Materials Science and Engineering C 37 (2014) 99–107
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Strategies to prepare TiO2 thin films, doped with transition metal ions, that exhibit specific physicochemical properties to support osteoblast cell adhesion and proliferation Marshal Dhayal a,⁎, Renu Kapoor a,1, Pavana Goury Sistla a,1, Ravi Ranjan Pandey b, Satabisha Kar a, Krishan Kumar Saini b, Gopal Pande a,⁎ a b
CSIR-Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India CSIR-National Physical Laboratory, Dr K S Krishnan Marg, New Delhi, India
a r t i c l e
i n f o
Article history: Received 16 August 2013 Received in revised form 28 October 2013 Accepted 27 December 2013 Available online 4 January 2014 Keywords: Biomaterials Electronegativity Atomic radius Osteoinduction Surface energy
a b s t r a c t Metal ion doped titanium oxide (TiO2) thin films, as bioactive coatings on metal or other implantable materials, can be used as surfaces for studying the cell biological properties of osteogenic and other cell types. Bulk crystallite phase distribution and surface carbon–oxygen constitution of thin films, play an important role in determining the biological responses of cells that come in their contact. Here we present a strategy to control the polarity of atomic interactions between the dopant metal and TiO2 molecules and obtain surfaces with smaller crystallite phases and optimal surface carbon–oxygen composition to support the maximum proliferation and adhesion of osteoblast cells. Our results suggest that surfaces, in which atomic interactions between the dopant metals and TiO2 were less polar, could support better adhesion, spreading and proliferation of cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Titanium (Ti) oxides and Ti alloys, with modified structural, electronic and chemical properties, have been extensively used for biomedical and other applications [1–4]. These materials were fabricated by techniques such as conventional sintering [5,6], spark plasma sintering and surface state modification [5,7], self-propagating high-temperature synthesis [8] and hot isostatic pressing [9]. Materials synthesized using these techniques tend to have large proportions of secondary crystallite phases that can adversely affect their bioactivity and mechanical stability. Hence, several prior investigations have aimed to optimize the physicochemical properties of these materials, such as obtaining uniform phase structure, surface roughness, energy and chemical constitution that would support tissue specific biological functions, such as cell attachment, proliferation, differentiation and migration [10–13]. Most of the currently available orthopedic implants have microtopography-altered rough surfaces and chemically added bioactive materials [14–18]. Numerous reports have shown that bone responses are related to different surface properties associated with microtopography and chemistry of these surfaces [19–23]. In addition
⁎ Corresponding authors. Tel.: +91 40 27192605; fax: +91 40 27160591. E-mail addresses: [email protected] (M. Dhayal), [email protected] (G. Pande). 1 Equal contribution. 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.035
to the microstructure, chemical modification of surfaces also focuses on metal coatings of potentially bioactive materials, such as hydroxyapatite (HA). It has been suggested that bone formation reactions are enhanced by such bioactive materials [24]. Several HA-coated implants are already being used for dental and orthopedic treatments [25,26]. Recent studies have shown that thin films (b 200 nm) of titanium dioxide (TiO2) are more suitable to control the size of the crystallite phases and surface morphology; in addition, due to their transparent nature, these films are also useful to study in vitro cellular properties under a light microscope [1]. Two strategies have been commonly used to modify the physicochemical and biological properties of TiO 2 in thin films: surface treatment [27–30] and doping [1,8]. It has been shown by others and us that doping of TiO2 with transition metals could improve its structural, chemical and surface charge stability [8]. This could be due to the unique properties of transition elements belonging to the same period of the periodic table which, due to a higher number of electrons in ‘d’ orbital, exhibit reduced atomic radius and increased electronegativity along with the rise in atomic number [31]. In contrast to this, in transition metals belonging to the VIIIB group and 3d or 4d periods of the periodic table, these properties do not change significantly in spite of increase in atomic number (see Fig. 1). The ionic radii (IR), atomic radii (AR) and electronegativity (EN) values in Fig. 1 were corresponding to the intrinsic values of doped metal atoms. This schematic diagram provides the justification for selection of different metal ions as dopants which may
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2. Experimental section 2.1. Materials Titanium tetrabutoxide, iron acetylacetonate, nickel acetylacetonate, ruthenium trichloride and palladium chloride were obtained from Sigma–Aldrich. The plasticware used for cell culture in this study was purchased from Nunc, USA. Culture media (α-MEM) was obtained from Invitrogen. All other tissue culture reagents, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Triton-X-100, BSA and Alexafluor 488-Phalloidin were all purchased from Sigma. All other analytical grade chemicals and solvents were purchased from local companies. Microscopic slides (25 × 76 mm2) used for dip coating were made of borosilicate glass and purchased from Blue Star Glass Company, Mumbai, India. 2.2. Thin film preparation
Fig. 1. Schematic diagram indicating the rationale of the selection of dopants of TiO2. (A) shows the ‘d Block’ part of the Periodic Table which includes elements of 3d and 4d periods and in the IVB and VIIIB groups. It is to be noted that the electronegativity values of elements in the same period of the VIIIB group are similar irrespective of their ionic radii or atomic number. Hence Fe and Ni from 3d period and Pd and Ru from the 4d period were selected for doping in TiO2. (B) shows the interaction between titanium and oxygen atoms to form TiO2; numbers in the parentheses indicate the atomic number. (C) Schematic representation of one of the possible covalent polar (dashed line) and non-polar (solid line) atomic interactions between TiO2 molecules and the four selected dopants. Atomic radii (AR), ionic radii (IR) and electronegative values (EN) of all doping elements are indicated; number in the parentheses indicates the atomic number.
control the polar and non-polar covalent interactions of the dopant with TiO2 in the colloidal suspension. Based upon this rationale we selected four elements from the VIIIB group – Iron (Fe) and Nickel (Ni) from the 3d period and Ruthenium (Ru) and Palladium (Pd) from the 4d period – and compared the physicochemical and biological properties of TiO 2 thin films doped respectively by these elements. This strategy, for the first time, allowed us to ask questions about the physicochemical consequences of doping TiO2 with or without changing its relative electronegative environment and correlate them with their cell biological response. In our experimental protocol we first prepared undoped and four types of doped TiO2 solutions by a sol–gel process standardized by us earlier and subsequently we used these solutions to prepare thin films on borosilicate glass by a dip coating method described elsewhere [6]. Our results indicate that depending on the chemical nature of the dopant used, we could induce subtle variations in the contents of surface carbon, oxygen and Ti composition of the films. These variations could predictably correlate with alterations in the crystallite phase, surface morphology and biological properties of the films. Based upon the analyses of cell adhesion, viability and proliferation assays of MC3T3 cells on these films, we propose that dopants with less polar interactions with TiO2 could generate surfaces that had the best biological activity. All these parameters are extensively characterized and included in this paper. A model describing the correlation between the atomic properties of the dopant and its influence on the electrochemical nature of the film surface has also been discussed.
TiO 2 films, on clean glass substrates, were prepared by sol–gel and dip coating process as described elsewhere [6]. Isopropanol and titanium tetra-butoxide mixture was used to prepare 0.5 M Ti sol. The Ti sol was partially hydrolyzed by adding 2% HNO3 and 1% water in the solution for overnight. This method results in the controlled hydrolysis of titanium tera-butoxide and yielded a partially hydrolyzed stable titania sol–gel for deposition as thin films [18]. Clean glass substrates were coated with titania sol–gel mixtures to obtain thin films of uniform thickness (about 160 nm) by a method optimized previously [1]. To prepare iron (Fe), nickel (Ni), ruthenium (Ru) and palladium (Pd) doped TiO2 films stock solutions of iron acetylacetonate (Fe3 + oxidation state), nickel acetylacetonate (Ni2 + oxidation state), ruthenium trichloride (Ru3 + oxidation state) and palladium chloride (Pd2 + oxidation state) respectively were prepared in isopropyl alcohol and calculated quantities of each were added to the 0.5 M Ti sol to achieve 5 mole % concentration of the respective dopant in Ti sol. Films from each of these solutions were deposited on clean glass in a similar manner as described earlier [1]. Thus, 5 types of thin films on glass i.e. undoped TiO2, and Fe3 +, Ni2 +, Ru3 + or Pd2 + doped TiO2 were obtained for further analysis. For cell culture studies, all coated substrates were cut to equal sizes (10 × 10 mm2) by using a diamond glass cutter and autoclaved at 120 °C for 30 min. 2.3. Surface characterization XRD was used to determine the changes in the crystallographic phases of the undoped TiO2 and TiO2 doped with Fe, Ni, Ru and Pd. XRD spectra of the samples were recorded using SIEMENS D-500 diffractometer with monochromatized CuKα radiation (λ = 1.541A°). To have better signal to noise ratio in XRD measurements, we have used powder samples which were prepared under conditions identical to those used for coating the films. The surfaces of undoped TiO2 and different metal doped TiO2 thin films were characterized using X-ray photoelectron spectroscopy (XPS) with AlKα (Al Kα = 1486.6 eV) on a Perkin Elmer 1257 model with hemispherical analyzer under high vacuum (b10−8 torr). Survey spectra were acquired at constant pass energy and the elemental quantification was carried out from wide scan spectra. Lower pass energy was used for obtaining highresolution scans of core levels of Ti, C and O atoms. Quantification of different surface states in the narrow scan XPS spectra of undoped TiO2 and different metal doped TiO2 thin films was carried out by specifying line shape (GL), relative sensitivity factor, position, full width half maxima and area constraints as described in our previous studies 7. The surface morphology of film was studied using Scanning Electron Microscopy (SEM), 3400 N Hitachi, Japan. We have used contact angle measurement system (OCA20, from Data Physics system) to measure contact angle between film surface and water droplet.
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(FBS) and 100U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml gentamycin at 37 °C in a humidified atmosphere of 5% CO2 . The cells were sub-cultured using trypsin-EDTA when they were 85–90% confluent. All experiments were done with cells when they were within five passages after revival from cryopreservation. Triplicate samples were used in each in vitro cell assay and all the experiments were repeated at least three times. For statistical analysis pair wise comparison of inter-experiment variation was done. MS Excel software was used for calculating mean and standard deviation values for every assay and significance of the values was determined using Student's t test where p values of b0.05 were considered significant. 2.4.1.1. Cell adhesion assay. The assay was carried out by plating 1 × 104 cells on the substrates in triplicate and incubated them for 4 h. After 4 h the non adherent cells were washed off and 25 μl of 2 mg/ml concentration MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to the remaining cells on each substrate and incubated at 37 °C and 5%CO2, 95% air conditions for 4 h. The reaction was stopped by adding acidified isopropanol and the complex was read at 490 nm spectrophotometer (Spectramax 190Molecular devices) to calculate the percentage of cell adhesion [32]. Fig. 2. XRD spectra of undoped TiO2 and TiO2 doped with Fe, Ni, Ru and Pd. (A) undoped TiO2, (B) TiO2 doped with 5 mol % Fe, (C) TiO2 doped with 5 mol % Ni, (D) TiO2 doped with 5 mol % Ru, and (E) TiO2 doped with 5 mol % Pd.
2.4.1.2. Cell viability assay. The assay was performed by plating 5 × 104 cells per substrate in triplicates and incubated for 24 h. At 24 h MTT assay was performed as described above to calculate the percentage of cell viability [32].
2.4. In vitro cell assays 2.4.1. Cell culture and statistical analysis The osteoblast cells (MC3T3) obtained from ATCC and subclone 4 were maintained in complete medium (CM) which was made of fresh α-MEM medium supplemented with 10% Fetal Bovine Serum
Fig. 3. SEM images of undoped TiO2 (A), Fe doped TiO2 (B), Ni doped TiO2 (C), Ru doped TiO2 (D) and Pd doped TiO2 (E).
Fig. 4. XPS analysis of undoped TiO2 thin film surface. (A) Peak fitted high resolution Ti2p spectra and (B) peak fitted high resolution C1s XPS spectra.
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2.4.1.3. Cell proliferation assay. 1 × 104 cells were plated per substrate in triplicates and were cultured for 72 h. At the end of the culture period the cell number on each substrate was calculated by MTT assay as described above [32]. 2.4.1.4. Cell spreading assay. 1 × 103 cells, in approximately 50 μl of CM, were plated on each substrate and incubated at 37 °C for 30 min for initial adherence. After this 3 ml CM was carefully added to each well without disturbing the cells and cells were further incubated at 37 °C for 3 h and 30 min; after this the substrates were washed with PBS in order to wash off non-adherent cells and the live adherent cells were stained with FDA (1 mg/ml) and incubated for 15 min at 37 °C and observed under fluorescence microscope (Axiovert 200 M) for cell morphology. Pictures with about 10 cells in each field were taken for judging the qualitative differences in the cell spreading among different substrates [32]. The data were not subjected to statistical analysis but each experiment was repeated thrice and in every experiment at least 10 cells were recorded. 3. Results 3.1. Crystallite phase and size distributions at the surface The powder X-ray diffraction patterns of undoped TiO2 and Fe, Ni, Ru and Pd doped TiO2 samples are shown in Fig. 2(A–E). Prominent diffraction peaks in the XRD pattern of undoped TiO2 sample (Fig. 2A) appear at 2θ values of 25.13, 36.94, 37.80, 38.66, 48.21, 53.94, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values (101), (103), (004), (112), (200), (105), (211), (204) & (116) respectively which confirms the formation of anatase phase of TiO2 as per JCPDS card No.832243. XRD spectra of Fe doped TiO2 sample showed the existence of rutile phase [33]. XRD peaks of this sample can be indexed corresponding to hkl values as A101, R110, R101, R200, R111, R210, R211, R220, R002, R310, A116 and R301 as per JCPDS card No. 03-1122. XRD peaks of Ni
doped TiO2 were corresponding to hkl values as A101, A004, A200, A105, A211, A204 and A116. Ni doped sample (Fig. 2C) confirms to the formation of anatase phase with very broad peaks confirming to the formation of very small crystallite size. XRD spectra of Ru3 + doped sample (Fig. 2D) show the presence of both the rutile and anatase phases of TiO2. Prominent diffraction peaks of Ru3+ doped samples can be assigned the miller indices as A101, R110, A004, A112, R200, R111, R210, A200, A105, A211, R220, A204, R002, A116 and R301; where letter A or R before the miller index represents the crystalline phase anatase or rutile to which that particular peak represent. Pd2+ doped sample shows anatase as major phase of TiO2 with hkl values as A101, A103, A004, A112, A200, A105, A211, A204 and A116. There are two unidentified peaks at 2θ values 33.7 & 52.0 degrees which cannot be assigned to any phase of TiO2. Crystallite size was calculated using the Scherrer's formula {D = (B× λ)/(β× cos )}, where; D — diameter of crystallite, B — constant (0.9 for TiO2), λ — wavelength of X-rays, β — full XRD peak width at half maxima and θ — angle corresponding to peak position. As per this calculation the crystallite size of nickel doped TiO 2 (~ 5 nm) was significantly lower from undoped TiO 2 (~ 11 nm) whereas in other doped films (Fe doped TiO 2 ~ 12 nm, Ru doped TiO 2 ~ 9 nm and Pd doped TiO 2 ~ 10 nm) only minor variations from undoped TiO2 were seen. The cell–surface interactions are highly complex phenomena and they can be significantly influenced by even slight variations in parameters such as surface roughness, wettability and surface charge distribution. In this study we have measured surface wettability (water contact angle) and surface morphology by scanning electron microscope. Surface morphology of these substrates was examined by SEM and the analysis showed a very uniform film surface for doped and undoped TiO2 (Fig. 3). TiO2 thin films doped with different metals showed only a slight change in the surface morphology indicating only an insignificant alteration in the effective surface area of these films after doping. Hence the respective biological responses of these films could be attributed to differences in the surface functional groups
Fig. 5. High resolution Ti2p XPS spectra of (A) Fe doped TiO2, (B) Ni doped TiO2, (C) Ru doped TiO2 and (D) Pd doped TiO2.
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and crystallite phases. The water contact angle of undoped TiO2 was 45 ± 3° and reduced after doping with Fe (28 ± 3°), Ni (42 ± 4°), Ru (32 ± 2°) and Pd (21 ± 4°). As can be seen the maximum reduction of ~ 24° was observed on Pd doped TiO2 whereas the least reduction was on Ni doped TiO2 surfaces. 3.2. Surface elemental content analyses Elemental and high resolution C1s and Ti2p XPS spectra were obtained from undoped and doped TiO2 thin film surfaces. The wide scan XPS spectra as well as high resolution carbon, titanium and oxygen XPS spectral peaks of undoped TiO2 are shown in Fig. 4. Detailed elemental analysis and peak fitting for surface state quantification from C1s and Ti2p were done as described in our previous studies [1,34]. The higher resolution C1s XPS spectra of undoped TiO2 film surface was fitted with six peaks of different carbon environments relative to C–C/C– H peaks as: hydrocarbon (C–H/C–C) at 284.6 ± 0.2 eV, (C–C(= O) OX) at 285 ± 0.1 eV, (C–OX) at 286.1 ± 0.2 eV, (C = O/O–C–O) at 287.6 ± 0.2 eV, (C(= O)OX) at 289.2 ± 0.2 eV and C–Ti at 282.9 ± 0.2 eV. Different titanium oxidation states were also quantified in high resolution Ti2p XPS spectra of undoped TiO2. The spectra were
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fitted with four peaks as Ti3 +2p3/2 at 452.6 ± 0.2 eV, Ti4 +2p3/2 at 454.6 ± 0.2 eV, Ti 3 +2p 1/2 at 457.8 ± 0.2 eV and Ti 4 +2p 1/2 at 460.2 ± 0.2 eV. Elemental quantification and the relative estimation of different surface states of carbon and titanium were carried out for Fe, Ni, Ru and Pd doped TiO 2 film surface XPS spectra which are shown in Figs. 5 and 6. Elemental and surface state quantification from C1s and Ti2p was done as described in our previous studies [1,34] for all the four doped substrates. As shown in Fig. 5, high resolution Ti2p XPS spectral analysis was done for all the dopant substrates and the spectra were fitted with four peaks as Ti3 +2p3/2, Ti4 +2p3/2, Ti3 +2p1/2, and Ti4 +2p1/2. Fig. 6 shows different carbon surface states identified from high resolution C1s XPS spectra of TiO2 doped with Fe, Ni, Ru and Pd film surface. The higher resolution C1s XPS spectra of TiO2 film surface was fitted with six peaks of different carbon environments relative to C–C/C–H peaks as: hydrocarbon (C–H/C–C) at 284.6 ± 0.2 eV, (C–C(=O)OX) at 285 ± 0.1 eV, (C–OX) at 286.1 ± 0.2 eV, (C = O/O–C–O) at 287.6 ± 0.2 eV, (C(=O)OX) at 289.2 ± 0.2 eV and C–Ti at 282.9 ± 0.2 eV. Ru and Pd doped TiO2 XPS had five different types of carbon bonding configurations, C/C–H, C–C(=O)OX, C–OX, C = O and C(=O)OX where one additional peak C–Ti was observed in Fe and Ni doped TiO2 samples. Surface state
Fig. 6. High resolution C1s XPS spectra of (A) Fe doped TiO2, (B) Ni doped TiO2, (C) Ru doped TiO2 and (D) Pd doped TiO2.
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quantification from C1s was done as described in our previous studies for all the four doped substrates. To have better comparison a relative variation (Δ) of the different surfaces states with undoped TiO2 samples is shown in Fig. 7. Δ of different % proportions (as indicated with relative change in comparison with undoped TiO2 samples); the + ve and − ve values of a particular element represent the % proportion of corresponding element either increased or decreased in that sample, respectively. These data show an increase in the Ti3 + surface states for all dopant elements but Fe doped TiO2 showed highest proportion of Ti3 + surface states, followed by Ru, Pd and Ni doped samples. These increases in Ti3 + states suggest the presence of sub-stochiometeric titanium oxide phases in these samples. Commonly five different types of carbon bonding configurations, C/C–H, C–C(= O)OX, C–OX, C = O and C(= O)OX were observed in TiO2 samples as shown in Fig. 6. Of these, the values for only three active functional groups C–OX, C = O and C(=O)OX are represented in Fig. 7 where the relative variation (Δ) in these active functional groups with respect to undoped TiO2 is represented. It has been observed that there was a considerable increase in the proportion of hydroxyl functional group at the surface in Pd doped samples whereas for Ni, Fe and Ru doped TiO2 samples did not show much change. Significant increase in the % proportion of carbon atoms as C = O in C1s at the surface of Fe doped TiO2 samples could be observed. Ni doped TiO2 samples have shown highest level of carboxylic functional group. In the current study we have not estimated the overall surface charge on these surfaces but XPS surface analysis confirms the presence of hydroxyl groups in significant proportions on surface of undoped TiO2 and doped with different metals which may influence the overall surface charge.
Thus, the overall analysis of surface elemental contents and their functional distribution, based on XPS data, indicates that the relative intensity of the various oxygen functionalities was significantly higher in doped TiO 2 in comparison to undoped TiO2 . Surfaces of Ru, Pd and Fe doped TiO 2 samples showed higher level of active oxygen and Ti3 + contents whereas surface carbon content was significantly lower in Ni doped TiO2 sample. 3.3. Cell adhesion, viability and proliferation assays Cell biological assays using MC3T3 osteoblast cells were done using protocols described in Materials and Methods and the results are shown in Figs. 8 and 9. In the adhesion assay at 4 h after plating the cells we could estimate that almost 100% cells were adherent on Ni doped TiO2 but only 65–80% cells were adherent on other surfaces (see Fig. 8). Results of cell viability assay, done at 24 h after plating the cells, were very different (Fig. 9A). Undoped and Fe or Ni doped TiO 2 showed N 80% cell viability but Ru and Pd coated surfaces showed b50% cell viability. Physicochemical surface properties had clearly indicated that Fe and Ni doping with TiO2 had induced higher positive surface charge and low surface carbon content. This suggests that negative surface charges on metal surfaces can suppress the cell viability whereas reduction of surface carbon content on these metal oxide surfaces can increase the cell viability. Cell counts at 72 h after plating 1 × 104 cells on doped and undoped TiO 2 surfaces were estimated and data are shown in Fig. 9B. As shown earlier by us [1] undoped and Ni doped TiO2 surfaces respectively showed a two and three fold increase in cell number after 72 h. However proliferation of cells on TiO 2 surfaces doped with other metals was lesser which could probably be due to poorer viability of cells on these surfaces. 4. Discussion In this paper we have demonstrated the physicochemical and cell biological properties of TiO2 thin films that had been doped with four different elements of the VIIIB group in the periodic table. The selection of these elements was done with the aim to understand the role of electronegativity and atomic size in determining the functional properties of TiO 2 in thin film surfaces. Our results indicate that physical properties of dopant elements can have a profound effect in determining the crystallite phase and size, and biological properties of the films. 4.1. Selection of metals for doping TiO2
Fig. 7. (A) Relative variation (Δ) of % atomic proportion in doped TiO2 samples relative to undoped TiO2 samples, (B) relative variation (Δ) of titanium surface states in Ti2p of doped TiO2 samples relative to undoped TiO2 samples, (C) relative variation (Δ) of oxygen surface states in O1s of doped TiO2 samples relative to undoped TiO2 samples and (D) relative variation (Δ) of active carbon functionalities in C1s of doped TiO2 samples relative to undoped TiO2 samples.
We had hypothesized that doping of colloidal solution of TiO 2 with group VIII B metals such as Fe, Ni, Ru and Pd can significantly alter the surface properties of doped TiO2 films. We had also predicted that electronegativity of dopant atoms would have a significant impact on the polar and non-polar inter-metallic interactions of doped TiO2 molecules. Electronegativity (χ) values of Fe, Ni, Ru, Pd, Ti and O atoms in Pauling scale are 1.83, 1.91, 2.2, 2.2, 1.54 and 3.44, respectively (Fig. 1). Hence, it is expected that inter-metallic interaction between Ti and the dopant atoms is non-polar for Fe and Ni (Δχ b 0.5) and polar for Ru and Pd (0.5 b Δχ b 1.7). These differences in polarity of interactions of the dopant containing colloidal solution of TiO2 regulate the surface oxygen and carbon content and the formation of mixed crystallite phases in thin films prepared by the respective doped metals. Based on this strategy we compared the biological responses generated by TiO 2 thin films having variable secondary crystallite phases and surface oxygen and carbon contents to support cell adhesion and proliferation. Based on the data described in Figs. 7 and 8 we can see that TiO2 thin films containing less secondary crystallite phase and lesser carbon/oxygen content
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Fig. 8. Cell adhesion assay done by using MTT. (A) The graph shows the percentage of cell adhesion on each substrate plated for 4 h. (B) A representation FDA staining on selected substrates after 4 h of cell plating. (a) Undoped TiO2, (b) TiO2 doped with 5 mol % Fe, (c) TiO2 doped with 5 mol % Ni, (d) TiO2 doped with 5 mol % Rd, and (e) TiO2 doped with 5 mol % Pd.
at the surface showed better adhesion and proliferation of osteoblast cells. 4.2. Interaction of metal dopants with TiO2 in thin film Based upon the characteristic of dopant elements we propose a model for a correlated variation in four physicochemical properties of TiO2 thin film surface. This model is represented in Fig. 10 where the effect of relative variation in atomic radius, ionic radius and electronegativity of dopant molecules (purple arrows) is shown on the polar and non-polar interactions, crystallite phase size and surface carbon–oxygen contents (green arrows) of the doped TiO2 surface. Based upon this analysis it can be concluded that TiO2 films doped with metals of lower electronegativity values showed lesser surface carbon and higher surface oxygen content at the film surface. Increase in electronegativity values of dopant metals reduced the % proportion of Ti 4 + surface state of Ti on the surface of films and caused corresponding reduction in the positive surface charge at film surfaces. Thus, the electronegative environment of doped metal played a significant role in the regulation of surface charges of TiO2 at thin film surface. Further analysis of the surface properties indicated the existence of secondary crystallite phases in TiO2 with decreasing ionic radii of the doped metal. Hence relative proportion of secondary crystallite phase of TiO2 can be reduced by increasing atomic number in the
same period. This analysis showed that the relative variation in ionic and atomic radii would significantly affect the size and phases of crystallites whereas relative electronegative environment of doped metal is less significant to modify these structural properties.
4.3. Biological outcomes of alteration of properties of TiO2 thin films The interaction between cells and tissue engineering substrates is a poorly understood phenomenon till date. Unlike tissue culture grade plastic, which is commonly used for the maintenance and growth of cells and has uniform distribution of negatively charged functional groups exposed at the surfaces, metal based substrates prepared by us, by a specified chemical process, do not have similar properties. It is therefore important to generate conditions that would result in uniform distribution of physico-chemical properties on which the cells can attach, spread and proliferate and differentiate optimally. We have developed a strategy for making doped TiO2 thin films and have shown that Ni doping of films creates the best biological responses from the osteoblast cells. This could be because Ni doped TiO 2 surfaces have a more uniform distribution of positive surface charges and have a reduced carbon and increased oxygen content. Thin films of Ni doped TiO2 showed better adhesion, viability and proliferation of osteoblasts and it correlated with positive surface charge and less carbon contents of these surfaces.
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impact on the biological behavior of osteoblast cells where the cells adhere and proliferate much more on the surface that has maximum positive charges exposed on it. We propose that dopant elements that have more apolar interactions with TiO2 and thus permit the positive charges of TiO2 to be retained on the surface, should be dopants of choice for TiO2 for improved biological applications. Acknowledgments We gratefully acknowledge the generous support of the Department of Science and Technology and the Department of Biotechnology, Government of India through grant numbers GAP220 and GAP311 to GP and GAP327 to MD. References
Fig. 9. (A) 24 h cell viability assay was done by using MTT. The viability was checked after plating the cells on each substrate and incubating for 24 h, later MTT assay was carried out to calculate the percentage of cell viability. (B) 72 h cell proliferation assay was done by using MTT. (a) Undoped TiO2, (b) TiO2 doped with 5 mol % Fe, (c) TiO2 doped with 5 mol % Ni, (d) TiO2 doped with 5 mol % Rd, and (e) TiO2 doped with 5 mol % Pd.
5. Conclusions For the first time we have shown that physical properties of dopant atoms can influence the electrochemical properties of TiO2 molecules coated as thin films on glass surface. This modulation can have an
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Fig. 10. Schematic diagram indicating the relative variation in crystallite phase, size, surface carbon content and surface charge with relative variation in atomic radii, ionic radii and electronegativity of dopant atoms. Top row shows relative representation of physicochemical properties of TiO2 doped with dopant from 3d- period with increasing atomic number. The centre part shows TiO2 and four different arrow directions indicate relative decrease in corresponding characterization of surface properties. Bottom row shows relative representation of physicochemical properties of TiO2 doped with dopant from 4d-period with increasing number.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Strategies to prepare TiO2 thin films, doped with transition metal ions, that exhibit specific physicochemical properties to support osteoblast cell adhesion and proliferation Marshal Dhayal a,⁎, Renu Kapoor a,1, Pavana Goury Sistla a,1, Ravi Ranjan Pandey b, Satabisha Kar a, Krishan Kumar Saini b, Gopal Pande a,⁎ a b
CSIR-Center for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India CSIR-National Physical Laboratory, Dr K S Krishnan Marg, New Delhi, India
a r t i c l e
i n f o
Article history: Received 16 August 2013 Received in revised form 28 October 2013 Accepted 27 December 2013 Available online 4 January 2014 Keywords: Biomaterials Electronegativity Atomic radius Osteoinduction Surface energy
a b s t r a c t Metal ion doped titanium oxide (TiO2) thin films, as bioactive coatings on metal or other implantable materials, can be used as surfaces for studying the cell biological properties of osteogenic and other cell types. Bulk crystallite phase distribution and surface carbon–oxygen constitution of thin films, play an important role in determining the biological responses of cells that come in their contact. Here we present a strategy to control the polarity of atomic interactions between the dopant metal and TiO2 molecules and obtain surfaces with smaller crystallite phases and optimal surface carbon–oxygen composition to support the maximum proliferation and adhesion of osteoblast cells. Our results suggest that surfaces, in which atomic interactions between the dopant metals and TiO2 were less polar, could support better adhesion, spreading and proliferation of cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Titanium (Ti) oxides and Ti alloys, with modified structural, electronic and chemical properties, have been extensively used for biomedical and other applications [1–4]. These materials were fabricated by techniques such as conventional sintering [5,6], spark plasma sintering and surface state modification [5,7], self-propagating high-temperature synthesis [8] and hot isostatic pressing [9]. Materials synthesized using these techniques tend to have large proportions of secondary crystallite phases that can adversely affect their bioactivity and mechanical stability. Hence, several prior investigations have aimed to optimize the physicochemical properties of these materials, such as obtaining uniform phase structure, surface roughness, energy and chemical constitution that would support tissue specific biological functions, such as cell attachment, proliferation, differentiation and migration [10–13]. Most of the currently available orthopedic implants have microtopography-altered rough surfaces and chemically added bioactive materials [14–18]. Numerous reports have shown that bone responses are related to different surface properties associated with microtopography and chemistry of these surfaces [19–23]. In addition
⁎ Corresponding authors. Tel.: +91 40 27192605; fax: +91 40 27160591. E-mail addresses: [email protected] (M. Dhayal), [email protected] (G. Pande). 1 Equal contribution. 0928-4931/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.12.035
to the microstructure, chemical modification of surfaces also focuses on metal coatings of potentially bioactive materials, such as hydroxyapatite (HA). It has been suggested that bone formation reactions are enhanced by such bioactive materials [24]. Several HA-coated implants are already being used for dental and orthopedic treatments [25,26]. Recent studies have shown that thin films (b 200 nm) of titanium dioxide (TiO2) are more suitable to control the size of the crystallite phases and surface morphology; in addition, due to their transparent nature, these films are also useful to study in vitro cellular properties under a light microscope [1]. Two strategies have been commonly used to modify the physicochemical and biological properties of TiO 2 in thin films: surface treatment [27–30] and doping [1,8]. It has been shown by others and us that doping of TiO2 with transition metals could improve its structural, chemical and surface charge stability [8]. This could be due to the unique properties of transition elements belonging to the same period of the periodic table which, due to a higher number of electrons in ‘d’ orbital, exhibit reduced atomic radius and increased electronegativity along with the rise in atomic number [31]. In contrast to this, in transition metals belonging to the VIIIB group and 3d or 4d periods of the periodic table, these properties do not change significantly in spite of increase in atomic number (see Fig. 1). The ionic radii (IR), atomic radii (AR) and electronegativity (EN) values in Fig. 1 were corresponding to the intrinsic values of doped metal atoms. This schematic diagram provides the justification for selection of different metal ions as dopants which may
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2. Experimental section 2.1. Materials Titanium tetrabutoxide, iron acetylacetonate, nickel acetylacetonate, ruthenium trichloride and palladium chloride were obtained from Sigma–Aldrich. The plasticware used for cell culture in this study was purchased from Nunc, USA. Culture media (α-MEM) was obtained from Invitrogen. All other tissue culture reagents, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), Triton-X-100, BSA and Alexafluor 488-Phalloidin were all purchased from Sigma. All other analytical grade chemicals and solvents were purchased from local companies. Microscopic slides (25 × 76 mm2) used for dip coating were made of borosilicate glass and purchased from Blue Star Glass Company, Mumbai, India. 2.2. Thin film preparation
Fig. 1. Schematic diagram indicating the rationale of the selection of dopants of TiO2. (A) shows the ‘d Block’ part of the Periodic Table which includes elements of 3d and 4d periods and in the IVB and VIIIB groups. It is to be noted that the electronegativity values of elements in the same period of the VIIIB group are similar irrespective of their ionic radii or atomic number. Hence Fe and Ni from 3d period and Pd and Ru from the 4d period were selected for doping in TiO2. (B) shows the interaction between titanium and oxygen atoms to form TiO2; numbers in the parentheses indicate the atomic number. (C) Schematic representation of one of the possible covalent polar (dashed line) and non-polar (solid line) atomic interactions between TiO2 molecules and the four selected dopants. Atomic radii (AR), ionic radii (IR) and electronegative values (EN) of all doping elements are indicated; number in the parentheses indicates the atomic number.
control the polar and non-polar covalent interactions of the dopant with TiO2 in the colloidal suspension. Based upon this rationale we selected four elements from the VIIIB group – Iron (Fe) and Nickel (Ni) from the 3d period and Ruthenium (Ru) and Palladium (Pd) from the 4d period – and compared the physicochemical and biological properties of TiO 2 thin films doped respectively by these elements. This strategy, for the first time, allowed us to ask questions about the physicochemical consequences of doping TiO2 with or without changing its relative electronegative environment and correlate them with their cell biological response. In our experimental protocol we first prepared undoped and four types of doped TiO2 solutions by a sol–gel process standardized by us earlier and subsequently we used these solutions to prepare thin films on borosilicate glass by a dip coating method described elsewhere [6]. Our results indicate that depending on the chemical nature of the dopant used, we could induce subtle variations in the contents of surface carbon, oxygen and Ti composition of the films. These variations could predictably correlate with alterations in the crystallite phase, surface morphology and biological properties of the films. Based upon the analyses of cell adhesion, viability and proliferation assays of MC3T3 cells on these films, we propose that dopants with less polar interactions with TiO2 could generate surfaces that had the best biological activity. All these parameters are extensively characterized and included in this paper. A model describing the correlation between the atomic properties of the dopant and its influence on the electrochemical nature of the film surface has also been discussed.
TiO 2 films, on clean glass substrates, were prepared by sol–gel and dip coating process as described elsewhere [6]. Isopropanol and titanium tetra-butoxide mixture was used to prepare 0.5 M Ti sol. The Ti sol was partially hydrolyzed by adding 2% HNO3 and 1% water in the solution for overnight. This method results in the controlled hydrolysis of titanium tera-butoxide and yielded a partially hydrolyzed stable titania sol–gel for deposition as thin films [18]. Clean glass substrates were coated with titania sol–gel mixtures to obtain thin films of uniform thickness (about 160 nm) by a method optimized previously [1]. To prepare iron (Fe), nickel (Ni), ruthenium (Ru) and palladium (Pd) doped TiO2 films stock solutions of iron acetylacetonate (Fe3 + oxidation state), nickel acetylacetonate (Ni2 + oxidation state), ruthenium trichloride (Ru3 + oxidation state) and palladium chloride (Pd2 + oxidation state) respectively were prepared in isopropyl alcohol and calculated quantities of each were added to the 0.5 M Ti sol to achieve 5 mole % concentration of the respective dopant in Ti sol. Films from each of these solutions were deposited on clean glass in a similar manner as described earlier [1]. Thus, 5 types of thin films on glass i.e. undoped TiO2, and Fe3 +, Ni2 +, Ru3 + or Pd2 + doped TiO2 were obtained for further analysis. For cell culture studies, all coated substrates were cut to equal sizes (10 × 10 mm2) by using a diamond glass cutter and autoclaved at 120 °C for 30 min. 2.3. Surface characterization XRD was used to determine the changes in the crystallographic phases of the undoped TiO2 and TiO2 doped with Fe, Ni, Ru and Pd. XRD spectra of the samples were recorded using SIEMENS D-500 diffractometer with monochromatized CuKα radiation (λ = 1.541A°). To have better signal to noise ratio in XRD measurements, we have used powder samples which were prepared under conditions identical to those used for coating the films. The surfaces of undoped TiO2 and different metal doped TiO2 thin films were characterized using X-ray photoelectron spectroscopy (XPS) with AlKα (Al Kα = 1486.6 eV) on a Perkin Elmer 1257 model with hemispherical analyzer under high vacuum (b10−8 torr). Survey spectra were acquired at constant pass energy and the elemental quantification was carried out from wide scan spectra. Lower pass energy was used for obtaining highresolution scans of core levels of Ti, C and O atoms. Quantification of different surface states in the narrow scan XPS spectra of undoped TiO2 and different metal doped TiO2 thin films was carried out by specifying line shape (GL), relative sensitivity factor, position, full width half maxima and area constraints as described in our previous studies 7. The surface morphology of film was studied using Scanning Electron Microscopy (SEM), 3400 N Hitachi, Japan. We have used contact angle measurement system (OCA20, from Data Physics system) to measure contact angle between film surface and water droplet.
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(FBS) and 100U/ml penicillin, 50 μg/ml streptomycin and 50 μg/ml gentamycin at 37 °C in a humidified atmosphere of 5% CO2 . The cells were sub-cultured using trypsin-EDTA when they were 85–90% confluent. All experiments were done with cells when they were within five passages after revival from cryopreservation. Triplicate samples were used in each in vitro cell assay and all the experiments were repeated at least three times. For statistical analysis pair wise comparison of inter-experiment variation was done. MS Excel software was used for calculating mean and standard deviation values for every assay and significance of the values was determined using Student's t test where p values of b0.05 were considered significant. 2.4.1.1. Cell adhesion assay. The assay was carried out by plating 1 × 104 cells on the substrates in triplicate and incubated them for 4 h. After 4 h the non adherent cells were washed off and 25 μl of 2 mg/ml concentration MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was added to the remaining cells on each substrate and incubated at 37 °C and 5%CO2, 95% air conditions for 4 h. The reaction was stopped by adding acidified isopropanol and the complex was read at 490 nm spectrophotometer (Spectramax 190Molecular devices) to calculate the percentage of cell adhesion [32]. Fig. 2. XRD spectra of undoped TiO2 and TiO2 doped with Fe, Ni, Ru and Pd. (A) undoped TiO2, (B) TiO2 doped with 5 mol % Fe, (C) TiO2 doped with 5 mol % Ni, (D) TiO2 doped with 5 mol % Ru, and (E) TiO2 doped with 5 mol % Pd.
2.4.1.2. Cell viability assay. The assay was performed by plating 5 × 104 cells per substrate in triplicates and incubated for 24 h. At 24 h MTT assay was performed as described above to calculate the percentage of cell viability [32].
2.4. In vitro cell assays 2.4.1. Cell culture and statistical analysis The osteoblast cells (MC3T3) obtained from ATCC and subclone 4 were maintained in complete medium (CM) which was made of fresh α-MEM medium supplemented with 10% Fetal Bovine Serum
Fig. 3. SEM images of undoped TiO2 (A), Fe doped TiO2 (B), Ni doped TiO2 (C), Ru doped TiO2 (D) and Pd doped TiO2 (E).
Fig. 4. XPS analysis of undoped TiO2 thin film surface. (A) Peak fitted high resolution Ti2p spectra and (B) peak fitted high resolution C1s XPS spectra.
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2.4.1.3. Cell proliferation assay. 1 × 104 cells were plated per substrate in triplicates and were cultured for 72 h. At the end of the culture period the cell number on each substrate was calculated by MTT assay as described above [32]. 2.4.1.4. Cell spreading assay. 1 × 103 cells, in approximately 50 μl of CM, were plated on each substrate and incubated at 37 °C for 30 min for initial adherence. After this 3 ml CM was carefully added to each well without disturbing the cells and cells were further incubated at 37 °C for 3 h and 30 min; after this the substrates were washed with PBS in order to wash off non-adherent cells and the live adherent cells were stained with FDA (1 mg/ml) and incubated for 15 min at 37 °C and observed under fluorescence microscope (Axiovert 200 M) for cell morphology. Pictures with about 10 cells in each field were taken for judging the qualitative differences in the cell spreading among different substrates [32]. The data were not subjected to statistical analysis but each experiment was repeated thrice and in every experiment at least 10 cells were recorded. 3. Results 3.1. Crystallite phase and size distributions at the surface The powder X-ray diffraction patterns of undoped TiO2 and Fe, Ni, Ru and Pd doped TiO2 samples are shown in Fig. 2(A–E). Prominent diffraction peaks in the XRD pattern of undoped TiO2 sample (Fig. 2A) appear at 2θ values of 25.13, 36.94, 37.80, 38.66, 48.21, 53.94, 55.15, 62.71 and 68.7 degrees which are assigned (hkl) values (101), (103), (004), (112), (200), (105), (211), (204) & (116) respectively which confirms the formation of anatase phase of TiO2 as per JCPDS card No.832243. XRD spectra of Fe doped TiO2 sample showed the existence of rutile phase [33]. XRD peaks of this sample can be indexed corresponding to hkl values as A101, R110, R101, R200, R111, R210, R211, R220, R002, R310, A116 and R301 as per JCPDS card No. 03-1122. XRD peaks of Ni
doped TiO2 were corresponding to hkl values as A101, A004, A200, A105, A211, A204 and A116. Ni doped sample (Fig. 2C) confirms to the formation of anatase phase with very broad peaks confirming to the formation of very small crystallite size. XRD spectra of Ru3 + doped sample (Fig. 2D) show the presence of both the rutile and anatase phases of TiO2. Prominent diffraction peaks of Ru3+ doped samples can be assigned the miller indices as A101, R110, A004, A112, R200, R111, R210, A200, A105, A211, R220, A204, R002, A116 and R301; where letter A or R before the miller index represents the crystalline phase anatase or rutile to which that particular peak represent. Pd2+ doped sample shows anatase as major phase of TiO2 with hkl values as A101, A103, A004, A112, A200, A105, A211, A204 and A116. There are two unidentified peaks at 2θ values 33.7 & 52.0 degrees which cannot be assigned to any phase of TiO2. Crystallite size was calculated using the Scherrer's formula {D = (B× λ)/(β× cos )}, where; D — diameter of crystallite, B — constant (0.9 for TiO2), λ — wavelength of X-rays, β — full XRD peak width at half maxima and θ — angle corresponding to peak position. As per this calculation the crystallite size of nickel doped TiO 2 (~ 5 nm) was significantly lower from undoped TiO 2 (~ 11 nm) whereas in other doped films (Fe doped TiO 2 ~ 12 nm, Ru doped TiO 2 ~ 9 nm and Pd doped TiO 2 ~ 10 nm) only minor variations from undoped TiO2 were seen. The cell–surface interactions are highly complex phenomena and they can be significantly influenced by even slight variations in parameters such as surface roughness, wettability and surface charge distribution. In this study we have measured surface wettability (water contact angle) and surface morphology by scanning electron microscope. Surface morphology of these substrates was examined by SEM and the analysis showed a very uniform film surface for doped and undoped TiO2 (Fig. 3). TiO2 thin films doped with different metals showed only a slight change in the surface morphology indicating only an insignificant alteration in the effective surface area of these films after doping. Hence the respective biological responses of these films could be attributed to differences in the surface functional groups
Fig. 5. High resolution Ti2p XPS spectra of (A) Fe doped TiO2, (B) Ni doped TiO2, (C) Ru doped TiO2 and (D) Pd doped TiO2.
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and crystallite phases. The water contact angle of undoped TiO2 was 45 ± 3° and reduced after doping with Fe (28 ± 3°), Ni (42 ± 4°), Ru (32 ± 2°) and Pd (21 ± 4°). As can be seen the maximum reduction of ~ 24° was observed on Pd doped TiO2 whereas the least reduction was on Ni doped TiO2 surfaces. 3.2. Surface elemental content analyses Elemental and high resolution C1s and Ti2p XPS spectra were obtained from undoped and doped TiO2 thin film surfaces. The wide scan XPS spectra as well as high resolution carbon, titanium and oxygen XPS spectral peaks of undoped TiO2 are shown in Fig. 4. Detailed elemental analysis and peak fitting for surface state quantification from C1s and Ti2p were done as described in our previous studies [1,34]. The higher resolution C1s XPS spectra of undoped TiO2 film surface was fitted with six peaks of different carbon environments relative to C–C/C– H peaks as: hydrocarbon (C–H/C–C) at 284.6 ± 0.2 eV, (C–C(= O) OX) at 285 ± 0.1 eV, (C–OX) at 286.1 ± 0.2 eV, (C = O/O–C–O) at 287.6 ± 0.2 eV, (C(= O)OX) at 289.2 ± 0.2 eV and C–Ti at 282.9 ± 0.2 eV. Different titanium oxidation states were also quantified in high resolution Ti2p XPS spectra of undoped TiO2. The spectra were
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fitted with four peaks as Ti3 +2p3/2 at 452.6 ± 0.2 eV, Ti4 +2p3/2 at 454.6 ± 0.2 eV, Ti 3 +2p 1/2 at 457.8 ± 0.2 eV and Ti 4 +2p 1/2 at 460.2 ± 0.2 eV. Elemental quantification and the relative estimation of different surface states of carbon and titanium were carried out for Fe, Ni, Ru and Pd doped TiO 2 film surface XPS spectra which are shown in Figs. 5 and 6. Elemental and surface state quantification from C1s and Ti2p was done as described in our previous studies [1,34] for all the four doped substrates. As shown in Fig. 5, high resolution Ti2p XPS spectral analysis was done for all the dopant substrates and the spectra were fitted with four peaks as Ti3 +2p3/2, Ti4 +2p3/2, Ti3 +2p1/2, and Ti4 +2p1/2. Fig. 6 shows different carbon surface states identified from high resolution C1s XPS spectra of TiO2 doped with Fe, Ni, Ru and Pd film surface. The higher resolution C1s XPS spectra of TiO2 film surface was fitted with six peaks of different carbon environments relative to C–C/C–H peaks as: hydrocarbon (C–H/C–C) at 284.6 ± 0.2 eV, (C–C(=O)OX) at 285 ± 0.1 eV, (C–OX) at 286.1 ± 0.2 eV, (C = O/O–C–O) at 287.6 ± 0.2 eV, (C(=O)OX) at 289.2 ± 0.2 eV and C–Ti at 282.9 ± 0.2 eV. Ru and Pd doped TiO2 XPS had five different types of carbon bonding configurations, C/C–H, C–C(=O)OX, C–OX, C = O and C(=O)OX where one additional peak C–Ti was observed in Fe and Ni doped TiO2 samples. Surface state
Fig. 6. High resolution C1s XPS spectra of (A) Fe doped TiO2, (B) Ni doped TiO2, (C) Ru doped TiO2 and (D) Pd doped TiO2.
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quantification from C1s was done as described in our previous studies for all the four doped substrates. To have better comparison a relative variation (Δ) of the different surfaces states with undoped TiO2 samples is shown in Fig. 7. Δ of different % proportions (as indicated with relative change in comparison with undoped TiO2 samples); the + ve and − ve values of a particular element represent the % proportion of corresponding element either increased or decreased in that sample, respectively. These data show an increase in the Ti3 + surface states for all dopant elements but Fe doped TiO2 showed highest proportion of Ti3 + surface states, followed by Ru, Pd and Ni doped samples. These increases in Ti3 + states suggest the presence of sub-stochiometeric titanium oxide phases in these samples. Commonly five different types of carbon bonding configurations, C/C–H, C–C(= O)OX, C–OX, C = O and C(= O)OX were observed in TiO2 samples as shown in Fig. 6. Of these, the values for only three active functional groups C–OX, C = O and C(=O)OX are represented in Fig. 7 where the relative variation (Δ) in these active functional groups with respect to undoped TiO2 is represented. It has been observed that there was a considerable increase in the proportion of hydroxyl functional group at the surface in Pd doped samples whereas for Ni, Fe and Ru doped TiO2 samples did not show much change. Significant increase in the % proportion of carbon atoms as C = O in C1s at the surface of Fe doped TiO2 samples could be observed. Ni doped TiO2 samples have shown highest level of carboxylic functional group. In the current study we have not estimated the overall surface charge on these surfaces but XPS surface analysis confirms the presence of hydroxyl groups in significant proportions on surface of undoped TiO2 and doped with different metals which may influence the overall surface charge.
Thus, the overall analysis of surface elemental contents and their functional distribution, based on XPS data, indicates that the relative intensity of the various oxygen functionalities was significantly higher in doped TiO 2 in comparison to undoped TiO2 . Surfaces of Ru, Pd and Fe doped TiO 2 samples showed higher level of active oxygen and Ti3 + contents whereas surface carbon content was significantly lower in Ni doped TiO2 sample. 3.3. Cell adhesion, viability and proliferation assays Cell biological assays using MC3T3 osteoblast cells were done using protocols described in Materials and Methods and the results are shown in Figs. 8 and 9. In the adhesion assay at 4 h after plating the cells we could estimate that almost 100% cells were adherent on Ni doped TiO2 but only 65–80% cells were adherent on other surfaces (see Fig. 8). Results of cell viability assay, done at 24 h after plating the cells, were very different (Fig. 9A). Undoped and Fe or Ni doped TiO 2 showed N 80% cell viability but Ru and Pd coated surfaces showed b50% cell viability. Physicochemical surface properties had clearly indicated that Fe and Ni doping with TiO2 had induced higher positive surface charge and low surface carbon content. This suggests that negative surface charges on metal surfaces can suppress the cell viability whereas reduction of surface carbon content on these metal oxide surfaces can increase the cell viability. Cell counts at 72 h after plating 1 × 104 cells on doped and undoped TiO 2 surfaces were estimated and data are shown in Fig. 9B. As shown earlier by us [1] undoped and Ni doped TiO2 surfaces respectively showed a two and three fold increase in cell number after 72 h. However proliferation of cells on TiO 2 surfaces doped with other metals was lesser which could probably be due to poorer viability of cells on these surfaces. 4. Discussion In this paper we have demonstrated the physicochemical and cell biological properties of TiO2 thin films that had been doped with four different elements of the VIIIB group in the periodic table. The selection of these elements was done with the aim to understand the role of electronegativity and atomic size in determining the functional properties of TiO 2 in thin film surfaces. Our results indicate that physical properties of dopant elements can have a profound effect in determining the crystallite phase and size, and biological properties of the films. 4.1. Selection of metals for doping TiO2
Fig. 7. (A) Relative variation (Δ) of % atomic proportion in doped TiO2 samples relative to undoped TiO2 samples, (B) relative variation (Δ) of titanium surface states in Ti2p of doped TiO2 samples relative to undoped TiO2 samples, (C) relative variation (Δ) of oxygen surface states in O1s of doped TiO2 samples relative to undoped TiO2 samples and (D) relative variation (Δ) of active carbon functionalities in C1s of doped TiO2 samples relative to undoped TiO2 samples.
We had hypothesized that doping of colloidal solution of TiO 2 with group VIII B metals such as Fe, Ni, Ru and Pd can significantly alter the surface properties of doped TiO2 films. We had also predicted that electronegativity of dopant atoms would have a significant impact on the polar and non-polar inter-metallic interactions of doped TiO2 molecules. Electronegativity (χ) values of Fe, Ni, Ru, Pd, Ti and O atoms in Pauling scale are 1.83, 1.91, 2.2, 2.2, 1.54 and 3.44, respectively (Fig. 1). Hence, it is expected that inter-metallic interaction between Ti and the dopant atoms is non-polar for Fe and Ni (Δχ b 0.5) and polar for Ru and Pd (0.5 b Δχ b 1.7). These differences in polarity of interactions of the dopant containing colloidal solution of TiO2 regulate the surface oxygen and carbon content and the formation of mixed crystallite phases in thin films prepared by the respective doped metals. Based on this strategy we compared the biological responses generated by TiO 2 thin films having variable secondary crystallite phases and surface oxygen and carbon contents to support cell adhesion and proliferation. Based on the data described in Figs. 7 and 8 we can see that TiO2 thin films containing less secondary crystallite phase and lesser carbon/oxygen content
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Fig. 8. Cell adhesion assay done by using MTT. (A) The graph shows the percentage of cell adhesion on each substrate plated for 4 h. (B) A representation FDA staining on selected substrates after 4 h of cell plating. (a) Undoped TiO2, (b) TiO2 doped with 5 mol % Fe, (c) TiO2 doped with 5 mol % Ni, (d) TiO2 doped with 5 mol % Rd, and (e) TiO2 doped with 5 mol % Pd.
at the surface showed better adhesion and proliferation of osteoblast cells. 4.2. Interaction of metal dopants with TiO2 in thin film Based upon the characteristic of dopant elements we propose a model for a correlated variation in four physicochemical properties of TiO2 thin film surface. This model is represented in Fig. 10 where the effect of relative variation in atomic radius, ionic radius and electronegativity of dopant molecules (purple arrows) is shown on the polar and non-polar interactions, crystallite phase size and surface carbon–oxygen contents (green arrows) of the doped TiO2 surface. Based upon this analysis it can be concluded that TiO2 films doped with metals of lower electronegativity values showed lesser surface carbon and higher surface oxygen content at the film surface. Increase in electronegativity values of dopant metals reduced the % proportion of Ti 4 + surface state of Ti on the surface of films and caused corresponding reduction in the positive surface charge at film surfaces. Thus, the electronegative environment of doped metal played a significant role in the regulation of surface charges of TiO2 at thin film surface. Further analysis of the surface properties indicated the existence of secondary crystallite phases in TiO2 with decreasing ionic radii of the doped metal. Hence relative proportion of secondary crystallite phase of TiO2 can be reduced by increasing atomic number in the
same period. This analysis showed that the relative variation in ionic and atomic radii would significantly affect the size and phases of crystallites whereas relative electronegative environment of doped metal is less significant to modify these structural properties.
4.3. Biological outcomes of alteration of properties of TiO2 thin films The interaction between cells and tissue engineering substrates is a poorly understood phenomenon till date. Unlike tissue culture grade plastic, which is commonly used for the maintenance and growth of cells and has uniform distribution of negatively charged functional groups exposed at the surfaces, metal based substrates prepared by us, by a specified chemical process, do not have similar properties. It is therefore important to generate conditions that would result in uniform distribution of physico-chemical properties on which the cells can attach, spread and proliferate and differentiate optimally. We have developed a strategy for making doped TiO2 thin films and have shown that Ni doping of films creates the best biological responses from the osteoblast cells. This could be because Ni doped TiO 2 surfaces have a more uniform distribution of positive surface charges and have a reduced carbon and increased oxygen content. Thin films of Ni doped TiO2 showed better adhesion, viability and proliferation of osteoblasts and it correlated with positive surface charge and less carbon contents of these surfaces.
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impact on the biological behavior of osteoblast cells where the cells adhere and proliferate much more on the surface that has maximum positive charges exposed on it. We propose that dopant elements that have more apolar interactions with TiO2 and thus permit the positive charges of TiO2 to be retained on the surface, should be dopants of choice for TiO2 for improved biological applications. Acknowledgments We gratefully acknowledge the generous support of the Department of Science and Technology and the Department of Biotechnology, Government of India through grant numbers GAP220 and GAP311 to GP and GAP327 to MD. References
Fig. 9. (A) 24 h cell viability assay was done by using MTT. The viability was checked after plating the cells on each substrate and incubating for 24 h, later MTT assay was carried out to calculate the percentage of cell viability. (B) 72 h cell proliferation assay was done by using MTT. (a) Undoped TiO2, (b) TiO2 doped with 5 mol % Fe, (c) TiO2 doped with 5 mol % Ni, (d) TiO2 doped with 5 mol % Rd, and (e) TiO2 doped with 5 mol % Pd.
5. Conclusions For the first time we have shown that physical properties of dopant atoms can influence the electrochemical properties of TiO2 molecules coated as thin films on glass surface. This modulation can have an
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Fig. 10. Schematic diagram indicating the relative variation in crystallite phase, size, surface carbon content and surface charge with relative variation in atomic radii, ionic radii and electronegativity of dopant atoms. Top row shows relative representation of physicochemical properties of TiO2 doped with dopant from 3d- period with increasing atomic number. The centre part shows TiO2 and four different arrow directions indicate relative decrease in corresponding characterization of surface properties. Bottom row shows relative representation of physicochemical properties of TiO2 doped with dopant from 4d-period with increasing number.
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