Cell proliferation on modified DLC thin films prepared by plasma enhanced chemical vapor deposition Adrian Stoica, Anton Manakhov, Josef Polčák, Pavel Ondračka, Vilma Buršíková, Renata Zajíčková, Jiřina Medalová, and Lenka Zajíčková Citation: Biointerphases 10, 029520 (2015); doi: 10.1116/1.4920978 View online: http://dx.doi.org/10.1116/1.4920978 View Table of Contents: http://scitation.aip.org/content/avs/journal/bip/10/2?ver=pdfcov Published by the AVS: Science & Technology of Materials, Interfaces, and Processing Articles you may be interested in Plasma enhanced chemical vapor deposition of wear resistant gradual a-Si 1 − x : C x : H coatings on nickeltitanium for biomedical applications J. Appl. Phys. 107, 053301 (2010); 10.1063/1.3310641 Effects of thermal annealing on the structural, mechanical, and tribological properties of hard fluorinated carbon films deposited by plasma enhanced chemical vapor deposition J. Vac. Sci. Technol. A 22, 2321 (2004); 10.1116/1.1795833 Room temperature synthesis of porous SiO 2 thin films by plasma enhanced chemical vapor deposition J. Vac. Sci. Technol. A 22, 1275 (2004); 10.1116/1.1761072 Oxygenated polymeric thin films deposited from toluene and oxygen by remote plasma enhanced chemical vapor deposition J. Vac. Sci. Technol. A 21, 1655 (2003); 10.1116/1.1597891 Hydrogenated amorphous silicon carbide deposition using electron cyclotron resonance chemical vapor deposition under high microwave power and strong hydrogen dilution J. Appl. Phys. 92, 2937 (2002); 10.1063/1.1500418

Cell proliferation on modified DLC thin films prepared by plasma enhanced chemical vapor deposition Adrian Stoicaa) and Anton Manakhov Plasma Technologies, CEITEC—Central European Institute of Technology, Masaryk University, Kotl arsk a, 2, Brno 61137, Czech Republic

k Josef Polcˇa Fabrication and Characterisation of Nanostructures, CEITEC—Central European Institute of Technology, Brno University of Technology, Technick a, 2, Brno, Czech Republic

 Pavel Ondracˇka and Vilma Bursıkova Plasma Technologies, CEITEC—Central European Institute of Technology, Masaryk University, Kotl arsk a, 2, Brno 61137, Czech Republic and Department of Physical Electronics, Faculty of Science, Masaryk University, Kotl arsk a 2, Brno 61137, Czech Republic

 Renata Zajıcˇkova Plasma Technologies, CEITEC—Central European Institute of Technology, Masaryk University, Kotl arsk a, 2, Brno 61137, Czech Republic

 Jirina Medalova Department of Experimental Biology, Faculty of Science, Masaryk University, Kamenice 5, Brno, Czech Republic

b) Lenka Zajıcˇkova Plasma Technologies, CEITEC—Central European Institute of Technology, Masaryk University, Kotl arsk a, 2, Brno 61137, Czech Republic and Department of Physical Electronics, Faculty of Science, Masaryk University, Kotl arsk a 2, Brno 61137, Czech Republic

(Received 11 February 2015; accepted 30 April 2015; published 12 May 2015) Recently, diamondlike carbon (DLC) thin films have gained interest for biological applications, such as hip and dental prostheses or heart valves and coronary stents, thanks to their high strength and stability. However, the biocompatibility of the DLC is still questionable due to its low wettability and possible mechanical failure (delamination). In this work, DLC:N:O and DLC: SiOx thin films were comparatively investigated with respect to cell proliferation. Thin DLC films with an addition of N, O, and Si were prepared by plasma enhanced CVD from mixtures of methane, hydrogen, and hexamethyldisiloxane. The films were optically characterized by infrared spectroscopy and ellipsometry in UV-visible spectrum. The thickness and the optical properties were obtained from the ellipsometric measurements. Atomic composition of the films was determined by Rutherford backscattering spectroscopy combined with elastic recoil detection analysis and by x-ray photoelectron spectroscopy. The mechanical properties of the films were studied by depth sensing indentation technique. The number of cells that proliferate on the surface of the prepared DLC films and on control culture dishes were compared and correlated with the properties of as-deposited and aged films. The authors found that the level of cell proliferation on the coated dishes was high, comparable to the untreated (control) samples. The prepared DLC films were stable and no decrease of the biocompatibility C 2015 American Vacuum Society. was observed for the samples aged at ambient conditions. V [http://dx.doi.org/10.1116/1.4920978]

I. INTRODUCTION Amorphous hydrogenated carbon, e.g., diamondlike carbon (DLC), defines a diverse group of hydrocarbon materials with outstanding properties. Amorphous carbon contains a mixture of predominantly sp2 hybridized carbon with varying amounts of sp3 carbon. DLC thin films have attracted great interest due to their intrinsic properties such as high hardness and wear resistance, low friction coefficients and chemical inertness, optical transparency in the infrared (IR) a)

Electronic mail: [email protected] Electronic mail: [email protected]

b)

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spectral range, and low electrical conductivities.1 Due to its combination of properties, DLC is nowadays intensively used on an industrial scale in applications like coatings of machinery tools,2 car engine components,3 electronic devices,4 and biological implants.5 Biomedicine is a relatively new area of applications of DLC thin films. They are reported to be suitable for use in medical devices such as hip and dental prostheses,6,7 heart valves and coronary stents,8 and intraocular and contact lenses.9 Nevertheless, there are a number of issues that need to be addressed, e.g., modification of surface wettability and bioactivity. The surface free energy and surface chemistry can

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C 2015 American Vacuum Society V

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be changed by introducing other elements, e.g., silicon, oxygen, or nitrogen, in the composition of the DLC surface10 or bulk.11 Additionally, the compositional changes in the bulk of the DLC material reflect on their mechanical properties.12,13 The high compressive stress characteristic to hard DLC thin films can be reduced, consequently decreasing the risk failure of the coating due to delamination. However, in order to avoid an excessive decrease of the hardness of the DLC, the doping element and its amount should be carefully selected. With regard to the DLC surface modification, May et al.14 demonstrated methods for spatially directing neural cell growth on oxygenated diamond surfaces. The neuronal cells were grown on oxygen-terminated CVD diamond after surface modification by UV/ozone treatment, with better results than on the standard tissue culture polystyrene substrate. Similar conclusions were obtained by Miksovsky et al.,15 where the high adhesion and growth of human osteosarcoma cells on ultrananocrystalline diamond and DLC films was achieved after the oxygen or ammonia plasma post-treatment of the surfaces. The weakness of surface treatment methods is a fast degradation of the unstable functional surface groups and quick recovery (within a few days) of the hydrophobic nature of the material.16,17 Much better stability of the surface chemistry and wettability can be achieved by the doping of DLC films with oxygen and/or nitrogen during the deposition process. It allows to achieve the water contact angle (WCA) of 60 as recommended in the literature for fibronectin adsorption and cell growth.18 From the point of view of mechanical properties, their influence on biological response has come recently to interest. Materials mechanical properties proved to be an important regulator of cellular processes such as proliferation, differentiation, and migration. In the work of Hopp et al., a technique is presented for controlled generation of gradients of surface elastic moduli. They showed that substrate mechanical properties strongly influence human dermal fibroblasts cell fate, stronger than the influence of chemistry and wettability.19 Plasma enhanced CVD (PECVD) is a robust method for deposition of DLC thin films, having the advantage of easily controlling the deposition parameters for tuning of coatings properties. Moreover, the doping elements are embedded in the bulk of the material, which brings an advantage over short term durability of the surface treatments. There are several works reporting the mechanical stability of DLC for biomedical applications. For example, Azzi’s approach was to employ interlayers.20 In the literature, it has been reported the use of PECVD as deposition method of DLC and doped DLC for bioapplications. Works such as the ones of Ou et al.,21 Ahmed et al.,22 and Yadav et al.23 deal with biomedical applications of the Si doped a-C:H prepared by radio frequency PECVD. The doping of DLC with nitrogen has also been reported in the literature.24 In this work, DLC thin films doped with N, O, and Si in order to enhance film stability and its adhesion to the Biointerphases, Vol. 10, No. 2, June 2015

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substrate were deposited by PECVD from gas mixtures of methane, hydrogen, and hexamethildisiloxane (HMDSO). A decrease in the internal compressive stress, which is a major issue in medical applications of DLCs, is achievable by changing the gas feed composition. The influence of surface chemistry, wettability, and mechanical properties of the prepared DLC:N,O and DLC:SiOx coatings are investigated with respect to cell proliferation. II. EXPERIMENTAL DETAILS A. Deposition arrangement

Different modified DLC thin films were deposited on silicon (Si), microscope glass slips, and tissue culture dishes. The substrates used are as follows: 1  0.5 cm double-side polished silicon single crystal h111i (N-type phosphorus doped) substrates with 0.5 X cm resistance, common microscope glass slides provided by Merci s.r.o. and tissue culture dishes (growth-enhanced treated) with a 34 mm internal diameter and a growth surface area of 9.2 cm2 supplied by TPP Techno Plastic Products AG. The gases employed in this work are as follows: argon: Ar (UN 1951) 99.999 vol. % stored in a refrigerated container, methane: CH4 (UN 1971) of purity 99.995 vol. %, hydrogen: H2 (UN 1049) purity 99.999 vol. %, and synthetic air UN 1002, all supplied by Messer Technogas s.r.o. The liquid precursor hexamethyldisiloxane (HMDSO—(CH3)3SiOSi(CH3)3) of purity 98.5% (GC) was supplied by Sigma-Aldrich Czech Republic. In a first step, the Si and glass substrates were cleaned in isopropyl alcohol (C3H8O) in an ultrasonic bath Bandelin ultrasound DT 103H and then blown-dry by compressed air. The plastic dishes were used directly from the manufacturers packaging. After coating, the dishes were sterilized in UV light before cell seeding. The preparation of the modified DLC thin films was done in a parallel-plate RF-PECVD reactor. The reaction chamber consists of a Pyrex glass cylinder of 274 mm inner diameter and 190 mm high closed by two stainless steel flanges. The electrodes used in this work are made of graphite and the distance between them was 55 mm. The powered bottom electrode is capacitively coupled to the RF generator and the upper electrode was grounded. A Dressler’s CESAR 136 RF power source operating at a frequency of 13.56 MHz and an electrically compatible matching network, Dressler Variomatch, were used. The supplied power was 50 W, and the negative self-bias voltage observed in the first minute of the deposition was 220 and increasing during the deposition depending on the gas mixtures and working pressure. The precursor gases were fed into the chamber through a glass torus with many outlets on its perimeter. Several gas compositions were used in this work and were fed into the reactor chamber: methane, methane with hydrogen, and methane with HMDSO. The deposition parameters are listed in Table I. In all the experiments, a flow of 0.2 sccm of synthetic air was added. For methane alone, the flow rate was 2 sccm. In admixture with hydrogen the flow rate of the methane was

029520-3 Stoica et al.: Cell proliferation on modified DLC thin films TABLE I. Deposition conditions.

Sample C1 C2 C3 C4

Q(CH4) (sccm) 2 1.8 2 2

Q(H2) (sccm)

Q(HMDSO) (sccm)

Q(air) (sccm)

P (Pa)

P (W)

Ub (V)

0.4 0.7

0.2 0.2 0.2 0.2

16 16 16 16

50 50 50 50

230 230 230 230

0.9

1.8 sccm and the flow rate of hydrogen was 0.9 sccm. For HMDSO, two flow rates were used: 0.4 and 0.7 sccm, while the methane flow rate was kept at 2 sccm. A pretreatment in argon plasma before each deposition was done for 5 min at 50 W power and negative self-bias voltage Ub ¼ 200 V. This pretreatment was used in order to sputter-clean the substrate surface and improve the adhesion of the coating–substrate system. After being exposed to argon plasma, the substrates were not in contact with air until after the deposition was complete. The deposition conditions were chosen based on our group’s previous work, as reported in our previous publications.25–27 B. Thin film characterization

Optical characterization in ultraviolet/visible range of 1.5–6.5 eV was done using a Jobin Yvon UVISEL ellipsometer at angle of incidence 65 . Transmittance (T) in the IR range of 400–4000 cm1 was measured with a Bruker Vertex 80v Fourier transform IR (FTIR) spectrophotometer equipped with a parallel beam transmittance accessory for correct absolute transmittance measurement. The spectra were recorded at the pressure of 250 Pa with the resolution of 4 cm1 and 500 scans. Ellipsometric and transmittance measurements were fitted simultaneously using the structural model of an ideal homogeneous film and a Kramers–Kronig consistent model combining a dispersion model parameterizing joint density of states for the description of interband transitions and a set of Gaussian peaks in the imaginary part of the film dielectric function, describing the phonon absorption in IR.28 This approach eliminated the influence of different film thicknesses on the analysis of IR data because it enabled to compare the film dielectric functions in the IR range.29 The atomic composition of the films was determined by a combination of Rutherford backscattering spectroscopy (RBS) and elastic recoil detection analysis (ERDA). RBS was performed using 1.74 and 2.2 MeV protons and 3.04 MeV alpha particles. At 2.2 MeV protons, the sensitivity for C, N, O, and Si is enhanced, and at 1.74, the strong resonance for C is used. The admixture of O is scanned by narrow resonance at 3.04 MeV alpha particles. ERDA was performed with 2.5 MeV alpha particles impinging at 75 to the surface normal. Forward recoiled protons under 75 are detected after passing through a foil stopping scattered alpha particles. ERDA needs precise estimation of impinged amount of alpha particles; we use RBS analyses of Au layer covered aluminum propeller disrupting the impinging beam Biointerphases, Vol. 10, No. 2, June 2015

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for one eleventh of measuring time. The dead time of RBS measurements must be very precisely controlled. For hydrogen detection ERDA was calibrated using a Si:H sample with 11.9% H. Absolute RBS errors can be assessed as 64%. X-ray photoelectron spectroscopy (XPS) for the surface (2–3 nm) chemical characterization of coated Si was carried out using an Omicron nonmonochromatic x-ray source (Al Ka, DAR400, output power 270 W) and an electron spectrometer (EA125) attached to a custom built ultrahigh vacuum system. No charge compensation was used. The quantitative composition was determined from detailed spectra taken at the pass energy of 25 eV and the electron take off angle 50 . The maximum lateral dimension of the analyzed area was 1.5 mm. The quantification was carried out using XPS MULTIQUANT software.30 In this work, the latest version CASAXPS 2.3.16 was used for the curve fitting and now it is specified in the experimental details. The shape of the curve was the convolution of 30% Lorentzian and 70% Gaussian shape because larger portion of Lorentzian led to better fitting of the data. However, we did not use larger portion of Lorentzian because maximum 30% is recommended for the XPS fitting of polymers by Beamson and Briggs.31 The FWHM was set in the range of 1.8–1.9 eV, in order to get the smallest as possible width of the peaks. The values of the width are quite high because of the nonmonochromatic x-ray source and nonregular chemistry of the layer, which is often observed for plasma polymers.32 In order to be consistent and to obtain the high quality of the fitting, the FWHM was varied in this narrow range from 1.8 to 1.9 eV for all samples. The WCA was measured by sessile drop method. The measurements were done using the surface energy evaluation device, SEE System, manufactured by Advex Instruments s.r.o. The value of the water contact angle was calculated as mean value from at least nine drops, where each drop volume was 2 ll. A Hysitron TI-950 triboindenter equipped with diamond Berkovich indenter was used in the evaluation of the hardness and elastic modulus of the deposited modified DLC thin films. The nanoscale measuring head with resolution of 1 nN and load noise floor less than 30 nN was used for the measurements. Two testing modes were used in the range of indentation loads from 0.5 to 10 mN, namely, quasistatic nanoindentation test and quasistatic nanoindentation with several partial unloading segments (PUL). The standard Oliver and Pharr33 approach was used to evaluate the hardness and elastic modulus of the studied films. The PUL technique was employed in order to create the profiles of the change of hardness and elastic modulus as functions of depth. Depth profiling is particularly advantageous on coated materials because it allows the detection of the substrate effect by observing local minima or maxima in the measured properties. As we measure at higher depths into the coated material, the substrate influence reflects in the changes in hardness or elastic modulus. In this work, a series of nine PUL tests with 20 unloading segments were performed in

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the above mentioned load range at different locations on each selected sample. The in situ scanning probe microscopy (SPM) imaging capability of the triboindenter was employed in order to get information about the surface topography before and after the tests. C. Cell cultivation

All the cell-related experiments were performed on the mouse myoblast C2C12 cell line (ATCC). Cells were cultivated (37  C, 5% CO2, and 95% humidity) for 24 h in high glucose DMEM (Dulbecco’s Modified Eagle Medium, Sigma Aldrich) with the addition of 10% of fetal bovine serum (HyClone, Thermo Scientific), 1.2 mM L-glutamine (Gibco), 100 U/ml of penicillin and streptomycin (HyClone, Thermo Scientific). Cell cultivation was performed on TPP plastic 1 ml dishes: without plasma coating (control test) or coated by plasma layer. The density of seeded cells was 0.2  106 cells per 1 ml. C2C12 myoblasts are surface adherent cells, and for their transfer to suspension, the enzymatic cleavage of adhesion molecules by 0.05% Trypsin-0.02% EDTA (PAA) was used. In 10–30 min of incubation with 400 ll Trypsin-EDTA was Trypsin neutralized by addition of 600 ll of fresh complete DMEM. One milliliter of final suspension was stored on ice and subsequently counted using Accuri C6 flow cytometer. The cell counts were used in the numerical comparison between coated and control samples. III. RESULTS A. Chemical structure 1. FT-IR

The film thicknesses determined by fitting the optical data were C1: 670 nm, C2: 575 nm, C3: 342 nm, and C4: 568 nm. These thicknesses were sufficient for the detection of IR absorption peaks that revealed the information about the chemical structure of the films. The complex parts of the IR dielectric functions of the films C1, C3, and C4 is compared in Fig. 1. The IR dielectric function of C2 was the same as C1 and, therefore, is not included in Fig. 1.

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All films exhibited a wide absorption peak in the range of 2750–3150 cm1, which arose by an overlapping of absorption peaks associated with the stretching of various spxCHy groups. This broad absorption band was expressed as the sum of nine Gaussians with the position fixed to the values used previously.29 In C1, C2, and C3, we can see another one or two peaks at higher wavenumbers. Those can be associated to OH groups.34 Lack of OH groups in C4 sample can be explained by a higher concentration of HMDSO during the deposition. Hence, a lower influence of air and water vapors on the film composition is observed after removing the samples from the deposition chamber and exposing them to ambient atmosphere. A sharp peak at 2340 cm1 is identified as CO2 trapped in the layer.35 Two peaks were required to fit Si–H absorption in region around 2150 cm1. This led to the conclusion that different types of SiHx groups are present in the layers. SiHx peaks are detectable even in silicon free films on c-Si substrate and were attributed to the presence of Si–H bonds in the interface region between substrate and coating.29 Carbon network vibrations can be seen in sample C1 and C2 as two broad peaks around 1300 and 1600 cm1,36 and one smaller peak around 1690 cm1.29 With addition of silicon oxide into carbon network (samples C3 and C4), new high intensity peaks corresponding to Si–O–Si vibrations appeared around 800 (bending) and 1000 (stretching) cm1, the peaks at 1300 and 1690 cm1 disappeared, the peak at 1600 cm1 decreased and shifted to lower wavenumbers. C-H vibrations are represented by the peak at around 1450 cm1 (sp3CH2 scissoring or sp3CH3 asym. bending34) and the weak peak at 1375 cm1 (sp3CH3 sym. bending37). CH3 symmetric (1255 cm1) and asymmetric (1410 cm1) bending vibrations in Si-(CH3)x groups38 can be observed in C3 and C4 samples. 2. XPS, RBS, and ERDA

The surface composition of the thin films was obtained by XPS as summarized in Table II. By the addition of 0.2 sccm synthetic air, it was possible to incorporate a relatively high

FIG. 1. Complex part of the film dielectric functions in IR range. For clarity, the sample C2 is not shown because the curve was completely identical to the sample C1. Biointerphases, Vol. 10, No. 2, June 2015

029520-5 Stoica et al.: Cell proliferation on modified DLC thin films TABLE II. Surface chemical composition as determined from XPS. Sample C1 as deposited C1 after 2 weeks C1 aged 6 months C2 as deposited C2 after 2 weeks C2 aged 6 months C3 aged 6 months C4 aged 6 months

C (%)

0 (%)

N (%)

Si (%)

O/C

N/C

Si/C

90 89 85 91 87 85 59 56

6 8 13 5 9 13 25 23

4 3 2 4 4 2 2 1

0 0 0 0 0 0 14 20

0.07 0.09 0.15 0.06 0.10 0.15 0.42 0.41

0.04 0.03 0.02 0.04 0.05 0.02 0.03 0.02

0 0 0 0 0 0 0.24 0.36

amount of oxygen and nitrogen in the as-deposited C1 and C2 film surfaces. The difference between the samples C1 and C2 deposited from CH4 and CH4þH2 mixtures, respectively, was negligible. The films deposited from HMDSOþCH4 mixtures exhibited significantly different chemical composition. A higher flow rate of HMDSO led to higher incorporation of silicon into the films as the C:Si:O:N ratio for C3 and C4 samples were equal to 59:14:25:2 and 56:20:23:1, respectively. The functional composition of the film surfaces was determined by the fitting of XPS C1s and Si2p peaks. The C1s

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signal for the layer grown in CH4þH2 mixture was fitted by sum of three peaks related to sp3carbon or hydrocarbon (CHx, BE  285 eV), carbon single-bonded to oxygen or nitrogen (C–O/C–N, BE  286.5 eV), and carbon double-bonded to oxygen (C¼O, BE  288.1 eV).32,39,40 The fitting of the layer obtained in CH4 required an additional contribution related to carboxyl or ester group (COOR, BE  289 eV).41–44 As shown in Fig. 2, the functional composition of C1 and C2 thin films (deposited from CH4 and CH4þH2, respectively) was very similar. The predominant contribution of carbon chemical environment was sp3 carbon and hydrocarbon CHx groups. The XPS cannot distinguish between C–H and C–C but the FTIR analyses did not show any difference between the absorption of hydrocarbon groups in C1 and C2 films. This conclusion was further confirmed by ion beam methods RBS and ERDA because the percentage of hydrogen was the same (Table III). Regarding the remaining carbon environment, slightly higher C–O/C–N and the presence of COOR contribution suggested a higher density of hydrophilic groups in the C1 film. A comparison of differences between the surface composition (determined by XPS) and the bulk (determined by RBS and ERDA) showed a similarity between the CH4 and

FIG. 2. XPS C1s curve fitting of the doped DLC grown in CH4: (a) sample C1 as deposited, (b) sample C1 aged 6 months; and in CH4þH2: (c) sample C2 as deposited, and (d) sample C2 aged 6 months. Biointerphases, Vol. 10, No. 2, June 2015

029520-6 Stoica et al.: Cell proliferation on modified DLC thin films TABLE III. Results of elemental analysis (including hydrogen) of DLC:N:O films by RBS and ERDA ion beam methods. Sample C1 C2

C (%)

H (%)

0 (%)

N (%)

O/C

N/C

62 64

31 30

2 2

5 4

0.03 0.03

0.08 0.06

CH4þH2 films. Even if the compositions from RBS and ERDA were recalculated as not including hydrogen, the O at. % was two times higher at the surface than in the bulk, whereas the N at. % was 1.5 lower at the surface. It is worth noting that the prepared DLC films were stable in air for very long periods. According to the XPS analysis, the concentration of oxygen at the film surfaces increased by 1–2 and 4–5 at. % after 2 weeks and 6 months storage in air, respectively. The functional composition changed very little after two weeks (not reported here) and a small indication of oxidation, increase of C¼O, was observed after 6 months (Fig. 2). The N1s and O1s signals were very similar regardless the plasma conditions and ageing (not reported here). The XPS O1s signal was centered at 533 eV, and its position was not changing with aging. The XPS N1s signal of as

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deposited and aged for 2 weeks C1 and C2 samples was fitted with one contribution with FWHM of 2.1 eV and BE of 399.5 eV. At this BE, the nitrile and secondary amine groups can be expected.31 The ageing of C1 and C2 for 6 months induced the increase of BE of N1s signal by 0.2–0.3 eV, i.e., the oxidation of amines to amides probably occurred during the long term aging. However, the observed changes are almost negligible. The fitting of the XPS C1s peak for samples C3 and C4 prepared from HMDSOþCH4 mixtures required additional contribution of the C–Si at 284 eV.45 In order to investigate the nature of silicon environment the Si2p peak was fitted with a sum of three contributions: silicon bonded to two oxygen atoms (Si2þ, BE  101.6 eV), silicon with three bonds to oxygen (Si3þ, BE  102.8 eV), and silicon with four bonds to oxygen (Si4þ, BE  103.4 eV).32,46 As shown in Fig. 3, the increase of the HMDSO flow rate led to a higher concentration of the Si2þ at the expense of Si4þ. The Si2þ corresponds to the initial structure of silicon in HMDSO as well as in the polydimethylsiloxane (PDMS) polymer, whereas Si4þ corresponds to the silicon oxide. Hence, at higher HMDSO flowrate, more organic nature of the silicon environment is expected.

FIG. 3. XPS C1s and Si2p curve fitting of the doped DLC films prepared from CH4þHMDSO: (a) C1s peak—sample C3, (b) C1s peak—sample C4, (c) Si2p peak—sample C3, and (d) Si2p peak—sample C4. Biointerphases, Vol. 10, No. 2, June 2015

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B. Wettability

The calculated average water contact angles did not show any significant differences among the films prepared. The WCA on C1 and C2 films presented similar values and varied within the limits 71 –81 . This similarity is in agreement with the XPS results which shows no significant difference between samples C1 and C2. WCA slightly increased by an addition of HMDSO into the deposition mixtures. It varied in the range of 77 –86 for lower HMDSO flow rate and even higher values, 81 –90 , were observed for 0.7 sccm of HMDSO. According to the chemical analyses of the film surface, the increase of WCA is related to higher concentration and hydrophobic PDMS-like environment of silicon. The influence of surface roughness on WCA was limited because optical measurements did not reveal any roughness on the films.29 C. Mechanical properties

The results of the mechanical characterization for all the prepared samples are listed in Table IV. Although the elemental composition of the coatings prepared from CH4 and CH4þH2, as revealed by RBS, ERDA, FTIR, and XPS, was very similar, they differed from the point of view of their mechanical properties. The main difference was in their adhesion to the silicon substrate. Sample C2 showed very low resistance against spontaneous delamination, while C1 was relatively stable. The hardness and elastic modulus values calculated from the indentation curves at low loads showed also a slight difference. Sample C1 exhibited higher hardness and elastic modulus comparing to sample C2. The main source of this difference is the lower interfacial stability of the C2 sample. The interface effects play an important role in the correct estimation of the mechanical properties. The indentation induced delamination at the coating/substrate interface may substantially influence the measured hardness and elastic modulus values. Also, the humidity is an important factor of influence during the nanoindentation tests mainly in case of the hydrogenated DLC coatings. This effect has been described previously in the literature by Sobota et al.47 It was found that the humidity may substantially influence not only the results of hardness, but also the results of tribological and dynamic wear tests. This effect was attributed to the adsorbed water on the film changing the properties at the near surface, which may cause changes also in the mechanical properties. The thin films covering the culture dishes did not show any sign of delamination, which is due to the different interface between coating and substrate. The pretreatment in argon plasma before the deposition was done in order to

FIG. 4. Cell proliferation on DLC:N:O films.

improve the adhesion of the films to the substrates. The films did not show any delamination even after the cells were removed and the culture dishes dried. The addition of HMDSO in the gas mixture led to the deposition of DLC:SiOx films with hardness and elastic modulus lower than in the case of sample C1. Moreover, it can be seen that hardness and elastic modulus decrease with increasing the HMDSO flow rate. This observation was previously reported by our group.48 This helps preparing a better adhesion of the thin films, as the high hardness of the DLC thin films prepared from CH4 and CH4þH2 is accompanied by high compressive stresses. D. Cell proliferation

The ratios between the cell count on coated dishes and the cell count on uncoated control dishes are presented in Figs. 4 and 5. The effect of the aging on cell proliferation was investigated. The graphs present the percentage increase [C (%)] in the number of cells counted on coated dishes (C1) relative to the number of cells counted on untreated (control)

TABLE IV. Mechanical properties of DLC:N:O and DLC:SiOx thin films. Sample

C1

C2

C3

C4

Hardness, HIT (GPa) 13.0 6 0.5 10.5 6 0.5 10.8 6 0.5 9.5 6 0.5 Reduced modulus, Er (GPa) 98 6 3 80 6 2 80 6 3 72 6 2 FIG. 5. Cell proliferation on DLC:SiOxthin films. Biointerphases, Vol. 10, No. 2, June 2015

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dishes (CC), described by the formula C (%) ¼ (C1/CC  1)  100. The error of the ratios was calculated using the standard uncertainty propagation laws taking into account that the counts themselves have Poisson distribution. As shown in Fig. 4, the number of cells counted on the coated dishes is as high as that on the unmodified (control) dishes. The cell counts show no significant difference between the DLC coatings prepared from methane alone or in admixture with hydrogen. The similarities in cell proliferation could be explained by the similar surface chemistry of the thin films surfaces revealed by XPS and WCA measurements. Moreover, the influence of ageing has a limited effect on cell count variation. We observed no negative effect of the coating on the cell proliferation within the range of experimental error. Similarly, the cell proliferation on DLC:SiOx thin films as presented in Fig. 5 shows no difference in the range of experimental error. The amount of HMDSO in the precursor gas mixture reveals that cells do not exhibit an affinity for one specific type of coating. The effect of the aging shows no significant influence on cell proliferation within a period of 1 month after deposition. It is worth noting that the classification of the biocompatibility of the DLC films was introduced in the work of Ohgoe et al.49 The authors define five groups of DLC coatings according to the number of cells attached to the surface of the coatings. They define levels 4 (high) and 5 (very high) with numbers of cells comparable to the control samples. Hence, the prepared DLC films exhibited high level of biocompatibility combined with the mechanical properties and the stability of the prepared DLC films. IV. CONCLUSIONS In this work, different modified types of DLC were prepared on silicon, glass, and cell culture dishes by using PECVD technique. The DLC coatings exhibited good adhesion on the polystyrene culture dishes, their composition was stable, and only a slow oxidation was observed by XPS analysis of layers stored in air for 6 months. The incorporation of N, O, and Si was affecting the mechanical properties of DLC as well as the wettability of the surface. However, regardless of the chemical composition of the films, the level of cell response was comparable to the control samples. According to the literature, the biocompatibility of the prepared DLC films can be classified as high or even very high. ACKNOWLEDGMENTS This work was supported by the project “CEITEC— Central European Institute of Technology” (CZ. 1.05/1.1.00/ 02.0068). The authors would like to thank Vratislav Perina, for the RBS/ERDA measurements conducted at the CANAM (Center of Accelerators and Nuclear Analytical Methods LM2011019) infrastructure with a funding from the Ministry of Education, Youth and Sports of the Czech Republic. Last but not least, the authors would like to thank Biointerphases, Vol. 10, No. 2, June 2015

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David Necˇas, for his advice on data analysis for cell culture results. 1

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