Thin-film nanocomposites of diamond-like carbon and titanium oxide; Osteoblast adhesion and surface properties

Diamond & Related Materials 19 (2010) 329–335

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Diamond & Related Materials j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d i a m o n d

Thin-film nanocomposites of diamond-like carbon and titanium oxide; Osteoblast adhesion and surface properties L.K. Randeniya a,⁎, A. Bendavid a, P.J. Martin a, M.S. Amin a, R. Rohanizadeh b, F. Tang c, J.M. Cairney c a b c

CSIRO Materials Science and Engineering, PO Box 218, Lindfield, NSW, 2070, Australia Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia The Australian Key Centre for Microscopy and Microanalysis, University of Sydney, Sydney, NSW, 2006, Australia

a r t i c l e

i n f o

Article history: Received 13 August 2009 Received in revised form 17 November 2009 Accepted 4 January 2010 Available online 11 January 2010 Keywords: DLC Nanocrystaline Titania Nanocomposite Osteoblast attachment

a b s t r a c t Thin films of a novel, nanocomposite material consisting of diamond-like carbon and polycrystalline/ amorphous TiOx (DLC-TiOx, x ≤ 2) were prepared using pulsed direct-current plasma enhanced chemical vapour deposition (PECVD). Results from Raman spectroscopy indicate that the DLC and TiOx deposit primarily as segregated phases. Amorphous TiO2 is found to be present on the surface region of the film and there is evidence for the presence of crystalline TiO in the bulk of the film. The hydrophilicity of the DLC-TiOx films increased with increasing titanium content. Culture studies with human osteoblasts revealed that the differences in three-day cell adhesion properties (count, morphology and area) between DLC and DLC-TiOx films containing up to 13 at.% Ti were not statistically significant. However, the cell count was significantly greater for the films containing 3 at.% of Ti in comparison to those containing 13 at.% of Ti. A post-plasma treatment with Ar/O2 was used to reduce the water contact angle, θ, by nearly 40° on the DLC-TiOx films containing 3 at.% of Ti. A cell culture study found that the osteoblast count and morphology after three days on these more hydrophilic films did not differ significantly from those of the original DLC-TiOx films. We compare these results with those for SiOx-incorporated DLC films and evaluate the long-term osteoblast-like cell viability and proliferation on modified DLC surfaces with water contact angles ranging from 22° to 95°. Crown Copyright © 2010 Published by Elsevier B.V. All rights reserved.

1. Introduction Following surgery, successful incorporation of an implant into the body depends on tissue integration and infection resistance, which is influenced by the adherence of autologous cells and bacteria to the surface [1–3]. It has been argued that there are important correlations between surface properties and initial cell attachment from serumcontaining cultures. Surface energy, surface chemistry and surface roughness are some properties that are widely investigated for their impacts on cell attachment [4–10]. Some studies have found a correlation between cell/protein attachment and surface energy on certain metal, polymer and hydroxyapatite surfaces [11–16]. Since surface energy, surface texture and surface chemistry are closely linked, the study of cell response to just one of these parameters has always been difficult [7]. Diamond-like carbon (DLC) is a metastable form of hydrogenated amorphous carbon with qualities such as chemical inertness, hardness, low coefficient of friction, bio-compatibility and haemocompatibility. DLC and doped DLC have been investigated extensively for possible bio-medical applications [17–22]. It has been demon-

⁎ Corresponding author. Tel.: +612 9413 7669; fax: +612 9413 7200. E-mail address: [email protected] (L.K. Randeniya).

strated that doping with elements such as Si, N, Ca, P and others improves the properties of DLC such as bio-compatibility, infection resistance and mechanical properties [23–28]. A few studies exist on mechanical properties of titanium incorporated DLC [29,30] and the changes in cellular response [31,32]. Films prepared by Schroeder et al. [31] showed increased proliferation and reduced osteoclast-like cell activity in rat bone marrow cell cultures. Films prepared by Thorwarth et al. [32] though not mechanically sound (nanohardness smaller than 2 GPa), showed significantly higher deposition of Ca compared to unmodified DLC suggesting better osteogenic differentiation on TiO2-incorporated DLC films. Studies with osteogenic precursor cells derived from rat bone marrow stroma by Dieudonne et al. [33] indicated that the sol–gel-derived TiO2 is likely to be compatible with bone cells and able to facilitate osteogenesis of bone precursor cells. Nanostructured TiO2 was also found to show normal growth and adhesion of primary and cancer cells with no need for coating with extracellular-matrix proteins [34]. In this paper we report the synthesis of a novel class of nanocomposite thin films consisting of nanocrystalline and amorphous TiOx and diamond-like carbon (DLC-TiOx, x ≤ 2). The hydrophilicity of these films increased with Ti content. A post-plasma treatment with Ar and O2 was then used to further increase the hydrophilicity on the DLC-TiOx films. We compare the cell culture results with the surface properties of these films and those for SiOx-

0925-9635/$ – see front matter. Crown Copyright © 2010 Published by Elsevier B.V.All rights reserved. doi:10.1016/j.diamond.2010.01.003

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incorporated DLC films and evaluate the long-term osteoblast-like cell viability and proliferation on modified DLC surfaces with water contact angle, θ, ranging from 22° to 95°. 2. Methods 2.1. Pulsed DC PECVD system The PECVD deposition system has been described in detail elsewhere [35,36]. Briefly, the substrate (semiconductor-grade Si, (100)) was attached to a steel substrate holder which functions as the electrode and was powered by a pulsed direct-current (DC) generator (Rübig Model MP120) operated at 420 V. No external heating of the substrate was used and the deposition of films occurred at the ambient temperature of the chamber. Methane (precursor for DLC, 50 sccm) and argon (30 sccm) were introduced into the chamber through a gas distributor using mass flow controllers. The glass vessel containing the titanium isopropoxide (purity 97%, Sigma-Aldrich) precursor was heated to 70 °C–90 °C in a water bath. The glass vessel was connected to the reaction chamber using stainless steel tubing that was heated to a temperature of about 20 °C greater than that of the glass vessel to prevent condensation of the metal–organic precursor. Increased temperature in the glass vessel correlated with increases in Ti concentrations in the films. Argon gas was used as a carrier gas for the metal–organic vapour. For Ar/O2 post-plasma treatment of DLC-TiOx films, Ar (30 sccm) and O2 (10 sccm) were used for a period of 2 min. The deposition pressure was maintained at 0.1 kPa for all experiments. Film thickness was measured using a stepedge profilometer (Sloan Instruments Dektak 3030). 2.2. Spectroscopic analysis The composition of the films was determined by X-ray photoelectron spectroscopy (XPS) using a SPECS 150 system operated with Mg Kα X-ray source (10 keV and 10 mA) [37]. Spectral resolution of the instrument is 0.1 eV. The fitting of the peaks utilised a Gaussian/ Lorentzian product formula with a Shirley background [38] and was carried out using the CASA-XPS V2.3.13 software. The peaks were referenced to adventitious C 1s peak at 284.5 eV. Raman spectroscopy was performed using a Renishaw In Via confocal Raman microscope system. Specimens were illuminated with 514 nm excitation radiation from an Ar+ laser at an incident power of ∼ 1 mW and a spot diameter size of approximately 1 μm. Cross-sectional transmission electron microscopy (TEM) specimens were prepared using both tripod polishing and a FEI Quanta 3D focused ion beam system. TEM investigations were carried out on a JEOL 3000F transmission electron microscope operating at 300 keV, using bright field and dark field imaging, high-resolution imaging, and selected area diffraction techniques. 2.3. Contact angle and surface roughness Contact angles were measured using the sessile drop method with an instrument equipped with a CCD-IRIS video camera, a laser light source and Rame-Hart 2001 imaging software. Water, diiodomethane and ethylene glycol were used as the test liquids. Prior to measurements with each liquid, the samples were cleaned with acetone, 95% ethanol and deionised water. A minimum of four measurements were taken for each sample. The procedure was then repeated to obtain a total of eight readings for each sample with each liquid. Standard deviations of the measurements were typically 2–3°. All measurements were made at 22 °C and ∼50% relative humidity. The average contact angles obtained from the three liquids were used in van Oss et al. [39] approach to obtain the dispersive component, γLW (Lifshitzvan der Waals component) and the polar components (acidic component/electron-acceptor component, γ+ and basic/electron-

donor component, γ−) of surface energy. The surface roughness of the films was measured using a non-contact Wyko Topo 3D digital phase-shifting interferometer surface profiler. The measurements were taken over an area of 117 × 154 μm. The root-mean-square (RMS) roughness value was an average of 3 measurements on each sample at different positions after the curvature and tilt from each profile was removed. 2.4. Cell attachment The osteoblasts were cultured using standard tissue culture protocols previously described [40,41]. All specimens were first cold sterilized in 70% ethanol for 30 min and exposed to UV light prior to being introduced into 15 mm diameter multi-well tissue culture plastic dishes. MG63 osteoblast-like cells were seeded onto the specimens along with Dulbecco's Modified Eagle medium containing calcium and supplemented with 10% foetal bovine serum (FBS), 1% Lglutamine, 2% HEPES buffer, 1% non-essential amino acids, and 2% penicillin and streptomycin. The cells were seeded at densities of 70,000 and 20,000 cells/well respectively for cell proliferation and morphology studies. The cells were then incubated for three days at 37 °C in a humidified atmosphere of 95% air and 5% carbon dioxide. In order to determine the cell morphology, cell layers were rinsed with pre-warmed phosphate buffered saline (PBS) solution and fixed using 2% glutaraldehyde in PBS. Cells were kept at room temperature for 1 h and then stored at 4 °C for 24 h. The following day, excess glutaraldehyde solution was removed and the cells were rinsed once more in PBS before being dehydrated progressively in higher concentrations of ethanol baths (50, 70, 80, 90, 95, and 100%). Samples were dried and gold sputter-coated for SEM. A Phillips XL30 SEM was used at high vacuum to view samples. To measure the number of cells after a three-day culture, the cells were rinsed with PBS solution and then detached by incubating for 9 min at 37 °C in 0.2% trypsin. Once all cells were detached from the surface, the solution was neutralised with FBS and then stained using Trypan Blue. The solution was flushed several times to suspend the cells evenly before pipetting 10 µL of suspended cells into a hemacytometer for cell counting. The statistical significance of the results was determined using an ANOVA (one-way analysis of variance) test with a Tukey– Kramer multiple comparisons test. Cell areas were measured using an Image J v1.36b program. A minimum of 25 cells were measured per sample type. 3. Results 3.1. XPS results Table 1 shows the atomic percentages of C, O and Ti for the films which are investigated in this study. Abbreviated sample names DCT1, DCT2 etc. are used for brevity. The Ti atomic percentage ranged from 0 to 13.0 at.% for these films. Note that the hydrogen atomic percentage is not included in the calculations shown in Table 1 as XPS is not able Table 1 Surface compositions (determined by XPS) for the films investigated in this study. DCT_O2 sample measured two weeks after post-plasma treatment just prior to cell culture studies. Sample name

DLC DCT1 DCT2 DCT3 DCT4 DCT5 DCT2_O2 (Ar/O2 treated)

Surface composition (at.%) C

O

Ti

89.5 82.6 76.0 70.0 58.6 40.7 53.9

10.5 17.2 21.0 25.0 31.4 46.3 39.8

0 0.2 3.0 5.0 10.0 13.0 6.3

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to determine H. We will use the surface atomic percentage of Ti determined by XPS as the label for DLC-TiOx films containing different amounts of Ti. Fig. 1 shows XPS spectra for Ti 2p, C 1s and O 1s for DCT4 (a sample containing 10 at.% of Ti, top panels) and DCT2_O2 (a post-plasmatreated sample, bottom panels). Also shown are the Gaussian fittings obtained using CASA software. The location of the Ti 2p3/2 peak at 458.8 eV (Fig. 1(a) and 1(a′)) corresponds to amorphous TiO2 [42]. From the higher-binding-energy residual after the fitting for TiO2, there is evidence for the presence of small amounts of suboxide/ oxycarbides. For Ti–C, the Ti 2p3/2 peak should appear below 456 eV [31,43] and therefore, the presence of titanium carbide in any significant amounts on the surface of the film can be ruled out. The C 1s peak for DCT4 (Fig. 1(b)) was resolved into two peaks with contributions from the main DLC sp2 network (284.5 eV) and C–OH bonds (286.5 eV). The O 1s peak for DCT4 (Fig. 1(c)) resolved into two peaks; one at 530.2 eV from TiO2, and another at 531.7 eV from Ti–OH [44] and/or C–OH bonds [45]. When the DLC-TiOx films are treated in Ar/O2 plasma for 2 min, the surface becomes richer in O as surface carbon atoms are oxidised. Some C is removed from the surface as evidenced by the increase in Ti atom percentage (Table 1). This is also associated with a substantial increase in the surface energy of the film (see below). The reason for this is the formation of additional hydroxyl (C–OH) and carboxyl acid (C–COOH) groups which increase the negative charge on the surface. This is evident in the XPS spectrum for C 1s for the Ar/O2 treated films (Fig. 1(b′)). The oxygen to carbon ratio is larger and the peak areas for C–OH (at 286.0 eV) and carboxylic groups (288.8 eV) are larger for DCT2_O2 (Fig. 1(b′)) in comparison to those for DCT4 (Fig. 1(b)). In Fig. 1(c′) it can be seen that the increase in O leads to an increase in the peak area at 532.3 eV which is assigned to hydroxyl groups. 3.2. TEM results TEM revealed a nanocrystalline structure consisting of nanoscale TiOx particles, in an amorphous DLC matrix. Fig. 2(a) is a bright field image of the cross-sectional specimen, taken approximately 100 nm below the surface of the film. The inset diffraction pattern is taken from approximately the selected area shown. The pattern has been

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indexed, and the positions of the rings are consistent with the expected crystal structure of titanium monoxide (TiO, Ref 00-0080117). The diffuse background of the diffraction pattern is thought to arise from an amorphous matrix phase. Fig. 2(b) is a high-resolution image of the same area. Crystal planes can be discerned in some areas (marked in the figure), revealing the approximate size of the crystallites (2–5 nm). These were often difficult to discern, due to their small size and the surrounding amorphous matrix. Further investigations are underway to fully characterise the bulk structure of these materials.

3.3. Raman spectroscopy results The Raman spectrum showed the characteristic G-peak (peak centred at 1558 cm− 1 which originates from C C stretching vibrations) and the D-peak (peak centred at 1387 cm− 1 which originates from breathing modes of six-fold rings) observed for amorphous hydrogenated carbon films [46,47]. Fig. 3 shows the variations in the location of the G-peak (4a) and the area ratio of the D-peak to G-peak, I(D)/I(G) (4b), as a function of the surface Ti concentration. Also shown are the results for SiOx-incorporated DLC (DLC-SiOx) for comparison purposes [36]. Except for very low concentrations of Ti, the I(D)/I(G) ratio, which is proportional to the size of carbon clusters, remains nearly constant for DLC-TiOx films. The value of I(D)/I(G) corresponding to this plateau is only about 20% smaller than the value for unmodified DLC. This small change in I(D)/I(G) ratio suggests that the cluster size and the sp3 fraction of DLC are only slightly modified when combined with TiOx during the formation of DLC-TiOx. For DLCSiOx, the ratio I(D)/I(G) and the G-peak location continue to decrease with increasing Si concentration. This pattern of change is associated with siloxane bond formation in the DLC-SiOx films [48]. Segregation of SiOx in DLC-SiOx films can be discerned from these diagrams for Si concentrations above 10% where data starts to level off [48]. From these results, we conclude that the DLC-TiOx films are primarily a nanocomposite of DLC and TiOx. This assertion is further supported by the small G-peak shift of less than 10 cm− 1 going from DLC to DLCTiOx containing 13 at.% of Ti. A significant presence of Ti–C bonds would have resulted in a larger downshift in the G-peak location [49].

Fig. 1. XPS spectra for Ti 2p, C 1s and O 1s core electrons for DCT4 (upper panels, a, b, c) and for DCT2_O2 (lower panels, a′, b′, c′).

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Fig. 2. TEM images taken approximately 100 nm below the surface of a DLC-TiOx film; bright field image with diffraction pattern inset (left) and high-resolution image (right).

3.4. Surface roughness The RMS surface roughness for unmodified DLC was found to be 0.5–1.0 nm. The RMS surface roughness for DLC-TiOx was found to be in the range of 1.0–4.0 nm. Note that the roughness measurements reported here were averaged over an area of 117 × 154 µm2 which is much larger than the areas which are typically used in atomic force microscopy. Roughness between 0.6 nm and 2.5 nm were obtained for DLC-SiOx films [36]. The measured roughness values for DLC-TiOx films are several factors larger than the unmodified DLC. They however, are much smaller than what are typically found for biopolymers or metallic substrates and DLC films prepared using radio frequency techniques [4,50]. It is generally believed that the rougher and textured surfaces promote cell adhesion and differentiation [4,14,51].

increased to ∼22° and measured Ti content was ∼6 at.% (see Table 1). Fig. 5 shows how the changes in θ correlates with the Lewis-base surface energy (γ−, electron-donating polar energy component) determined using the van Oss et al. approach [39]. For Ar/O2 treated sample, γ− increases by more than a factor of two. As deduced earlier from the XPS data, this increase in the surface energy originates from the increase in hydroxyl and carboxyl groups on the surface. The dispersive component (not shown) remained in the range of 43– 46 mJ/m2 for all films studied here. 3.6. Cell count

As seen from Fig. 4, the measured contact angle for water (θ) on DLC-TiOx films reduced from 63° to 47° when the Ti content in the film increased from 0 to 13 at.%. For DLC-SiOx, θ increased up to 95° for Si concentrations above 10 at.%. When a film containing 3 at.% of Ti was exposed to Ar/O2 plasma for 2 min (DCT_O2), the value for θ reduced to 15°. It then increased with time, returning to its original value (∼60°) after 6 weeks. The osteoblast adhesion experiments were carried out after two weeks from synthesis and by then θ has

Fig. 6 shows the cell count for tissue culture polystyrene (TCPS), DLC and modified DLC films after three days of cell culture. The results are shown for films with surface titanium concentrations of 3 at.% (DCT2) and 13 at.% (DCT5). Also shown is the result for the films which contained 3 at.% Ti and treated with Ar/O2 plasma to enhance the surface hydrophilicity (DCT2_O2). Although in comparison to unmodified DLC the cell count for DCT5 was lower, and the cell count for DCT2 was higher, the differences were not statistically significant either between DLC and DCT2 or between DLC and DCT5 (number of samples, n = 5; P N 0.05). Therefore, the addition of TiOx to DLC is neither beneficial nor detrimental for osteoblast viability. However, the cell count on DCT5 was significantly lower than that of DCT2 (P b 0.001) which indicates that the osteoblasts prefer samples containing smaller Ti concentration over those containing higher Ti

Fig. 3. The variations of (a) G-peak location and (b) I(D)/I(G) ratio for DLC-TiOx and DLC-SiOx films as a function of Ti and Si atomic percentages.

Fig. 4. Variation of water contact angle, θ, for DLC-TiOx and DLC-SiOx films as a function of Ti and Si contents in the films. Also shown is the result for DCT2_O2 film two weeks after post-plasma treatment with Ar/O2.

3.5. Surface free energy

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Fig. 5. Variation of the Lewis-base surface energy component, γ−, as a function of Ti content and Si content for the DLC-TiOx and DLC-SiOx films. Also shown is the result for DCT_O2 sample two weeks following the post-plasma treatment with Ar/O2.

concentrations. The difference in cell count between DCT2 and postplasma treated sample, DCT2_O2, is not statistically significant either. Therefore, the increase in water contact angle and/or increase in OH/ COOH concentrations on the surface do not have a significant impact on cell proliferation and viability in these particular cases. Fig. 7 shows the cell counts after three days for samples with water contact angle ranging from 22° to 95° and the surface roughness ranging from 0.8 nm to 4.0 nm. For θ larger than 63°, the cell counts are markedly similar although the surface roughness changed by nearly a factor of four from 0.8 nm to 3.0 nm. Except for the result obtained for DCT5 with high Ti concentration, the data indicates a trend for increased cell counts for surfaces with θ ≤ 63°; additional studies using surfaces with smaller contact angles are necessary to determine whether the trend is statistically significant. 3.7. Cell areas and morphology The SEM images taken after 90 min and three days of cell culture confirmed similar size and morphologies for cells growing on DLC and DLC-TiO x samples (Fig. 8). Images generally showed multiple microvilli and spherical structures on the surface indicating continuous exchange between the cell surface and the environment. The long and fine cytoplasmic extensions in multiple directions indicate excellent adhesion and large lamellipodes indicate homogeneous colonisation. The cells on DLC-TiOx appear to exhibit more flattening on the substratum. After 90 min of culture the cell areas for DLC samples were 320 ± 40 μm2. For DLC-TiOx samples, the areas were slightly larger, 400 ±

Fig. 6. Osteoblast live-cell count after three days for DLC and DLC-TiOx films; DCT2 (3 at. % of Ti), DCT5 (13 at.% of Ti) and DCT2_O2 (originally 3 at.% Ti and post-plasma treated with Ar/O2 for 2 min, see Table 1 and text for details). Cell count on tissue culture polystyrene (TCPS) is also shown for comparison. Statistically different pairs (P b 0.001) are marked with an asterisk.

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Fig. 7. Osteoblast live-cell count after three days for DLC-based films. Water contact angle and RMS roughness are shown in parenthesis. Si content in the DLC-SiOx films with water contact angles 76° and 95° is 11 at.% and 13 at.% respectively.

30 μm2, but not statistically significant (P N 0.05). After the 3-day culture averaged cell area for the osteoblasts growing on DLC was found to be 900 ± 200 μm2. For all DLC-TiOx samples (including the post-plasma-treated samples), the average cell area was found to be 1200 ± 250 μm2. Again the cell areas are larger on DLC-TiOx samples but not statistically significant. 4. Discussion The XPS results suggest that the surface layer (∼ 5 nm) of the DLCTiOx films used in the current study consists primarily of a composite of amorphous TiO2 and DLC. There is no evidence for the presence of Ti–C bonds on the surface. The mechanical properties of these films were reported previously [52]. The compressive stress and the hardness of the films reduced with the increasing Ti content. It was expected from the recent results in the literature that the presence of TiO2 would facilitate an increase in the osteoblast cell adhesion in the modified DLC films [33,34]. In addition it was expected that the increase in hydrophilicity (surface energy) in DLCTiOx in comparison to that of pure DLC would benefit the osteoblast proliferation [14,53]. We did not find evidence to support these hypotheses. However, TiO2 on the surface appears biocompatible and the cell morphologies show healthy proliferation of cells on all DLCTiOx surfaces studied here. Our previous study found that the osteoblast proliferation properties were unchanged by the incorporation of SiOx to the DLC films [36]. In those films Si and C bond chemically to form siloxane structures [48]. The surface becomes more hydrophobic due to the presence of these polymer-like structures. In the case of DLC-TiOx, the surface of the films consists of TiO2 and DLC as a composite. The hydrophilicity is enhanced due to the presence of TiO2. Together with the information from the post-plasma treated samples of DLC-TiOx, we can use the data for DLC-TiOx and DLC-SiOx to compare osteoblast proliferation on surfaces with water contact angles ranging from 22° to 95°. The surface energy of DLC-TiOx was found to increase with the increasing titanium content. Although the increase in roughness can have a contribution, the systematic increase in surface energy with Ti content could also have been caused by surface chemistry. In the normal ambient, there is a small fraction of ultraviolet radiation which can lead to the generation of electrons (e) and holes (e+) in TiO2 [54]. The holes lead to the generation of Ti3+ from Ti4+ and the electrons generate O2 from O− 2 . Oxygen is ejected and vacancies are created on the surface making the surface hydrophilic. Since we primarily studied the cell counts and morphologies after 3 days, it is not possible to derive direct conclusions regarding the early cell attachment process and the influence of substratum in terms of physical forces involved. The data however, is useful for evaluating

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Fig. 8. SEM images for osteoblasts growing on DLC (left panels (a) and (c)) and on DLC-TiOx (right panels, (b) and (d)) films. Two top panels represent typical SEM images for cells after a 90 min culture and bottom panels are those taken after a 3 day culture.

the potential of these materials as coatings for prosthesis. The cell culture results suggest that for the DLC-TiOx films studied here, the viability and proliferation of osteoblasts were significantly greater in films containing lower Ti concentrations (3 at.% of Ti) in comparison to films with higher Ti concentration (13 at.%). In addition, though not statistically significant, the cell count was found to be smaller on films containing 13 at.% Ti than on unmodified DLC. A possible explanation for these differences could be the impacts of surface chemical composition where osteoblasts prefer DLC over TiO2 under these conditions. Unfortunately, cell culture studies on films with greater Ti concentration (N13 at.%) were not possible due to poor film quality. The post-plasma treatment was found to induce over a two-fold increase in the Lewis-base (electron-donating) surface energy (see Fig. 5) and an increase in the surface hydroxyl (C–OH) and carboxyl (C–COOH) concentrations in comparison to films that were not postplasma treated. However, the modifications did not lead to a significant change in cell viability and proliferation. The DLC-SiOx film with much lower electron-donating surface energy (a factor of five smaller compared to DLC) also showed similar cell counts and morphologies (Fig. 7). In the past, there has been some linking of increased cell adhesion to increased surface energy of metal, polymer and hydroxyapatite materials [11,12,14,55]. There is some evidence which supports a hypothesis that the early attachment phase for certain bone cell types is driven by the surface physical forces [53]. Surfaces with higher energy (θ b 65°) were found to be more compatible for cell growth. Our study showed enhanced cell count (although not statistically significant) on samples with θ b 63° (Fig. 7) except for DCT5. We already speculated that the low cell count on DCT5 could be a result of greater TiO2 concentration on the surface. We hypothesise that the

relatively small influence of substratum surface energy on the threeday cell counts and morphology observed in this study is due to the possibility that even on poorly compatible surfaces, the cells may naturalise their environment by various mechanisms and the differences in cell proliferation as a function of surface energy become less important with time. Lim et al. [15] studied integrin expression and the secretion of osteopontin and type I collagen in human foetal osteoblasts growing on hydrophobic and hydrophilic surfaces. They argued that the surface energy affected the integrin-mediated osteoblast adhesion via the regulation of specific integrin expression αvβ3. The expression of this integrin subunit was higher on hydrophilic surfaces (θ b 65°) where initial cell attachment was more favourable than on hydrophobic surfaces. However, although the hydrophobic surfaces decreased the production of αvβ3, they displayed greater steady-state levels of osteopontin, an extracellularmatrix (ECM) protein containing Arg-Gly-Asp (RGD) integrin recognition sequence. The authors raised the possibility that the cells, by increased secretion of osteopontin-containing RGD integrin recognition sequences, are conditioning the hydrophobic surface for optimal cell growth. Their assertion was also partly supported by the observation that the differences in αv and β3 integrin subunits, vinculin, and osteopontin expression in these particular cells as a function of surface energy decreased with culture time. Further consideration may be given to the impact of surface charge and the associated chemistry of the surface on osteoblast proliferation. Recent studies have indicated that the osteoblasts attach preferentially to surfaces with a positive charge, followed by surfaces with a negative charge and lastly to surfaces that are neutral [56]. The initial attachment was postulated to be facilitated by the electrostatic attraction between positively-charged surface and the negatively-

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charged pericellular hyaluronan matrix [57]. In the present experiment, both the incorporation of Ti and the treatment with Ar/O2 plasma increased the C–OH and C–COOH tending the surfaces more negatively-charged than DLC and DLC-SiOx. Therefore, the expectation again is that the DLC-TiOx surfaces and in particular DCT2_O2 would show enhanced osteoblast proliferation. The fact that we observed only a weak trend in this direction (statistically non-significant) after three days of culture may mean that although the early phase of attachment is weaker on surface neutral films, as pointed out by previous studies, the long-term cell population and morphology is relatively insensitive to surface charge for the DLC-based films studied here.

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