A. Fadl-allah, A. Montser, Improvement in antibacterial properties of Ti by electrodeposition of biomimetic Ca–P apatite coat on anodized titania, Appl. Surf. Sci. 261 (2012) 1–7. [46] V.M. Wu, S. Tang, V. Uskoković, Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: the antibacterial effect, ACS Appl. Mater. Interfaces 10 (2018) 34013–34028. [47] C.-W. Chan, L. Carson, G.C. Smith, A. Morelli, S. Lee, Enhancing the antibacterial performance of orthopaedic implant materials by fibre laser surface engineering, Appl. Surf. Sci. 404 (2017) 67–81. [48] H. Zhou, J. Lee, Nanoscale hydroxyapatite particles for bone tissue engineering, Acta Biomater. 7 (2011) 2769–2781. [49] M. Gross, S.E. Cramton, F. Götz, A. Peschel, Key role of teichoic acid net charge in Staphylococcus aureus colonization of artificial surfaces, Infect. Immun. 69 (2001) 3423–3426. [50] H. Ai, H. Meng, I. Ichinose, S.A. Jones, D.K. Mills, Y.M. Lvov, X. Qiao, Biocompatibility of layer-by-layer self-assembled nanofilm on silicone rubber for neurons, J. Neurosci. Methods 128 (2003) 1–8. [51] N. Mitik-Dineva, J. Wang, V.K. Truong, P. Stoddart, F. Malherbe, R.J. Crawford, E.P. Ivanova, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus attachment patterns on glass surfaces with nanoscale roughness, Curr. Microbiol. 58 (2009) 268–273. [52] N. Beyth, Y. Houri-Haddad, L. Baraness-Hadar, I. Yudovin-Farber, A.J. Domb, E.I. Weiss, Surface antimicrobial activity and biocompatibility of incorporated polyethylenimine nanoparticles, Biomaterials 29 (2008) 4157–4163. [53] K. Wallat, Elektrophoretische Abscheidung von Thermochromen und Bioaktiven Nanopartikeln, Universitätsbibliothek Duisburg-Essen, 2014. [54] S. Agnihotri, S. Mukherji, S. Mukherji, Size-controlled silver nanoparticles synthesized over the range 5–100 nm using the same protocol and their antibacterial efficacy, RSC Adv. 4 (2014) 3974–3983. [55] R.A. Bapat, T.V. Chaubal, C.P. Joshi, P.R. Bapat, H. Choudhury, M. Pandey, B. Gorain, P. Kesharwani, An overview of application of silver nanoparticles for biomaterials in dentistry, Mater. Sci. Eng. C 91 (2018) 881–898. [56] H. Geng, G. Poologasundarampillai, N. Todd, A. Devlin-Mullin, K.L. Moore, Z. Golrokhi, J.B. Gilchrist, E. Jones, R.J. Potter, C. Sutcliffe, Biotransformation of silver released from nanoparticle coated titanium implants revealed in regenerating bone, ACS Appl. Mater. Interfaces 9 (2017) 21169–21180. [57] X. Zhang, L. Wang, E. Levänen, Superhydrophobic surfaces for the reduction of bacterial adhesion, RSC Adv. 3 (2013) 12003–12020. [58] L.V. Stebounova, E. Guio, V.H. Grassian, Silver nanoparticles in simulated biological media: a study of aggregation, sedimentation, and dissolution, J. Nanopart. Res. 13 (2011) 233–244.

M. Mulier, J. Schrooten, H. Weinans, A.A. Zadpoor, Bone regeneration performance of surface-treated porous titanium, Biomaterials 35 (2014) 6172–6181. E.A. Chudinova, M.A. Surmeneva, A.S. Timin, T.E. Karpov, A. Wittmar, M. Ulbricht, A. Ivanova, K. Loza, O. Prymak, A. Koptyug, Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles, Colloids Surf. B: Biointerfaces, 176 130–139. S. Bakhshandeh, S.A. Yavari, Electrophoretic deposition: a versatile tool against biomaterial associated infections, J. Mater. Chem. B 6 (2018) 1128–1148. S. Bakhshandeh, Z. Gorgin Karaji, K. Lietaert, A.C. Fluit, C.E. Boel, H.C. Vogely, T. Vermonden, W.E. Hennink, H. Weinans, A.A. Zadpoor, Simultaneous delivery of multiple antibacterial agents from additively manufactured porous biomaterials to fully eradicate planktonic and adherent Staphylococcus aureus, ACS Appl. Mater. Interfaces 9 (2017) 25691–25699. R. Saginur, M. StDenis, W. Ferris, S.D. Aaron, F. Chan, C. Lee, K. Ramotar, Multiple combination bactericidal testing of staphylococcal biofilms from implant-associated infections, Antimicrob. Agents Chemother. 50 (2006) 55–61. S. Chernousova, M. Epple, Silver as antibacterial agent: ion, nanoparticle, and metal, Angew. Chem. Int. Ed. 52 (2013) 1636–1653. C. Greulich, D. Braun, A. Peetsch, J. Diendorf, B. Siebers, M. Epple, M. Köller, The toxic effect of silver ions and silver nanoparticles towards bacteria and human cells occurs in the same concentration range, RSC Adv. 2 (2012) 6981–6987. J. Helmlinger, C. Sengstock, C. Gross-Heitfeld, C. Mayer, T. Schildhauer, M. Köller, M. Epple, Silver nanoparticles with different size and shape: equal cytotoxicity, but different antibacterial effects, RSC Adv. 6 (2016) 18490–18501. J.S. Kim, E. Kuk, K.N. Yu, J.-H. Kim, S.J. Park, H.J. Lee, S.H. Kim, Y.K. Park, Y.H. Park, C.-Y. Hwang, Antimicrobial effects of silver nanoparticles, Nanomedicine 3 (2007) 95–101. A.A. Ivanova, R.A. Surmenev, M.A. Surmeneva, T. Mukhametkaliyev, K. Loza, O. Prymak, M. Epple, Hybrid biocomposite with a tunable antibacterial activity and bioactivity based on RF magnetron sputter deposited coating and silver nanoparticles, Appl. Surf. Sci. 329 (2015) 212–218. D. Kozlova, S. Chernousova, T. Knuschke, J. Buer, A.M. Westendorf, M. Epple, Cell targeting by antibody-functionalized calcium phosphate nanoparticles, J. Mater. Chem. 22 (2012) 396–404. A.D. Edelstein, M.A. Tsuchida, N. Amodaj, H. Pinkard, R.D. Vale, N. Stuurman, Advanced methods of microscope control using μManager software, J. Biol. Methods 1 (2014) 1–18. J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, Fiji: an open-source platform for biological-image analysis, Nat. Methods 9 (2012) 676. R.C. Team, R: A Language and Environment for Statistical Computing, (2013). S. Lê, J. Josse, F. Husson, FactoMineR: an R package for multivariate analysis, J. Stat. Softw. 25 (2008) 1–18. H. Wickham, Elegant Graphics for Data Analysis (ggplot2), Springer-Verlag, New York, NY, 2009. F.M. Alboukadel Kassambara, cran.r-project.org. Y. Kok, X. Tan, S.B. Tor, C.K. Chua, Fabrication and microstructural characterisation of additive manufactured Ti-6Al-4V parts by electron beam melting: this paper reports that the microstructure and micro-hardness of an EMB part is thickness dependent, Virtual and Physical Prototyping 10 (2015) 13–21. X. Tan, Y. Kok, Y.J. Tan, M. Descoins, D. Mangelinck, S.B. Tor, K.F. Leong, C.K. Chua, Graded microstructure and mechanical properties of additive manufactured Ti–6Al–4V via electron beam melting, Acta Mater. 97 (2015) 1–16. S. Banerjee, K. Loza, W. Meyer-Zaika, O. Prymak, M. Epple, Structural evolution of silver nanoparticles during wet-chemical synthesis, Chem. Mater. 26 (2014) 951–957. M. Dinu, A.A. Ivanova, M.A. Surmeneva, M. Braic, A.I. Tyurin, V. Braic, R.A. Surmenev, A. Vladescu, Tribological behaviour of RF-magnetron sputter deposited hydroxyapatite coatings in physiological solution, Ceram. Int. 43 (2017) 6858–6867. S. Amin Yavari, L. Loozen, F.L. Paganelli, S. Bakhshandeh, K. Lietaert, J.A. Groot, A.C. Fluit, C. Boel, J. Alblas, H.C. Vogely, Antibacterial behavior of additively manufactured porous titanium with nanotubular surfaces releasing silver ions, ACS Appl. Mater. Interfaces 8 (2016) 17080–17089. P.-G. De Gennes, Wetting: statics and dynamics, Rev. Mod. Phys. 57 (1985) 827. B. Feng, J.Y. Chen, S.K. Qi, L. He, J.Z. Zhao, X.D. Zhang, Characterization of surface oxide films on titanium and bioactivity, J. Mater. Sci. Mater. Med. 13 (2002) 457–464. B. Feng, J. Weng, B.C. Yang, J.Y. Chen, J.Z. Zhao, L. He, S.K. Qi, X.D. Zhang, Surface characterization of titanium and adsorption of bovine serum albumin, Mater.

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