Materials Science and Engineering C 46 (2015) 409–416

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Materials Science and Engineering C

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Graphitic carbon in a nanostructured titanium oxycarbide thin film toimprove implant osseointegration

R. Zanoni a,⁎, C.A. Ioannidu b, L. Mazzola b, L. Politi b, C. Misiano c, G. Longo d,e, M. Falconieri f, R. Scandurra b

a Dipartimento di Chimica, Università di Roma ‘La Sapienza’ p.le Aldo Moro 5, 00185 Rome, Italyb Dipartimento di Scienze Biochimiche, Università di Roma ‘La Sapienza’, p.le Aldo Moro 5, 00185 Rome, Italyc Romana Film Sottili, Anzio, Rome, Italyd Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italye Ecole Polytechnique Fédérale de Lausanne, SB IPSB LPMV, BSP 409 (Cubotron UNIL), R.te de la Sorge, CH-1015 Lausanne, Switzerlandf ENEA, Unità Tecnica Applicazioni delle Radiazioni, via Anguillarese 301, 00123 Rome, Italy

⁎ Corresponding author.E-mail address: robertino.zanoni@uniroma1.it (R. Zan

http://dx.doi.org/10.1016/j.msec.2014.10.0730928-4931/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 20 March 2014Received in revised form 22 October 2014Accepted 27 October 2014Available online 28 October 2014

A nanostructured coating layer on titanium implants, able to improve their integration into bones and to protectagainst the harsh conditions of body fluids, was obtained by Ion Plating Plasma Assisted, a method suitable forindustrial applications. A titanium carbide target was attached under vacuum to a magnetron sputtering sourcepoweredwith a direct current in the 500–1100W range, and a 100W radio frequencywas applied to the sampleholder. The samples produced at 900 W gave the best biological response in terms of overexpression of somegenes of proteins involved in bone turnover.We report the characterization of a reference and of an implant sam-ple, both obtained at 900W. Differentmicro/nanoscopic techniques evidenced themorphology of the substrates,and X-ray Photoelectron Spectroscopy was used to disclose the surface composition. The layer is a 500 nm thickhard nanostructure, composed of 60% graphitic carbon clustered with 15% TiC and 25% Ti oxides.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Titanium (Ti) is a very popular material to build orthopedic anddental prostheses for its peculiar characteristic properties, such asdensity (about half that of stainless steel), high biocompatibilityand high cost/performance ratio. However, Ti is also the most easilyoxidized metal. Oxidation gives high biocompatibility to Ti, but, inthe harsh conditions of biological tissues, the oxide layer in contactwith the bones may fracture, exposing bulk Ti. This may lead toinflammation with consequent osteolysis and mobilization of theimplant [1,2]. Protection can be obtained with a suitable overlayer,which can also induce a better interaction with the bone tissue, in-creasing its biocompatibility and integration into the bone [3]. Coat-ings with carbon are especially attractive, since the layers producedmay induce, in the early stages of osteogenesis, a stimulating effecton osteoblast growth and proliferation [4].

We have long been investigating compositional effects on thin layerdeposits containing carbon as TiC, able to stimulate the integration of anorthopedic prosthesis into the bone tissue [5–7]. We tested two

oni).

different techniques, Pulsed Laser Deposition (PLD) and Ion Plating Plas-ma Assisted (IPPA), to coat Ti prostheses. In both cases, we determinedthat TiC is the best coating for Ti prostheses, for its hardness and itschemical structure composed by Ti, a known biocompatible material,and carbon, themost diffuse element in biological tissues. The PLD tech-nology gave good results using a TiC target [5]. However, the PLD depo-sition is unfit for industrial application, since the apparatus allows oneto coat only one substrate at a time. In IPPA, first proposed by Mattox[8,9], and further developed byMisiano [10], the samples are introducedin a high-vacuum chamber containing a Ti target attached to a magne-tron sputter source; a radiofrequency (RF) is applied to the sample hold-er, and ethylene is used as the reactive gas [6,7]. This sequenceproducedcoating layers with unprecedented preliminary results in biologicaltests in vitro, which are predictive of good results in vivo and mayhave an industrial application. We have obtained full evidence that os-teoblasts grown on the coated samples have a higher proliferationrate, an increased formation of filopodia, and a positively improvedmRNA synthesis of proteins involved in bone formation, when com-pared to those grown on uncoated ones [6,7].

Starting from the good results reported in the literature for car-bon species [4,11–13] and on the basis of our results with TiC [5–7],we tried to build a nanostructured coating layer for implants, mainly

Fig. 1. A general scheme (left) of the IPPA device and a pictorial representation (right) of the sample holder. The sample holder (A) containing the substrates to be treated (disks and/orimplant screwsmoved bymotor B), is biased by a Radio Frequency (RF) produced by the generator, while themagnetron sputtering source (C) onwhich a TiC target is placed, is poweredbyDC. Vacuum is applied to the chamber through the pump (D). Plasma clouds are generated in front of the TiC target and the sample holder, and deposition is produced on the substrateswhose composition varies with the value of power applied to the magnetron, at constant RF applied to the sample holder. Deposition may be shielded by the shutter (S).

410 R. Zanoni et al. / Materials Science and Engineering C 46 (2015) 409–416

composed of sp2 carbon. Such a layer should be an ideal anchoringmaterial both for Ti carbide, which can give hardness to the Ti im-plant, and for Ti oxides, which increases biocompatibility, alongwith graphitic carbon. With the aim of obtaining a deposition pro-cess both simpler and richer in TiC and in carbon, we report hereon the results of changing the Ti target into one of pure TiC. The pro-posed approach reproduces the conditions used in PLD, but with thereliability of the IPPA technology, with an easy scaling to the indus-trial level.

2. Experimental

2.1. Ti substrates

The Ti samples (disks and dental implants) were kindly supplied byOrvit (Bologna, Italy) using Ti grade 2 (Ti% = 99.85; C% = 0.0006;Ni% = 0.01; O% = 0.1; H% = 0.003; Se% = 0.03), produced as rods byCerac, WI, USA. The rods were cut into disks of 13 mm diameter,2 mm thick, and blasted with 120 μm diameter zirconia microspheres.The implants were obtained as 4 mm × 8 mm screws, blasted in the

Fig. 2. Gene expression by Q-RT-PCR of alkaline phosphatase (ALP), collagen 1A2(COLL1A2) and osteocalcin (OC) in human primary osteoblasts, grown for 6 h on uncoated(Ti) and TiC-coated Ti disks at 500, 700, 900, 1100W, as a function of DC power applied tothe magnetron source in the deposition process. Results are reported as relative mRNA(messenger RNA) level and presented as the mean ± standard error of the mean(S.E.M.): p ≤ 0.05 was considered significant (*).

same way. The substrates were thoroughly cleaned by immersion for30 min in a 0.1% solution of Triton X-100 (Sigma-Aldrich, St. Louis,MO, USA), followed by sonication and rinse with ultrapure water andthree times with hexane, followed by air drying.

2.2. Ion Plating Plasma Assisted

The apparatus used for Ion Plating Plasma Assisted deposition withMagnetron Sputtering source (IPPA), sketched in Fig. 1, is composedof a high-vacuum chamber hosting a TiC target on a magnetronsputtering source, powered by a direct current (DC).

Ti disks were located in the IPPA chamber at 10 cm from the TiC tar-get. A final substrate cleaningwas performed in the chamber, by RF etchsputtering in Ar. The sample holder was maintained insulated from theground and 100WRF powered, while themagnetron sputtering sourcewas powered at 500, 700, 900, and 1100W. The ion plating effect of thisdepositionwas obtained both by energizing a percentage of condensingmaterial and by bombarding the growing filmwith the plasma ions sur-rounding the substrate. The thickness of the layer and the depositionrate were dynamically controlled by a quartz crystal monitor (Inficon,Switzerland) that, when properly calibrated, can guarantee a precisionin the range of 2–4 nm.

2.3. Atomic force microscopy (AFM) imaging

Scanning probe characterizationwas performed with a NanoWizardIII microscope (JPK, Berlin, Germany), operating in contact mode in aphosphate buffered saline (PBS) environment. The measurementswere performed with a scanning speed of ~1 s/row. No noise filterwas applied to the raw data, which were treated only by a softwarebackground subtraction and, when necessary, by a plane alignment,performed using the image analysis software Gwyddion (www.gwyddion.net). The detailed approach used is described in detail inour previous works [14–16].

2.4. Surface chemical analysis

The surface atomic composition of differently obtained TiC coatingswas analyzed by XPS, using both achromatic (MgKα and AlKα) andmonocromatized (AlKα1) radiation sources. All the Ti disks weremounted on amagnetic stainless steel tips covered by a Teflon film.The Ti implant consisted of a screw, which was machined in order topresent a flat side to be fixed to the tip. Electrical conductance was

Fig. 3. A collection of morphological, physical and chemical characterization of the nanostructured TiC surface layer in a 900 W sample. (a) SEM image. (b) Large-scale topography and(c) high-resolution error signal AFM images. (d) Analysiswith FIB-FEG and TEM-SAED. (e) EDS elemental analysis in terms of C, Ti and O content at points 1–5marked in red in the image.

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givenby thedirect contact between thedown face of the sample and thetip through a small hole in the Teflon layer. The layer of Teflon hasallowed separating in energy the C 1s contributions due to the substratefrom the ones due to the holder. Peak assignment was given by refer-ence to previous literature references [17], and to the NIST StandardXPS Reference Database 20, version 3.5.

Raman spectroscopy was performed using a home-built micro-Raman system based on a liquid-nitrogen cooled CCD(Charge CooledDetector), a 550 mm monochromator equipped with a 1800 grooves/mm grating, and a notch filter for improved rejection of elasticallyscattered light. The monochromator was fiber-coupled to a micro sam-pling head equipped with a 32× long-working distance objective usedto deliver the 532 nm laser excitation onto the sample with a spot sizeapproximately equal to 10 μm, and to collect the signal in a backscatter-ing configuration.

2.5. Focused Ion Beam

Helios Nanolab 600 Dual Beam Focused Ion Beam/Scanning ElectronMicroscope (FIB/SEM, FEI, Hillsboro, Oregon, USA) used is a SEM withtwo columns: an electron column for imaging, equippedwith an EnergyDispersion Spectrometer (EDS), and an ion-beam column (FEG, Field-Emission Gun) for micro- and nano-manufacturing. The Ion Columnwas used to realize a thin lamella to be observed with a TransmissionElectron Microscope (TEM, FEI CM120) equipped with a Selected AreaElectron Diffractometer (SAED).

2.6. Scratch tests

The TiC coating adhesion was investigated following UNI-EN-1071-3,with a CSM REVETEST Xpress Plus scratch tester. A progressive load was

Table 1XPS atomic ratios for the sample obtained by powering the magnetron at 900 W in thedeposition process.

Atomic % BE (eV) BE (literature)a

Ti 2p3/2 componentsTiC 35.2 454.8 454.6TiO 24.4 455.6 455.34 ± 0.39Ti2O3 13.5 457.1 457.13 ± 0.35TiO2 26.9 458.6 458.66 ± 0.22

C 1s componentsC (TiC) 11.5 281.8 281.5C-interf 7.4 282.4C-graphite 64.6 284.8 284.6C\X 10.8 285.8C(O)_O 5.7 288.8

a Binding Energy (BE) values (eV) for Ti oxides are taken from the collection of data inref. [19], while values for TiC (Ti 2p and C1s) and graphite from ref. [17] The label ‘C_interf’stands for the carbon component at the interface between TiC and amorphous carbon.

454 456 458 460 462 464 466

Ti 2p

Inte

nsity

/arb

. u.

Binding Energy/eV

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applied from 1 to 15 N, with a 100 N/m loading rate and a 10 mm/minscratch speed.

Fig. 5. Ti2p3/2,1/2 XPS spectrum of the layer deposited by IPPA on a Ti clean disk (sample900 W), taken with Al kα monochromatized radiation. The four spin-orbit split compo-nent commented in the text are clearly evidenced.

2.7. Cell culture and RNA extraction

Primary human osteoblasts (hOB)were isolated from bone fragmentsobtained from surgical procedures and cultured, as described elsewhere[5–7], by using McCoy culture medium supplemented with 10% FetalBovine Serum (FBS). Test substrates (untreated and treated Ti disks13 mm × 2 mm) were seeded with hOB at a density of 2 × 104/cm2 andcultured in McCoy supplemented with 1% FBS, for 6 h at 37 °C in 5%CO2. After incubation, cells washed with cold PBS were scraped, and

Fig. 4. Plot of theXPS relative atomic ratios among Ti (bottom) and carbon (top) species asa function of DC power applied to the magnetron source in the deposition process.

total RNA was extracted using 1 mL of Trizol reagent (Invitrogen, Carls-bad, CA, USA), according to the manufacturer's instruction. RNA wasstored at−20 °C until used. Complementary DNA (cDNA) was synthe-sized from 1 μg of total RNA, using reverse transcriptase Improm II(Promega Corporation, Madison, WI, USA), and analyzed by QuantitativeReverse Transcriptase-PCR (Q-RT-PCR) using an AB Prism 7300 (AppliedBiosystems, Foster City, CA, USA), as described previously [5–7]. Signalswere normalized for glyceraldeheide-3-phosphate dehydrogenase(GAPDH). Gene expression of cells grown on uncoated Ti disks wasconsidered as 1. Each experiment was repeated at least three times. Thestatistical significance of the differences betweenmean valueswas deter-mined by a two-tailed t-test; p≤ 0.05 was considered significant. Resultsare expressed as the mean ± standard error of the mean (S.E.M.).

3. Results

The highest expression of genes of ALP (alkaline phosphatase),COLLA2 (collagen1A2) and OC (osteocalcin), was reached for samplesobtained at a magnetron sputtering source power of 900 W (Fig. 2).

281 283 285 287 289 291

C 1s

Inte

nsity

/arb

. u.

Binding Energy/eV

Fig. 6. C1s XPS spectrum taken with Al kαmonochromatized radiation for the same sam-ple reported in Fig. 2. Five components result from curve-fitting,with carbidic and graphit-ic carbon respectively falling at about 281.8 and 284.8 eV.

280 284 288 292 296 300

C

A

B

C 1s

Inte

nsity

/arb

. u.

Binding Energy/eV

Fig. 7. XPS spectra of the C 1 s region for: (A) 900W sample; (B) Ti screw coated at 900Wwith TiC, and (C) clean Ti disk which was not coated with TiC.

413R. Zanoni et al. / Materials Science and Engineering C 46 (2015) 409–416

This was then considered the reference for an optimized biological re-sponse, to be associatedwith themost suitable surface chemical compo-sition, as will be shown later. The morphology of this surface wasobserved with SEM (Fig. 3a), which showed a rough and patchy aspect,with defective areas homogeneously distributed on the entire surface.

Large-scale AFM images (Fig. 3b) confirmed suchmorphology, hintingat a complete and uniform coating. At higher resolution (Fig. 3c), AFM im-ages revealed a granular nanostructure with diameter of the grains in theorder of hundreds of nanometers. The root-main-square (rms) roughnessmean values calculated on several tens of different 2 μm×2 μm images ofthe uncoated (24.3±4.3 nm) and the 900W(27.5±10.4 nm) substratesshowed that the TiC coating proceduremodified only by ~3 nm themeanroughness of the samples, a further indication that the film has uniformlycovered the substrates.

Nano- and micro-scratch tests revealed that the layer is stronglybound to bulk Ti, with about five times the hardness of Ti (26.9 ± 7.2vs. 4.44 ± 0.58 GPa), and twice its elastic modulus (299.7 ± 79.8 vs.129.5 ± 14.6 GPa) [7]. These results may be due to the presence ofhigh amounts of Ti carbide connected with graphitic carbon, and tothe compressive residual stress induced by the high density of thelayer formed by the plasma technology [15]. The morphology of thelayer was further studied by TEM, which operated on a thin lamellataken from a transversal section of the 900 W sample. The TiC coating

Fig. 8. (Left) Raman spectrum in the graphitic band region taken from the TiC coating layer prodclarity. (Right) C KLL X-ray-excited first derivative Auger spectra of: Ti screw coated at 900 W

(Fig. 3d) appears compact without porosity and well adhered to thesubstrate, with an overall thickness of ~500 nm.

While the near-interface region confirms a good adhesion of thecoating, a less dense layer is present near the surface. SAED analysis per-formedon the lamella has shown that the upper bright layer has a nano-crystalline structure, probably due to a higher concentration of TixOywith respect to the lower TiC layer, which is amorphous and has a nano-structure in which two different phases can be identified: a first,brighter, is where Ti and O are bound in TixOy, while the second is theregion of Ti carbide. The lowest bulk Ti has a crystalline structure.

EDS elemental analysis shows a bottom layer on the Ti substrate(marked with dot 1) with a thickness of about 30 nm (dot 2), whereTi and oxygen are the most and least abundant element, respectively.The 2nd layer (dots 3 and 4) of about 430 nm represents the real TiCcoating. The last layer (dot 5) has a thickness of about 30 nm, and ahigh concentration of oxygen, calling for Ti preferentially bound to oxy-gen, with respect to carbon.

To chemically define each layer, EDS semi-quantitative spot elemen-tal analysis was performed on the regions identified by the red pointsshown in Fig. 3e. The layer closer to the substrate (marked as 2) has athickness of ~30 nm, and is the layer where Ti and O are respectivelypresent at the highest and lowest percentage. A second layer (markedas 3 and 4), of about 430 nm, identifies the TiC coating. The top layerof the coating (red point 5) has a thickness of ~30 nm, is the brightestone and is rich in oxygen, hinting at a Ti oxide region with a nanocrys-talline structure quite distinct from the TiC layer but with TiC stillpresent at high concentration.

The thin layer object of the present report, hereon referred to as900W,was obtained by powering themagnetron at 900W, a conditionwhich gave the best biological response in the optimization process ofthe deposition, as previously shown in Fig. 2.

XPS afforded the binding energy (B.E.) values and the atomic ratiosof the various Ti and carbon species present in the 900 W layer, whichare reported in Table 1. The close correspondence, within experimentalerrors, of the various components to the values reported in the relevantliterature [14], allows for a straightforward assignment.

In Fig. 4a,b we report the chemical composition obtained from curvefitting the 900W sample: The TiC and carbon contents are the highest inthe series, while the content in TiO2 is the least (Fig. 4).

Interestingly, in all the deposition conditions tested itwas found thatthe major amount of carbon is not bound to Ti to give TiC, but goes intocarbon species. Among these last, graphitic carbon was found in all thetreated samples, at its highest amount in the sample deposited at900 W.

Fig. 5 shows the Ti 2p3/2,1/2 XPS spectra takenwithmonochromatizedradiation. Four spin-orbit split components are present in the complex Ti

uced on a TiC 900W sample. A flat backgroundwas subtracted anddatawere smoothed forwith TiC (A); TiC deposit obtained at 900 W on a Ti clean disk (B); Pure graphite (C).

Fig. 9.XPS spectra takenwith Al ka photons of the 900Wsample. Spectra A andB are taken at two distinct spots on the sample, roughly corresponding to a first half and a secondhalf of thesurface area, while spectrum C was taken by varying the angle of photoelectron escape from 11 (curve B) to 71°. (Left) C 1 s region; (Right) Ti 2p region.

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2ppeak,which can be assigned, in order of increasing BE, to TiC, TiO, Ti2O3

and TiO2 [17,19]. TiC and TiO2 are the two largely prevailing components.The C 1 s spectral region reported in Fig. 6 is a composite peak,

formed by a minimal number of five components (Table 1). From lowto high BE's, we found the carbide due to TiC (281.5–281.8 eV) and asecond component at 282.4 eV, whichmaywell correspond to the com-ponent reported in the literature for a similar system at 282.5 eV, andpreviously assigned to the carbon species at the interface between TiCand amorphous carbon in nanocomposite films [18,20]. A third compo-nent can be located at 284.8 eV, which we assign to graphitic carbon onthe basis first of all of its BE (also in a comparison with an untreated Tidisk, see Fig. 7), and of the results coming from angle-dependent XPSand Raman measurements (see below for the discussion of the abovepoints). The further two components may be respectively assigned, inthe same sequence of increasing BE values, respectively to C\X bonds,with X being a heteroatom as O or N, and to C_O and C(_O)\Ogroups, commonly found in surface contaminants coming from CO2.

Some carbonaceous contamination is likely to be present because ofmanipulation in air of all the investigated samples after their prepara-tion. Within the above limits, we do not ascribe the component at284.8 eV to contamination because the XPS spectra taken on a Ti diskwhich was not subjected to the deposition of TiC (and stored in thesame conditions of the coated samples) presents a completely differentC 1 s peakwith respect either to the 900W sample and to the Ti implantafter TiC deposition. This is shown in Fig. 7, where the carbon complexpeak can be ascribed to usual contamination deposits due to carbonand oxygen-containing carbon species.

Moreover, on passing from 11° to 71° photoelectron collection angle(i.e., in conditions where the surface contribution is enhanced over the

Fig. 10. Comparison between XPS spectra of C 1 s (left) and Ti 2p (rig

subsurface and bulk ones, see Fig. 5), the O content remains, within theuncertainty, the same, (from 25.8 to 25.0%) and only a quite modestrelative increase results from the C content (from 58.0 to 62.8%), mainlyat the expenses of Ti (from 16.3 to 12.2%).

Raman spectroscopy confirmed the presence of graphitic carbon inthe 900 W sample. As the color of the film was not homogeneous underthe optical microscope, data were taken on a relatively dark area, whichis supposed to be carbon rich. The Raman spectrum in the region of gra-phitic bands is reported in Fig. 8 (left), where a high intensity band at1580 cm−1 (G band) and a lower one at 1350 cm−1 are clearly shown.

The first band (G band) is a well-known fingerprint of graphitic car-bon, being attributed the bond stretching of all pairs of sp2 atoms in bothrings and chains of graphite [21]. The second band (D band) originatesfrom a hybridized vibrational mode associated with graphite edges orsmall crystallites, and is therefore indicative of partial disorder in thegraphite structure, likely generated by the presence of Ti oxides andTiC, bound to the layers of graphitic carbon of the nanostructure.We re-call here that stoichiometric TiC is not Raman active.

We report the C KLL Auger spectra in Fig. 8 (right).The results for the TiC coating obtained at 900Won a Ti implant and

on a Ti disk are very similar in the region relating to the carbide peaks[22], which further confirms the reproducibility of the treatment. Thepeak at ~274 eV, characteristic of graphite [21], is shifted at 264 eV inthe spectra of both nanostructured layers. On the basis of a recent liter-ature report on graphene grown on titania [23], we speculate that thisshift could be related to the peculiar structural electronic environmentdefined by graphitic carbon surrounded by nanostructured Ti oxides,which could favor a mechanism of electron transfer from graphite toTiO2 deposits.

ht) regions for the Ti screw and the Ti disk, both coated at 900 W.

Fig. 11. Pictorial model of the TiC coating layer, where Ti oxides (TiO2), TiC and graphiteare intimately connected as a consequence of the deposition conditions adopted.

415R. Zanoni et al. / Materials Science and Engineering C 46 (2015) 409–416

Consistent with the above experimental findings for carbon, thesequence of Ti 2p spectra reported in Fig. 9 shows small differences be-tween the spectra taken at 11° (Fig. 9B) and 71° (Fig. 9C) photoelectroncollection angles,mainly due to a relative reduction of the TiC component.

We note on passing the close correspondence of peaks A and B in theabove two sequences, which calls for a homogeneous coverage of thedisk, in the explored surface range (a few mm2).

A Ti implant was subjected to the same experimental conditions inwhich the 900 W sample was obtained from a Ti disk. We note that acomparison between the C 1 s and the Ti 2p peak components in thetwo samples reveals closely comparable peak components, as shownin Fig. 10. XPS quantitative analysis shows the presence of a comparableamount of Ti in the two samples: 15% on the disk vs. 16% on the implant,with a relative increase in O (from 25 to 36%), and decrease in C (from60 to 48%).

4. Discussion

SEM and AFM showed that the morphology of the layer obtained isuniform. The chemical composition, defined by XPS, relates the chemi-cal structure of each layer both with its surface composition and withthe biological response. In agreement with previous literature reports[17], the narrow C peak at 284.8 eV (Fig. 6) was assigned to graphiticcarbon, and the ones at 282.4, 285.8 and 288.4 eV, respectively to inter-stitial carbon, carbon bound to a heteroatom, carboxylate groups. Wenote that the layer is rich in graphitic carbon.

In a previous work on closely related systems, based on a differentexperimental approach, we already found that the layer obtained wasrich in carbon [7]. We made use of ethylene as the reactive gas, and ofTi as the target, andwe assigned the carbon species to amorphous or al-iphatic carbon. In the present work, where the Ti source used was TiC,

the presence of aliphatic carbon can be confidently excluded in favorof graphitic carbon, on the basis of Raman and XPS findings. In orderto rationalize the presence of graphitic carbon, we first recall here thatwhen IPPA is used to produce a coating layer, the deposition is obtainedby energizing a part of the condensing material and by bombarding thegrowing film with energetic particles. The samples are then depositedwith both neutral particles and accelerated ions of various species con-stituting the plasma surrounding the substrate, which can react withthe environment. A portion of the TiC particles reaches the Ti substrateunmodified, while another fraction is dissociated by ion bombardmentand by contact with DC and RF plasmas, possibly giving Ti+ and C− re-active ions. We propose that on the surface of the substrate C− reacts toform mainly graphitic rings, either directly or by condensation of C2

molecules present at the high temperature of the plasma cloud [24].The formation of graphitic rings is thermodynamically favored, andfrom such rings graphitic clusters are likely to result inside the nano-structure, during the deposition. Ti+ present on the substrate surfaceeasily reactswith residual oxygen to give the series of Ti oxides reportedin Table 1, due to the high affinity of Ti for oxygen [25].

Taking into account the above comments, we propose amodel of thelayer obtained by the IPPA technology in Fig. 11, where graphitic carbon,which is the major component of the layer obtained, is clustered insidethe nanostructure and in close contact with TiC and Ti oxides to give avery hard coating layer, strongly bound to the Ti substrate.

The above considerations and model described for the nanostruc-tured TiC layer can be immediately extended to the 3-dimensional de-posit on the Ti implant. Considering that the explored layer in XPS isreduced to a few nm, the close correspondence experimentally foundfor the 900W sample, obtained as a disk and as Ti implant, is a good pre-mise for an extension to real samples of the process outlined here.

The AFM results indicate that the coating procedure results in a uni-form layer, one of the most important parameters for successfulosseointegration [26]. These data allow associating any difference inthe biological response of the cells grown on the treated and untreatedsamples only to the chemical and physical properties of the nanostruc-tured layer.

5. Conclusions

The morphological and XPS analysis of thin protective layers of TiCproducedby IPPAon reference samples of Ti and onTi screws for implantshas contributed a detailed knowledge of such flat and three-dimensionalcoatings. The innovative use of magnetron-activated TiC as the source ofboth Ti and carbon allowed for the formationof highly reproducible nano-structured thin films. A general model of the surface layer compositionfavors the presence of graphitic carbon, which is the major componentof the layer obtained, clustered inside the nanostructure and boundwith TiC and Ti oxides to give a very hard coating layer, strongly boundto the Ti substrate.

Acknowledgments

This work has been supported by the Italian Department of Healththrough the grant n.GR-2009-1305007. Financial support fromUniversitàdi Roma La Sapienza is gratefully acknowledged.

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Robertino Zanoni is full professor in General and InorganicChemistry at Università La Sapienza, Rome from 2005. Au-thor/coauthor of N150 papers and a patent. Visiting scientistat University of Wisconsin–Madison in 1987, '88 and '89(Fulbright). Local responsible in a Science (1992–95) and aHuman Capital & Mobility (1994–'96) ERP. Responsible ofseveral MIUR National Interest Projects. A member of theELETTRA synchrotron review committee (2004–09); expertevaluator in Framework Programmes of EU. He leads a groupin surface science at La Sapienza active in the application of

conventional and synchrotron-excited photoemission andrelated techniques to research topics in surface chemistry,mainly in surface–molecule interaction and bonding.

Caterina Alexandra Ioannidu was born in Rome, Italy, in1984. Bachelor Degree in Biological Science in 2007, Special-istic Degree in Genetics and Molecular Biology in 2010 andPhD in “Pathophysiology and Clinic of Skeletal Apparatus”in 2014. During the PhD course she studied the effects of ananostructured titanium carbide coating on human primaryosteoblasts and Saos2 cells on gene modulation by Real-Time PCR techniques and cell adhesion by immunofluores-cence and electron microscopy.

Luca Mazzola is senior scientist at the Italian Aerospace Re-search Centre (CIRA). He holds aMaster degree in AerospaceEngineer (2006) and a PhD degree in Materials Science(2011), both achieved at University of Rome “La Sapienza”.Post-doc at Astronautical, Electrical and Energetic Depart-ment of “La Sapienza” University (2012). His main interestsare in surface science, with an accent on adhesion phenome-na between coatings and substrates and innovative charac-terization techniques. He is the founder and President ofthe European Association on Material Surface Science &

Technology — E.M.A.S.S.T. He is referee for several interna-tional journals, amongwhich: “Journal of IntelligentMaterialSystems and Structures”, “Fresenius”, “Measurements”.

Engineering C 46 (2015) 409–416

Laura Politi is Associate Professor ofMolecular Biology at theFaculty ofMedicine of University of Rome La Sapienza,whereshe was involved for a long time in the research on structureand function of proteins and in the Biochemistry of renal fail-ure, and in particular in developing devices to improve thepurification of blood in uremic patients. In the last yearsher interest is in the intracellular signaling in the chondro-cyte, in toxic effects at a molecular level of heavy metalsand in the stimulating effect of some nanostructures on oste-

oblasts.

CarloMisianowas born in Rome and obtained the doctoratein Physics at Univeristy La Sapienza, Rome (1967). Early inthe 1970s he started his research in the applications of RadioFrequency Sputtering Deposition and Sputter Etching and,early in the 1980s, Ion Plating Plasma Assisted. From 1988he is external professor at University of L'Aquila and Univer-sity of Roma 3. He was awarded by the Society of VacuumCoaters in 2005 with the “Mentors Award” and in 2006 withthe “Nathaniel Sugerman Memorial Award”.

Giovanni Longo is associate researcher at the EPFL in Lau-sanne, Switzerland and at the Istituto Superiore di Sanità inRome, Italy. He is an expert in scanning probe microscopiesand high-resolution characterization techniques, with mainfocus in the study of samples of interest in biology, microbi-ology andmedicine. His recent work is focused on the inves-tigation of living cells and bacteria, and he has developedinnovative techniques based on atomic force microscopy tocharacterize biological systems.

Mauro Falconieri is senior scientist (physicist) at ENEA, theItalian National Agency for New Technologies, Energy andSustainable Economic Development, where he holds a staffposition since 1990. From 1990 to 2000 his research activitywas devoted to spectroscopy of active media for eye-safe la-sers, and to third-order nonlinear optical measurements.From 2000 to present he is running an optical and laser spec-troscopy laboratory oriented to materials science research.Hismain interests are inmaterials for applications inphoton-ics, renewable energies and bio-imaging.

Roberto Scandurra retired in 2010 from full Professor ofChemistry at the Faculty of Medicine of University of RomeLa Sapienza, where he directed the PhD Course on Physiopa-thology and Clinics of Skeletal Apparatus (1984–2010). Hisactivity was addressed to the metabolism of sulfur com-pounds and to structure and function of proteins as transam-inases, decarboxylases, dehydrogenases and proteins ofextremophilic bacteria, to study the molecular basis of pro-tein thermostability by methods of biochemistry, biophysicsand molecular biology. He is presently involved in studying

some molecules active on osteoarthritis and on thin filmswith stimulating activity on osteoblasts.