A scanning probe investigation

Materials Science and Engineering C 62 (2016) 444–449

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Magnetic hydroxyapatite coatings as a new tool in medicine: A scanning probe investigation A. Gambardella a,⁎, M. Bianchi a, S. Kaciulis b, A. Mezzi b, M. Brucale b, M. Cavallini c, T. Herrmannsdoerfer d, G. Chanda d, M. Uhlarz d, A. Cellini e, M.F. Pedna e, V. Sambri e,g, M. Marcacci a,f, A. Russo a,f a

Laboratorio di NanoBiotecnologie (NaBi), Istituto Ortopedico Rizzoli, Via di Barbiano 1/10, I-40136 Bologna, Italy Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche, Via Salaria km 29.300, P.O. Box 10, 00015 Monterotondo Staz, Roma, Italy Magnetic Nanostructures for Spintronics and Nanomedicine, CNR-ISMN, Via Gobetti 101, 40129 Bologna, Italy d Dresden High Magnetic Field Laboratory (HLD-EMFL), Helmholtz-Zentrum Dresden-Rossendorf, D-01314 Dresden, Germany e Unità Operativa Microbiologia Laboratorio Unico del Centro Servizi AUSL della Romagna, Pievesestina, Cesena, Italy f Laboratorio di Biomeccanica ed Innovazione Tecnologica, Istituto Ortopedico Rizzoli, Via di Barbiano1/10, I-40136 Bologna, Italy g Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale (DIMES), Università degli Studi di Bologna, Via Zamboni 33, 40126 Bologna, Italy b c

a r t i c l e

i n f o

Article history: Received 9 November 2015 Received in revised form 21 December 2015 Accepted 27 January 2016 Available online 29 January 2016 Keywords: Magnetic biomaterials Scanning probes Nanomedicine Tissue engineering Percolation Escherichia coli adhesion

a b s t r a c t Hydroxyapatite films enriched with magnetite have been fabricated via a Pulsed Plasma Deposition (PPD) system with the final aim of representing a new platform able to disincentivate bacterial adhesion and biofilm formation. The chemical composition and magnetic properties of films were respectively examined by X-ray photoelectron spectroscopy (XPS) and Superconducting Quantum Interference Device (SQUID) measurements. The morphology and conductive properties of the magnetic films were investigated via a combination of scanning probe technologies including atomic force microscopy (AFM), electrostatic force microscopy (EFM), and scanning tunneling microscopy (STM). Interestingly, the range of adopted techniques allowed determining the preservation of the chemical composition and magnetic properties of the deposition target material while STM analysis provided new insights on the presence of surface inhomogeneities, revealing the presence of magnetite-rich islands over length scales compatible with the applications. Finally, preliminary results of bacterial adhesion tests, indicated a higher ability of magnetic hydroxyapatite films to reduce Escherichia coli adhesion at 4 h from seeding compared to control hydroxyapatite films. © 2016 Elsevier B.V. All rights reserved.

1. Introduction More than 250,000 joint replacements are performed yearly only in the United States. Implant infection, which occurs for 1–2% of primary replacements and 3–4% of revisions of previously infected prostheses, is a common and fearsome complication [1]. Antibiotic-loaded implant coatings present a straightforward approach for the prevention of implant-associated infections, as they allow the local delivery of the high antibiotics concentration, thus minimizing systemic drug exposure that can lead to increased toxicity and increased antibiotic resistance. The utilization of a bioactive hydroxyapatite (HA) ceramic coating as antibiotic carriers offers the added value of providing the physiochemical environment and structural scaffold required for bone-implant integration thanks to excellent biocompatibility, osteoconductivity, lack of cytotoxicity and nonimmunogenicity. In vitro-release of antibiotics from HA-coated implants has been reported for chlorhexidine, vancomycin, gentamicin, tobramycin and several ⁎ Corresponding author. E-mail address: [email protected] (A. Gambardella).

http://dx.doi.org/10.1016/j.msec.2016.01.071 0928-4931/© 2016 Elsevier B.V. All rights reserved.

other antibiotics [2]. However, calcium phosphates suffer of limited or no ability to be loaded with organic molecules via intercalation, which limits loading mechanism only to physisorption and makes prolonged release difficult to achieve [3]. Thus, recent studies have been focused on the incorporation of inorganic factors, such as metal nanoparticles and ions into the HA structure [4]. Magnetic iron oxide nanoparticles have been recently attracted great attention due to their unique ability to kill bacteria by easily penetrating the biofilm membrane, especially under the effect of an external magnet [5]. However, only few reports about the fabrication of a magnetite/hydroxyapatite composite coatings for medical purposes are available in literature [6–9] and, to our knowledge, no one realized by an industrially-scalable deposition technology such as a physical vapor deposition (PVD) or plasma-spraying techniques. Recently, we reported the first deposition of HA thin films by the Pulsed Plasma Deposition (PPD) method, an innovative PVD technique showing as a valid alternative to conventional plasma sprayed coatings [10]. In this work, we have fabricated for the first time magnetic HA coatings (Mag HA) by ablating a HA/magnetite target, with the aim of providing a new platform to control bacterial adhesion on the micro- and nanoscale. In order to fully characterize the deposited

A. Gambardella et al. / Materials Science and Engineering C 62 (2016) 444–449

coatings, we carried out a surface analysis focusing on chemical, magnetic, morphological and electronic characterizations. In particular, our attention has been focused on conductive atomic force microscopy (C-AFM) and scanning tunneling microscopy and spectroscopy (STMSTS) techniques, as complementary and powerful tools for the detection of the magnetic component at scale lengths comparable with the applications, i.e. the bacterial characteristic dimensions. Finally, we carried out some preliminary bacterial adhesion inhibition tests to compare the ability of Mag HA coatings compared to HA to block the bacterial adhesion. 2. Experimental 2.1. Materials HA/Magnetite (90:10 wt.%) cylindrical targets (10 × 8 mm2, generously provided by the Institute of Science and Technology for Ceramics of the Italian National Research Council (ISTEC-CNR of Faenza, Italy) were used as target material for the deposition of magnetic hydroxyapatite thin films. Briefly, commercial HA powders (Finceramica Faenza Spa, Italy) were calcined at 1000 °C for 2 h in order to decrease the specific surface area value and prepare stable suspensions with higher solid loading. The optimized suspension was prepared by ball mixing deionized water, 1.5 wt.% of dispersant Dolapix CE- 64 (Zschimmer & Schwarz, Lahnstein, Germany) and HA and magnetite nanopowders (50–100 nm particle size (TEM), 97% trace metals basis, Sigma Aldrich) having a specific surface area value of 3.93 m2/g and 10.41 m2/g, respectively, to achieve a 90/10 wt.% final composition. Finally, the suspension was sintered at 1200 °C for 1 h in controlled atmosphere of Ar/H2 so that to maintain high magnetization value (associated with the starting amount of magnetite powder) and to reach the highest consolidation extent. According to the producer, the hysteresis temperature curve at 309 K shows a saturation magnetization of 0.26 emu/g, 90% of it was reached at 0.08 T (saturation field). Pure HA cylindrical targets have been also realized according to a similar protocol in order to deposit HA coatings to be used as control. Silicon wafers (p-type doped monocrystalline (100) silicon, size of (10 × 10 × 2) mm3 (Bruno Kessler Foundation, Trento, Italy) were used as substrates. 2.2. Film deposition A Pulsed Electron Ablation system in the PPD version [11–13] was used to deposit thin films of Mag HA by the ablation of the target described in the Section 2.1. The vacuum chamber for the deposition was initially voided down to a pressure of 1 × 10− 6 mbar by using a turbo-molecular pump EXT255H (Edwards, Crawley, England). Then the pressure was raised to the working value by introducing in the chamber the working gas (oxygen, purity level: 99.999%). After a preliminary deposition set, aimed to optimize deposition parameters, the working gas pressure, the beam acceleration voltage and the shot frequency were set at 5 × 10−5 mbar, 17.0 kV and 5 Hz, respectively. The deposition was performed at room temperature, i.e. without heating the substrate. 2.3. Atomic force microscopy Film thickness, morphology and roughness were evaluated by AFM operating in semi-contact mode at ambient conditions (Stand-Alone SMENA AFM, NT-MDT, Moscow, Russia). The AFM was equipped with a SFC050 head mounting silicon probes with a guaranteed curvature radius of b 10 nm and resonance frequencies of 190–330 kHz (NSG series probes, NT-MDT, Moscow, RU). The reported values for the average roughness RA were obtained by averaging the values obtained upon at least three non-overlapping sample regions, whose dimensions ranged from (50 × 50) μm2 to (2 × 2) μm2. Image resolution was (512 × 512) pixels.

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2.4. X-ray photoemission spectroscopy The chemical composition of the films was evaluated by X-ray photoelectron spectroscopy (XPS) using an ESCALAB 250Xi spectrometer (Thermo Fisher Scientific, UK), equipped with monochromatic Al Kα excitation source and a 6-channeltron spectroscopic detection system. Photoelectron spectra were collected at 20 eV constant pass energy of the analyzer and a base pressure in the analysis chamber of about 5 × 10− 8 Pa. XPS measurements were carried out at 90° take-off angle, by using a large spot of X-ray source (diameter of 0.9 mm) and electromagnetic lens mode resulting in 0.5 mm diameter of analyzed sample area. The sample charging was suppressed by using in-lens electron flood gun at low energy of 1 eV. The accuracy of experimental binding energy (BE) scale was ±0.1 eV. Spectroscopic data were processed by the Avantage v.5 software. Shirley background and mixed Lorentzian/Gaussian peak shape (30%) were used for the peak fitting. The spin-orbit splitting values used for Fe 2p and Ca 2p spectra were of about 13.0 and 3.6 eV, respectively. The ratio between 2p3/2 and 2p1/2 peak areas was kept at about 2: 1. 2.5. SQUID magnetometry Magnetization measurements were performed using a SQUID magnetometer, that measures the total magnetic moment of a sample including all atomic and molecular magnetic contributions. The sample was fixed to a custom-made sample holder which ensures a canceling of background contributions. The magnetic field was swept at constant temperature of 310 K. Having reached predetermined values of the field, the sample was consistently moved through a pick-up coil system connected to the SQUID via a flux transformer. The magnetization of Mag HA films, as a function of applied magnetic field, was measured in both ascending and descending fields in the field range 0 to ±7 T. 2.6. Electrostatic force microscopy Electrostatic force microscopy (EFM) was performed on a Bruker Multimode8 AFM (Bruker Nano Surface, Milan, Italy) equipped with a Nanoscope V controller, a JV type scanner and conductive Ultrasharp NSC36/Ti–Pt probes (MikroMasch, Sofia, Bulgaria). The microscope was operated in double-pass Lift Mode with lift heights ranging from 30 to 500 nm and a tip bias of ±(100 ÷ 500) mV. The EFM images (both topographies and phase signals) were acquired at 256 × 256 pixel resolution. 2.7. Scanning tunneling microscopy Scanning tunneling microscopy (STM) was performed at ambient conditions by using a Omicron UHV system at a background pressure in the low 10−10 mbar regime, operating with PtIr tips. With the aim of obtaining specimens that can be successfully investigated by STM, a 5 μm-thick Mag HA film was deposited and then scratched with a diamond pencil, obtaining plate-like fragments of size variable around (0.2 ÷ 0.5) mm (the thickness of the sample shown in the Figs. 5 and 6 was (3.1 ± 0.1) μm, measured by a Zeiss Scanning Electron Microscope), that were directly glued on the sample holder by using a conducting paint. Topographies were acquired at the tunneling parameters set at tunnel current I T = (60–120) pA and bias voltage VB = (0.5–1.2) V (grounded sample). Tunneling Spectra (TS) were acquired using the Current Imaging Tunneling Spectroscopy technique (CITS), in which the tip is scanned in the constant current mode to maintain a constant distance from the sample. TS were recorded on several sample's regions, recorded simultaneously with the corresponding topographic image. To limit the effects of the thermal noise, each spectrum was sampled at an energy resolution of about 20 meV, i.e. 400 sampled points/curve, then spectra were averaged over some tens of curves on the same area and numerically differentiated to

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estimate the Local Density Of States (LDOS). STM topographies were acquired at 512 × 512 pixel resolution and unfiltered, apart from subtraction of a 2nd order plane by using Scanning Process Image Analysis and Processing (SPIP®) software. 2.8. Bacterial adhesion Initially, Mag HA and HA samples were placed in 24 well cell culture plates (n = 3 repetitions for each sample). A 0.5 McF (~1 × 108 CFU/ml) bacterial suspension of Escherichia coli (wild type isolate obtained by a urine sample of a patient at the Unit of Microbiology, The Greater Romagna Hub Laboratory, Pievesestina, Cesena, Italy) was diluted in Brainheart infusion broth (Biomerieux, Marcy L'Etoile, France) in order to obtain a final bacterial concentration of 1 × 105 CFU/ml. 500 μl of this diluted bacterial suspension were placed into each individual well of plates containing the Mag HA and HA samples. The plates were then incubated at (36 °C ± 1) °C for 4 h. In order to evaluated the effect of external static magnetic field on the bacterial adhesion, half of the samples was tested with a cylindrical NdFeB magnet (10 mm high × 2 mm diameter; 1.45 T; Italfit Magneti s.r.l., Udine, Italy) placed during the incubation period under each well of the plate. The distance between the top of the magnet and the specimen was set as 3 mm, thus the effective magnetic gradient at the film level, estimated by finite element analysis calculations (not shown) was ~6 T/m. After the incubation time, the bacterial suspension was removed and replaced with 400 μl of glutaraldehyde (2.5% in PBS 1 ×) for sample fixation in dark-incubation over night at 4 °C. After dark-incubation, the fixation agent was removed and samples washed 3 times (20 min each), with 450 μl of phosphate-buffered saline PBS (1 × solution). The so fixed samples were then observed under optical microscope (Eclipse digital 80i, Nikon, equipped with a Hg-Lamp and BP filter LP 475 nm) and bacteria enumerated by exploiting the autofluorescence of E. coli. To this aim, images were acquired over up to 100 different areas on each sample and statistically processed to estimate the bacteria number. Results are provided as mean value standard deviation (SD). Student t-test was used to locate significant (p b 0.05) differences between means. 3. Results and discussion 3.1. Surface morphology Mag HA films showed a nanostructured surface constituted of randomly distributed grains with widths ranging from 50 to 400 nm (Fig. 1a). The films exhibited moderate values of RA, in the range of 20 to 100 nm, which increased with film thickness, with thinner samples exhibiting higher homogeneity (Fig. 1b). There is a wide consensus on the fact that macroporous and highly rough materials promote bacterial adhesion, as grooved and pitted surfaces provide a ‘shelter’ from external forces (i.e. hydrodynamic turbulence), which may otherwise dislodge adhering cells [14]. However, the effect of sub-micrometer roughness is still poorly understood. The role of surface topography on bacterial adhesion has been recently deeply investigated. In a recent study, quantitative force measurements between a staphylococcus and a patterned microgel surface show that the adhesion strength decreases significantly at intergel spacings comparable to bacterial dimensions [15]. Importantly, the adhesion and spreading of osteoblast-like cells was preserved. In another study, the comparison between the adhesion of Staphylococcus aureus to standard micro-rough commercial and flat electropolished titanium was carried out. The results showed a significant decrease in the amount of bacteria adhering to the electropolished compared to standard micro-rough surfaces [16]. The Mag HA films deposited by PPD exhibited a much lower roughness when compared to conventional micro-rough plasma-sprayed coatings; according to this fact, keeping all the other parameters constant, one may expect lower bacterial adhesion compared to conventional coatings.

Fig. 1. a) 5 × 5 μm2 AFM topography of a (790 ± 30) nm-thick sample. b) RA values vs X–Y scale for a set of three samples. Film thickness is: (2100 ± 100) nm (blue), (790 ± 30) nm (red) and (280 ± 30) nm (black curve).

3.2. Chemical composition The investigation of the surface composition by XPS confirmed the presence of Ca2p, P2p and O1s photoemission peaks characteristic for hydroxyapatite [17,18], and of Fe2p containing at least two components (Table 1). The spectra of elemental core levels are depicted in Fig. 2. For a detailed investigation of the chemical states, the results of peak fitting for experimental spectra are also reported. The interpretation of the Fe2p spectrum is the following: two components of Fe (A and B) with their spin-orbit splitting by about 13 eV and following shake-up satellites are present at the binding energy (BE) values that can be assigned to the different chemical states [19,20]. The first Fe2p3/2 component (A) at BE = 710.0 eV corresponds to Fe(+ 3), whereas the second one (B) at higher BE ≈ 713 eV can be attributed to magnetite Fe3 O 4 and/or Fe hydroxide [19,20]. XPS quantification was done by taking into account the atomic sensitivity factors and the dependence of attenuation length on the kinetic energy of photoelectrons. The results of quantitative analysis are reported in the Table 1. Firstly, the atomic ratio of Ca/P = 1.67 confirmed the preservation of the HA stoichiometry, secondly, the atomic ratio of Fe/P confirmed the content of about 10% of Fe, as expected from the composition of the starting deposition target.

Table 1 Quantitative XPS results. Atomic Ca/P and Fe/P ratios confirm the preservation of the hydroxyapatite stoichiometry and the presence of Fe according with the starting target composition. Name

BE [eV]

FWHM [eV]

at%

wt.%

Bond

C1s — 1 C1s — 2 C1s — 3 Ca2p3 Fe2p3 O1s — 1 O1s — 2 P2p

285.0 288.9 286.8 347.0 710.1 530.6 532.3 133.3

2.1 2.1 2.1 2.0 3.5 2.0 2.0 2.2

9.5 4.9 2.8 18.5 1.3 45.4 6.2 11.4

5.2 2.7 1.5 33.7 3.3 33.0 4.5 16.0

Aliphatic –COO C–O Ca2+ Fe3+ PO4 OH− PO4


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Fig. 2. XPS core level spectra are shown along with their related fit curves. The spectra a) b) and c) are characteristics for hydroxyapatite while Fe2p peaks (d) can be attributed to the presence of magnetite.

3.3. Magnetic characterization Mag HA films exhibited a saturation magnetization of about 0.14 emu/g at 0.4 T, with a hysteresis ferromagnetic behavior around the zero-field as shown in Fig. 3 and its inset. Remarkably, this value is compatible with the saturation magnetization of the deposition target

of 0.26 emu/g at 0.08 T, and likely reflects the reduction of the magnetism for mass unit due to clustering or different oxidation of the iron states. Full magnetization saturation being reached at fields of about 0.4 T is thus accessible by state of the art implantable permanent magnets. The coercive field of Mag HA film is negligible (about 50 Oe) which supports the expectation of a super-paramagnetic character of our sample nanoparticles, which is determined by very weak magnetic interactions between the nanoparticles. 3.4. Electrostatic force microscopy

Fig. 3. SQUID measurement showing the magnetization curve vs applied field, with ferromagnetic hysteresis around zero field (inset) and a saturation magnetization of about 0.14 emu/g.

Scanning probe techniques can be employed to achieve a more complete characterization of surface micrometer and sub-micrometer electrostatic/magnetic inhomogeneities, which are not evaluable with large-area-averaging techniques. A variety of signals (amplitude and/ or phase) measurable via double pass/interleave mode atomic force microscopy using conductive and/or magnetic probes can be exploited to provide a qualitative mapping of the electrostatic charges and eventually magnetic fields at the nanoscale, although it is expected that the relatively low concentration of the magnetic fraction may result in small contrasts [21]. For example, our magnetic force measurements on Mag HA detected phase variations smaller than a few degrees at lift distances ranging from 500 to 30 nm, thus making data interpretation an issue. Nevertheless, a valuable signal contrast (voltage distribution) in Electrostatic Force mode is detectable. At polarizing biases ranging from 100 to 500 mV, the surface exhibits both topographic and non– topographic contrast, in correspondence of regions of micrometers size as well as single grains (Fig. 4a–b). Importantly, a complementary

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Fig. 4. a) AFM morphology — (13 × 13) μm2 large image — recorded in semi-contact mode (z-scale 0–280 nm) and b) its corresponding EFM (z–scale 0–60 mV). The observed inhomogeneities in the charge distribution suggest a compositional variation and/or local fluctuations of conductivity due to percolation.

EFM image is observed (not shown here) when the polarization is switched to negative, indicating that the contrast is originated by a potential variation instead of variation of the tip-sample capacitance [21]. These findings add information to the XPS results, suggesting that surface inhomogeneities occur over microscopic length scales, hence still comparable with the bacteria dimension. Nevertheless, at this stage increasing both spatial and energy resolution necessarily requires the investigation of electron surface states, i.e. to operate in conducting regime. 3.5. Scanning tunneling microscopy and spectroscopy We thus employed STM-STS as a tool for elucidating the origin of the inhomogeneities suggested by the EFM findings and reported in the Section 3.4. Simply, we take advantage of the conducting nature of the Fe3O4 with respect to the other sample components. The percolating tunneling transport of electrons in a random network of nanoclusters made of conductive material embedded in a dielectric matrix has been object of several studies. Whether macroscopic in-plane conduction is prevented because of the overall poor film conductivity, in the conducting regime vertical tunneling can occur above the percolation threshold [22,23]. This circumstance depends on the relative fraction of the conductor within the sample bulk, and hence in Mag HA films it should be driven importantly by the good bulk conductivity (102– 103 Ω cm−1) of the Fe3O4. Recently developed models, despite some strong assumptions, are able to predict critical behavior of the current density also at small volume fractions of conducting nanoclusters [22,

24]; such models predict a near-critical variation of the conductance which is rather close to the universal power-law behavior of a directcontact composite material. The success rate of tip approaches on the specimens used in this experiment was about (10–30)% for each, at setpoint values ranging between VB = (0.6–1.2)V and IT = (20–100) pA. This “patchy” conduction, for which a stable vertical tunneling takes place only at random surface positions, is compatible with a scenario in which only a few regions over the magnetite-rich surface are effectively connected to a complete percolating path throughout the bulk. Indeed, an STM observation shows that the surface is patched up of b1 μm2-wide conducting domains, randomly-located, formed by nanoclusters 10–20 nm wide and arranged in plate-like stacks (Fig. 5a). The apparent topography does not reflect significant changes with the bias voltage, indicating that local variations in the DOS occur at energies far from eVB. For our STS experiments, predetermined regions with moderate zcorrugation (z b 10 nm) were selected within larger scale images. Averaged TS exhibited no correlation with the topographic features. These curves are generally featureless and their semi-metallic behavior suggests that, in agreement with the XPS results, characteristic electronic states of Fe3O4 were detected (Fig. 5b). The magnitude of the bandgap near EF obtained from the dI/dV curves is (0.20 ± 0.05) eV, that compares well with the expected bandgap of magnetite obtained by early electrical-PES measurements, as well as tunneling experiments [25]. Occasionally semiconducting-like TS with (2–2.5) eV large bandgaps were detected, although they do not appear associated to a definite sample area. Likely, this behavior results from the convolution of the

Fig. 5. a) (250 × 250) nm2 large STM image (VB = 1.2 V, IT = 60 pA, area, z-scale = 0–42 nm). and (b) averaged room temperature TS recorded upon the same region. The presence of magnetite-rich states resulted in a homogeneous semi-metallic behavior with small bandgap, as shown in the corresponding dI/dV curve (inset).


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distributed surface areas exhibiting inhomogeneous charge distribution, which in turn indicates that a compositional variation occurs over distances of a few micrometers or less. The STM analysis supported these findings revealing b 1 μm2 large islands featuring homogeneous smallbandgap states that suggest the presence of Fe3O4-rich regions. Finally, preliminary bacterial adhesion tests suggested that Mag HA coatings are able to hamper the bacterial adhesion capability in comparison with the conventional HA coatings. In conclusion, our study represents a first step toward the knowledge of magnetic coatings as a new tool in medicine, supporting the idea of using them as a new platform able to decrease bacterial adhesion on the implant surface, while at the same time preserving the ability of the implant to integrate with the surrounding bone tissue.

Fig. 6. Adhesion of E. coli to magnetic and non-magnetic hydroxyapatite films at 4 h from seeding.

electronic states of magnetite with insulating states closer to the tip apex, thus a comprehensive semiconducting-like behavior is observed, as characteristic of certain nanogranular-mixed compounds [26]. 3.6. Evaluation of bacterial adhesion The above characterisations suggest an overall scenario in which a fraction of the magnetic component is randomly distributed at the surface, over length scales compatible with the bacteria dimensions, these latter typically ranging from a few hundred of nanometers to a few microns square. Thus, in order to preliminary assess the ability of magnetic films to interfere with the bacterial adhesion, we evaluated the adhesion of E. coli after 4 h of incubation in individual wells containing separately magnetic and not-magnetic HA films. Further, to provide evidence about the possible contingent synergistic effect due to the presence of an external magnetic field, we performed bacterial adhesion tests in the presence or in the absence of permanent NdFeB magnets externally allocated below the samples. Notably, the number of bacteria that adhered after 4 h of incubation to the substrate was lower (p b 0.05) for Mag HA compared to HA samples, independently from the presence of the permanent magnet below the sample (Fig. 6). Further on, the anti-adhesive effect was even enhanced (p b 0.05) when the films were coupled with an external permanent magnet. Even if further studies about the bacterial adhesion to these novel biomaterials (i.e. longer experimental times, different bacterial species and isolates) will be required in order to better elucidate the effect of the magnetic content in the film onto bacterial adhesion capability, as well as the role of the external magnetic field, these preliminary results suggest that the magnetic coatings is likely to interfere with the bacterial adhesion, when compared to non-magnetic HA films. 4. Conclusions The PPD technique described herein was shown to yield nanostructured magnetic coatings with surface composition and magnetic moment similar to the deposition target. More specifically, the quantitative XPS analysis strongly suggested the presence of a dominant magnetite phase at the surface, together with other satellite iron oxide phases. According to this scenario, the EFM showed the presence of randomly-

Acknowledgments Part of this work was supported by Dresden High Magnetic Field Laboratory (HLD), an Institute of the Helmholtz-Zentrum DresdenRossendorf (HZRD, Science center of the Helmholtz association), member of the European Magnetic Field Laboratory (EMFL).

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