Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Cytocompatibility of Si-incorporated TiO2 nanopores films Shi Qian, Xuanyong Liu ∗ State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China
a r t i c l e
i n f o
Article history: Received 16 March 2015 Received in revised form 27 May 2015 Accepted 2 June 2015 Available online 8 June 2015 Keywords: TiO2 nanopores Anodization Silicon Cytocompatibility
a b s t r a c t Si-incorporated TiO2 nanopores films were prepared by anodization and silicon plasma immersion ion implantation. The microstructure and phase composition of the films were investigated by scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The hydrophilicity of the films was evaluated using water contact angle measurement and MG63 cells were cultured on the films to investigate the cytocompatibility. The results showed that the concentration and depth of silicon on the Si-incorporated TiO2 nanopores films increased with the duration time of implantation. Both the as-annealed and Si-incorporated nanopores films exhibited good hydrophilicity and cytocompatibility, while the TiO2 nanopores films implanted silicon for 1.0 h showed higher proliferation rate and vitality of MG63 cells than others, indicating a great potential application for titanium implants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the surface properties of implants have been widely investigated for improving their biological applications and understanding the interaction of materials and cells [1,2]. Titanium and its alloys have been used extensively as orthopedic implants due to the low elastic modulus, corrosion resistance and biocompatibility [3]. However, because of their bioinert nature, they usually get encapsulated by fibrous tissues and the insufficient osseointegration may result in implant failures [4]. Therefore, surface modification of titanium has been recognized as a potential approach to overcome these defects. For instance, in situ generated TiO2 nanotubes on titanium substrates have drawn enormous attention in biology [5,6], which can be attributed to the simple anodization process and the adjustable dimensions in nanoscale. Oh et al. [7] investigated the growth of MC3T3 osteoblast cells on nanotubes and found that the cell adhesion was improved with the filopodia going into the nanotube to produce interlocked connection, suggesting that the tubular structure induced a significant acceleration in the cell growth. Popat et al. [8] demonstrated the ability of TiO2 tubular nanostructures to promote osteoblast differentiation and matrix production, and enhance short- and long-term osseointegration. Moreover, the adhesion, growth and differentiation of stem cells were illustrated to be critically dependent on the tube diameter [9]. It was concluded
∗ Corresponding author. Tel.: +86 21 52412409; fax: +86 21 52412409. E-mail address: [email protected] (X. Liu). http://dx.doi.org/10.1016/j.colsurfb.2015.06.007 0927-7765/© 2015 Elsevier B.V. All rights reserved.
that small nanotube (about 30 nm in diameter) mainly promoted stem cell adhesion and larger nanotube (about 70 nm) could induce the selective differentiation into ostoblast-like cells with cell elongation [10]. In addition, other nanostructures, such as nanorods [11] and nanowires [12] have also been synthesized on titanium surfaces. It was revealed that cell proliferation was increased and osteogenic related genes and proteins were up-regulated [12], and the enhanced cell activities were attributed to the higher surface energy of nanostructures [13]. It is interesting that nanopores structures have been identified as the common constituents of tissues in vivo, such as basement membrane of the cornea, the aortic heart valve and the vascular system [14]. Therefore, producing the nanopores on titanium that could emulate the tissue structures are supposed to regulate the cell behavior and function. On the other hand, silicon (Si) has been considered as an essential element for normal growth and development of bone and cartilage [15]. Moreover, numerous researches have demonstrated that the presence of Si is vital to enhance the bioactivity of some bioactive glasses and ceramic [16,17]. The Si-containing TiO2 coating or films have also been investigated and proven to be favorable for the adhesion and spreading of osteoblast cells on the surface [18,19]. Thus, the incorporation of Si is thought to be effective for chemical modification of titanium. Here, an interest that the effects of Si on the composition and structure of TiO2 nanopores films has been taken due to rarely researches been reported. Plasma immersion ion implantation (PIII) is a versatile process for the surface modification of biomaterials [20]. In this work, Siincorporated TiO2 nanopores films were prepared using a two-step
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
anodization process and PIII, and its surface structure, phase composition and cytocompatibility were investigated. 2. Materials and methods 2.1. Samples preparation Commercially available pure titanium plates (purity > 99.85%, Grade 1, Baoji Shenghua Nonferrous Metal Materials Co, Ltd, China) were used in dimensions of 10 × 10 × 1 mm3 . They were polished mechanically and ultrasonically cleaned in acetone, ethanol and deionized water, and then dried in a dust proof condition. Anodization was conducted in a two electrode cell with a graphite plate used as the cathode. An ethylene glycol solution was adopted as the electrolyte supplemented with 0.25 wt% NH4 F and 1 vol% H2 O. The TiO2 nanopores film was fabricated by a twostep anodization process. The first step was carried out at 50 V for 1 h using a WYJ-5A-150 V DC power supply. By separating the formed oxide layer in the first stage from titanium substrate, the second step was conducted in the same electrolyte at 50 V for 1 h, and TiO2 nanopores films were produced. Then, the asprepared films were annealed at 450 ◦ C in atmosphere for 2 h using a Muffle furnace with rates of 1 ◦ C/min and cooled down with the furnace. Si-incorporation TiO2 nanopores films were prepared by PIII using the annealed TiO2 nanopores as substrates. The detailed preparation was similar to that described previously [19]. Briefly, a PIII system was used with a filtered cathodic arc plasma source, and the 99.99% pure silicon rod was adopted as the cathode. During PIII, a pulsed negative voltage applied to the samples was 20 kV, the pulse duration was 450 s, and pulse duration of the cathodic arc current was 800 s. The pulsed negative voltage and cathodic arc current were synchronized at a pulsing frequency of 8 Hz. The working pressure was 4.0 × 10−3 Pa. The as-annealed TiO2 nanopores films were labeled as “TS0”, and the Si-implanted films for 0.5 h and 1.0 h were labeled as “TS0.5” and “TS1.0”, respectively.
215
2.4. Cell morphology Four kinds of samples were placed in 24-well plates. A 1 ml cell suspension with a density of 7 × 103 cells/ml was seeded onto each sample and cultured under standard cell culture condition. After 1, 4 and 7 days of culture, samples were washed with phosphate buffer saline (PBS) twice and fixed with 2.5% glutaraldehyde. Then, the samples were successively dehydrated in gradient ethanol solutions (30, 50, 70, 90 and 100%) and critical point-dried. After sputter-coated with gold, samples were examined using SEM (Hitach S3400, Japan). 2.5. Cytoskeleton staining Cells were seeded on the surfaces of samples at a cell density of 7 × 103 cells/ml. After being cultured for 7 days, the samples with cells were washed twice with the PBS, fixed with 4% paraformaldehyde solution (Sigma, USA) for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 (Sigma, USA) for 2 min. Then, cells were washed with PBS, and stained with FITC-Phalloidin (Sigma, USA) in darkness at room temperature for 1 h and further stained with DAPI (Sigma, USA) for 5 min. The cell cytoskeleton and nuclei were examined by a fluorescence microscopy (Olympus, Japan). 2.6. Cell proliferation Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Dojindo, Japan) with measuring dehydrogenases’ metabolic activity through a tetrazolium reaction [21]. Cells were seeded on samples at a cell density of 7 × 103 cells/ml. After cultured for 1, 4 and 7 days, samples were washed twice with PBS to eliminate non-viable cells. Each well was incubated with 1 ml of medium containing 100 l of CCK-8 solution for 3 h in the incubator. Then the optical density was measured by a microplate reader (Multiskan MK3, Thermo) at excitation wavelength of 450 nm. Cell proliferation was calculated according to the manufacturer’s instruction.
2.2. Surface characterizations
2.7. Alkaline phosphatase (ALP) activity
The surface morphologies of titanium and films were examined by scanning electron microscopy (SEM, Hitach S3400, Japan). The phase compositions of films were analyzed by X-ray diffraction (XRD, D/MAX-2550, Rigaku, Japan) using Cu K␣ radiation at 40 kV and 100 mA. The elemental depth profiles and Si chemical states were determined by X-ray photoelectron spectroscopy (XPS, Physical electronics PHI 5802). The surface wettability of samples was conducted by water contact angle measurements (SL200B, Solon Information Technology Co, Ltd, China) using 2 l of sessile distilled water droplets. The average contact angle value was obtained by measuring the angles of droplets on three samples, and three different positions were tested at each one.
ALP activity was evaluated quantitatively through pnitrophenol formed from the enzymatic hydrolysis of p-nitrophenylphosphate (pNPP) [21]. Cells were seed at 7 × 103 cells/ml on samples and cultured for 1, 4, and 7 days, and then washed twice with PBS. Further, samples were incubated with pNPP (Sigma, USA) at 37 ◦ C for 30 min. The ALP activity was determined by measuring the optical density at a wavelength of 405 nm on a microplate reader (Multiskan MK3, Thermo). The ALP levels were normalized to the total protein content, which was measured by the BCA protein assay, and the experiments were carries out in triplicates. 2.8. Statistical analysis
2.3. Cell culture The osteoblast-like cell line MG63 (Shanghai Institute of Biological Science, Chinese Academy of Sciences, China) was seeded on the Ti control, TS0, TS0.5 and TS1.0 films to evaluate the cytocompatibility. Cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Life Technologies, USA) and 1% penicillin–streptomycin at 37 ◦ C in a moist 5% CO2 atmosphere. The culture medium was replaced every 3 days. After reaching confluence, the cells were subcultured into new dishes using trypsin–ethylenediamine tetra-acetic acid (EDTA, Gibco).
The one-way analysis of variance (ANOVA) and Student–Newman–Keuls post hoc tests were used to determine the level of significance by using GraphPad Instant Software (GraphPad Software, Inc., USA). The statistical analysis significance level was set to p < 0.05. 3. Results and discussion Fig. 1 shows the surface views of the titanium, the as-annealed and Si-implanted TiO2 nanopores films. The surface of the titanium substrate appeared as a flat topography without structures,
216
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
Fig. 1. Surface views of Ti (a), TS0 (b), TS0.5 (c) and TS1.0 (d) samples.
as shown in Fig. 1a. After anodization and heat treatment, the prepared film displayed a clean and uniform surface with the regular nanopores presenting, of which the diameter was about 70 nm (Fig. 1b). The similar surface structures with slight smooth were also observed in both Fig. 1c and d, in which the TiO2 nanopores were treated by Si-PIII with 0.5 h and 1.0 h, respectively. It meant that the surface nanostructures of films were unchanged with the Si implantation. The XRD patterns of the TS0, TS0.5 and TS1.0 films are shown in Fig. 2A. The peaks of films were corresponding to crystalline anatase and titanium substrates. The peaks of silicon were not found from the XRD patterns of Si-implanted samples due to the low content of silicon in the films. Fig. 2B shows the XPS spectra of these films. The feature peaks of Si 2p and 2s were observed in the survey spectra of Si-implanted films, especially in the inset higher magnification, indicating that the Si-incorporated TiO2 nanopores were prepared successfully. Fig. 3a and b shows the high-resolution spectra of Si 2p obtained from the surface of TS0.5 and TS1.0, respectively. The Si 2p spectra of TS0.5 can be deconvoluted into two different chemical bonding that were corresponded to SiOx (100.4 eV) and Si (99.6 eV) [22,23] (Fig. 3a). In Fig. 3b, the peak at 102.35 eV was in agreement with Si4+ in titanium silicon oxide due to the range from 101.85 eV to 103.20 eV ascribed to titanium silicon oxide with different
stoichiometric ratio [24]. The silicon depth profiles are plotted in Fig. 3c. The silicon depth of TS0.5 was about 70 nm and the maximum content was 3.15% at the surface with a decreasing amount toward the interior. For TS1.0, the silicon depth was about 100 nm and the maximum amount was about 4.24% and the peak was located at about 20 nm. Fig. 3d shows the peak positions of Si 2p spectra obtained from each depth scale. The bonding energies of silicon in various depths were nearly constant in both films except the value at the surface, which thought to be resulted from the adsorption of oxygen and the shift of the peak. The results indicated that the silicon formed the same compound in TiO2 nanopores, meaning the formation of titanium silicon oxide in TS1.0 and silicon oxide in TS0.5. In Si-PIII, silicon ions with high energy were generated and accelerated toward to the TiO2 nanopores substrate, which was loaded a negative bias. The incoming silicon ions bombard the substrate and created thermal spike to facilitate the nucleation of crystalline phase [25]. With the PIII time increases, more silicon ions reached on the nanopores films surface, indicating a higher amount of silicon and much more energy supported for the atomic scale diffusion [26] and the nucleation of compounds. Therefore, compared to TS0.5, TS1.0 had a deeper profile and higher concentration of silicon and was composed of titanium silicon oxide, which was formed feasibly.
Fig. 2. XRD patterns (A) and XPS spectra (B) of TS0 (a), TS0.5 (b) and TS1.0 (c) films. The inset in (B) is the enlarged section of the spectra with the range from 0 to 200 eV.
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
217
Fig. 3. High-resolution XPS spectra of Si 2p obtained from TS0.5 (a) and TS1.0 (b) films; silicon depth profiles (c) and Peaks position of Si 2p spectra in different depths (d) of TS0.5 and TS1.0 films.
The wettability of titanium, as-annealed and Si-implanted TiO2 nanopores films were evaluated by water contact angle measurement and the results are presented in Fig. 4. The water contact angle of Ti was 87.14 ± 1.78◦ and those of TS0, TS0.5 and TS1.0 films were 27.89 ± 0.85◦ , 26.88 ± 0.45◦ and 27.45 ± 0.54◦ , respectively, meaning that the as-annealed and Si-implanted TiO2 nanopores were hydrophilic and there was no significant difference among them. It was indicated that the nanopores structures were beneficial for improving the surface wettability. The views of MG63 cells cultured on Ti, TS0, TS0.5 and TS1.0 films for 1, 4, 7 days were observed by SEM and typical images are presented in Fig. 5. After being cultured for 1 day, cells on TS0, TS0.5 and TS1.0 films showed in a larger number, elongated
Fig. 4. Water contact angles of Ti, TS0, TS0.5 and TS1.0 samples, all the data are expressed as means ± SD and n = 9.
morphologies and numerous of filopodia extensions compared to Ti control. At day 4, the cells numbers on all samples increased significantly. It was noticeable that cells on TS1.0 spread completely and attached closely to the substrate surface. After 7 days of incubation, cells covered the entire surface of the all samples. Moreover, larger cell spreading area and prominent filopodia and lamellipodia extension were apparent on the nanopores films (TS0, TS0.5 and TS1.0), indicating that nanostrucured films possessed excellent cytocompatibility compared to Ti control. The proliferation of MG63 cells on Ti, TS0, TS0.5 and TS1.0 films were determined by CCK-8 assay, and Fig. 6 shows the result of the cells cultured for 1, 4 and 7 days. At day 1, the CCK-8 values on all samples appeared similar. After cultured for 4 days, cell proliferation on TS0.5 and TS1.0 films was higher than that on Ti control and TS0 film (p < 0.05). With the culture time prolonged to 7 days, cell numbers on TS0.5 and TS1.0 films were still significantly higher than those on TS0 and Ti (p < 0.05), indicating that the Si-incorporated films were more favorable to the proliferation of MG63 cells. The ALP activity of MG63 cells on samples at various time points were displayed in Fig. 7. After being cultured 1 day, there was no significant difference in ALP activity on four groups of samples (p > 0.05). At day 4, it was clear that the ALP activity was significantly higher on TS0.5 and TS1.0 films than that on Ti (p < 0.05). After 7 days of incubation, the ALP activity was also significantly higher on TS0.5 and TS1.0 films than that on Ti (p < 0.05) and TS1.0 film showed significantly higher ALP activity that those on other groups (p < 0.05). Moreover, it could be seen that the ALP activity was increasing with the increased silicon content at day 4 and 7, although statistically significant difference was not observed among all of them. ALP has been recognized as a marker expressed at the early stage of osteogenic differentiation, and the level of ALP
218
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
Fig. 5. SEM views of MG63 cells cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4, 7 days; insets are the higher magnification images.
activity is an important indicator of osteoblast differentiation [27]. ALP expression in this work was significantly upregulated on TS0.5 and TS1.0 films compared with Ti control, and the ALP activity of cells on the films increased with the increase of Si concentration in the films, indicating the beneficial effects of silicon on the differentiation of MG63 cells. Cytoskeleton staining experiment was also carried out to evaluate the cell adhesion and proliferation activity by staining with FITC and DAPI, respectively, and the results are shown in Fig. 8. After cultured for 7 days, the number of cells was large and the material surface was covered completely by cell cytoskeleton for all samples. The cytoskeletons of cells cultured on Ti and TS0 could be observed clearly; meanwhile those on TS0.5 and TS1.0 were fuzzy, which might be result in an image of multilayer cells. It is in good
agreement with the SEM results in Fig. 5, indicating that the Siincorporated nanopores films could be excellent platforms for cells culture. Surface structure and chemical composition of titanium are the key factors to influence the biological performances. In this work, based on titanium substrates, we prepared TiO2 nanopores films by anodization firstly. This nanostructured surface shows hydrophilicity, which is believed to improve the protein adhesion and facilitate the cell behaviors such as cell adhesion, proliferation and differentiation [13]. Their effects are consistent with some other nanostructures, like TiO2 nanotubes [8], nanorods [11], and nanowires [12]. Then, we modified nanopores films using Si-PIII for the incorporation of silicon, which has been proven to benefit
Fig. 6. Cells proliferation shown by CCK-8 assay of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4 and 7 days, # p < 0.05 vs. Ti and $ p < 0.05 vs. TS0.
Fig. 7. ALP activity of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4 and 7 days, # p < 0.05 vs. Ti and $ p < 0.05 vs. TS0.
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
219
Fig. 8. Fluorescence images of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 7 days, scale bar = 200 m.
the growth of bone. The Si-implanted films showed better cytocompatibility than the Ti control and as-annealed nanopores film. Moreover, the TS1.0 exhibited the best cell proliferation and the ALP activity. It could be attributed to the highest silicon concentration of the films and the formation of titanium silicon oxide, which could react with H2 O to form Si OH and Ti OH for good bioactivity [19]. 4. Conclusion Si-incorporated TiO2 nanopores films were prepared by anodization and silicon plasma immersion ion implantation. The concentration and depth of silicon in TiO2 nanopores films increased with the duration time of implantation. Silicon oxide was formed on the surface of TiO2 nanopores films implanted silicon for 0.5 h, while titanium silicon oxide was found when the implantation time up to 1.0 h. Both the as-annealed and Si-incorporated nanopores films exhibited good hydrophilicity and cytocompatibility, while the TiO2 nanopores films implanted silicon for 1.0 h showed higher proliferation rate and vitality of MG63cells than others. Acknowledgements Financial support from the National Basic Research Program of China (973 Program, 2012CB933600), National Natural Science Foundation of China (51401234 and 81271704), Shanghai Committee of Science and Technology, China (13441902400 and 14XD1403900) and Innovation Fund of SICCAS (Y46ZC3130G) are acknowledged.
References [1] K. Anselme, et al., The interaction of cells and bacteria with surfaces structured at the nanometre scale, Acta Biomater. 6 (10) (2010) 3824–3846. [2] S. Qian, et al., Selective biofunctional modification of titanium implants for osteogenic and antibacterial applications, J. Mater. Chem. B 2 (43) (2014) 7475–7487. [3] M. Geetha, et al., Ti based biomaterials, the ultimate choice for orthopaedic implants—a review, Prog. Mater. Sci. 54 (3) (2009) 397–425. [4] Y.T. Sul, et al., The bone response of oxidized bioactive and non-bioactive titanium implants, Biomaterials 26 (33) (2005) 6720–6730. [5] K. Das, et al., TiO2 nanotubes on Ti: influence of nanoscale morphology on bone cell–materials interaction, J. Biomed. Mater. Res. A 90A (1) (2009) 225– 237. [6] K. Das, et al., Titanium surface modification to titania nanotube for next generation orthopedic applications, Adv. Bioceram. Porous Ceram. 29 (7) (2009) 37–41. [7] S. Oh, et al., Significantly accelerated osteoblast cell growth on aligned TiO2 nanotubes, J. Biomed. Mater. Res. A 78A (1) (2006) 97–103. [8] K.C. Popat, et al., Influence of engineered titania nanotubular surfaces on bone cells, Biomaterials 28 (21) (2007) 3188–3197. [9] J. Park, et al., Nanosize and vitality: TiO2 nanotube diameter directs cell fate, Nano Lett. 7 (6) (2007) 1686–1691. [10] S. Oh, et al., Stem cell fate dictated solely by altered nanotube dimension, Proc. Natl. Acad. Sci. U.S.A. 106 (7) (2009) 2130–2135. [11] T. Amna, et al., TiO2 nanorods via one-step electrospinning technique: a novel nanomatrix for mouse myoblasts adhesion and propagation, Colloids Surf. B: Biointerfaces 101 (2013) 424–429. [12] X.L. Ding, et al., Titanate nanowire scaffolds decorated with anatase nanocrystals show good protein adsorption and low cell adhesion capacity, Int. J. Nanomed. 8 (2013) 569–579. [13] L.C. Xu, C.A. Siedlecki, Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces, Biomaterials 28 (22) (2007) 3273– 3283. [14] M.J.P. Biggs, et al., Nanotopographical modification: a regulator of cellular function through focal adhesions, Nanomed.-Nanotechnol. 6 (5) (2010) 619–633. [15] E.M. Carlisle, Silicon: a possible factor in bone calcification, Science 167 (1970) 279–280.
220
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
[16] L. Bao, et al., Preparation and characterization of TiO2 and Si-doped octacalcium phosphate composite coatings on zirconia ceramics (Y-TZP) for dental implant applications, Appl. Surf. Sci. 290 (2014) 48–52. [17] X. Qiu, et al., Preliminary research on a novel bioactive silicon doped calcium phosphate coating on AZ31 magnesium alloy via electrodeposition, Mater. Sci. Eng. C 36 (2014) 65–76. [18] X.B. Zhao, et al., Delicate refinement of surface nanotopography by adjusting TiO2 coating chemical composition for enhanced interfacial biocompatibility, ACS Appl. Mater. Inter. 5 (16) (2013) 8203–8209. [19] S. Qian, et al., Effect of Si-incorporation on hydrophilicity and bioactivity of titania film, Surf. Coat. Technol. 229 (2013) 156–161. [20] P.K. Chu, Surface engineering and modification of biomaterials, Thin Solid Films 528 (2013) 93–105. [21] B. Wang, et al., Proliferation and differentiation of osteoblast cells on silicondoped TiO2 film deposited by cathodic arc, Biomed. Pharmacother. 66 (8) (2012) 633–641.
[22] W.A.M. Aarnink, et al., Angle-resolved X-ray photoelectron-spectroscopy (ARXPS) and a modified Levenberg–Marquardt fit procedure—a new combination for modeling thin layers, Appl. Surf. Sci. 45 (1990) 37–48. [23] G.E. Franklin, et al., Interface formation and growth of Insb on Si(1 0 0), Phys. Rev. B: Condens. Matter 45 (7) (1992) 3426–3434. [24] M. Anpo, et al., Photocatalysis over binary metal-oxides-enhancement of the photocatalytic activity of TiO2 in titanium silicon-oxides, J. Phys. Chem. 90 (8) (1986) 1633–1636. [25] Y. Lifshitz, et al., The mechanism of diamond nucleation from energetic species, Science 297 (2002) 1531–1533. [26] A. Anders, Atomic scale heating in cathodic arc plasma deposition, Appl. Phys. Lett. 80 (6) (2002) 1100–1102. [27] L.Z. Zhao, et al., Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation, Biomaterials 33 (9) (2012) 2629–2641.
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Cytocompatibility of Si-incorporated TiO2 nanopores films Shi Qian, Xuanyong Liu ∗ State Key Laboratory of High Performance Ceramics and Superfine Microstructures, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, China
a r t i c l e
i n f o
Article history: Received 16 March 2015 Received in revised form 27 May 2015 Accepted 2 June 2015 Available online 8 June 2015 Keywords: TiO2 nanopores Anodization Silicon Cytocompatibility
a b s t r a c t Si-incorporated TiO2 nanopores films were prepared by anodization and silicon plasma immersion ion implantation. The microstructure and phase composition of the films were investigated by scanning electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The hydrophilicity of the films was evaluated using water contact angle measurement and MG63 cells were cultured on the films to investigate the cytocompatibility. The results showed that the concentration and depth of silicon on the Si-incorporated TiO2 nanopores films increased with the duration time of implantation. Both the as-annealed and Si-incorporated nanopores films exhibited good hydrophilicity and cytocompatibility, while the TiO2 nanopores films implanted silicon for 1.0 h showed higher proliferation rate and vitality of MG63 cells than others, indicating a great potential application for titanium implants. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In recent years, the surface properties of implants have been widely investigated for improving their biological applications and understanding the interaction of materials and cells [1,2]. Titanium and its alloys have been used extensively as orthopedic implants due to the low elastic modulus, corrosion resistance and biocompatibility [3]. However, because of their bioinert nature, they usually get encapsulated by fibrous tissues and the insufficient osseointegration may result in implant failures [4]. Therefore, surface modification of titanium has been recognized as a potential approach to overcome these defects. For instance, in situ generated TiO2 nanotubes on titanium substrates have drawn enormous attention in biology [5,6], which can be attributed to the simple anodization process and the adjustable dimensions in nanoscale. Oh et al. [7] investigated the growth of MC3T3 osteoblast cells on nanotubes and found that the cell adhesion was improved with the filopodia going into the nanotube to produce interlocked connection, suggesting that the tubular structure induced a significant acceleration in the cell growth. Popat et al. [8] demonstrated the ability of TiO2 tubular nanostructures to promote osteoblast differentiation and matrix production, and enhance short- and long-term osseointegration. Moreover, the adhesion, growth and differentiation of stem cells were illustrated to be critically dependent on the tube diameter [9]. It was concluded
∗ Corresponding author. Tel.: +86 21 52412409; fax: +86 21 52412409. E-mail address: [email protected] (X. Liu). http://dx.doi.org/10.1016/j.colsurfb.2015.06.007 0927-7765/© 2015 Elsevier B.V. All rights reserved.
that small nanotube (about 30 nm in diameter) mainly promoted stem cell adhesion and larger nanotube (about 70 nm) could induce the selective differentiation into ostoblast-like cells with cell elongation [10]. In addition, other nanostructures, such as nanorods [11] and nanowires [12] have also been synthesized on titanium surfaces. It was revealed that cell proliferation was increased and osteogenic related genes and proteins were up-regulated [12], and the enhanced cell activities were attributed to the higher surface energy of nanostructures [13]. It is interesting that nanopores structures have been identified as the common constituents of tissues in vivo, such as basement membrane of the cornea, the aortic heart valve and the vascular system [14]. Therefore, producing the nanopores on titanium that could emulate the tissue structures are supposed to regulate the cell behavior and function. On the other hand, silicon (Si) has been considered as an essential element for normal growth and development of bone and cartilage [15]. Moreover, numerous researches have demonstrated that the presence of Si is vital to enhance the bioactivity of some bioactive glasses and ceramic [16,17]. The Si-containing TiO2 coating or films have also been investigated and proven to be favorable for the adhesion and spreading of osteoblast cells on the surface [18,19]. Thus, the incorporation of Si is thought to be effective for chemical modification of titanium. Here, an interest that the effects of Si on the composition and structure of TiO2 nanopores films has been taken due to rarely researches been reported. Plasma immersion ion implantation (PIII) is a versatile process for the surface modification of biomaterials [20]. In this work, Siincorporated TiO2 nanopores films were prepared using a two-step
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
anodization process and PIII, and its surface structure, phase composition and cytocompatibility were investigated. 2. Materials and methods 2.1. Samples preparation Commercially available pure titanium plates (purity > 99.85%, Grade 1, Baoji Shenghua Nonferrous Metal Materials Co, Ltd, China) were used in dimensions of 10 × 10 × 1 mm3 . They were polished mechanically and ultrasonically cleaned in acetone, ethanol and deionized water, and then dried in a dust proof condition. Anodization was conducted in a two electrode cell with a graphite plate used as the cathode. An ethylene glycol solution was adopted as the electrolyte supplemented with 0.25 wt% NH4 F and 1 vol% H2 O. The TiO2 nanopores film was fabricated by a twostep anodization process. The first step was carried out at 50 V for 1 h using a WYJ-5A-150 V DC power supply. By separating the formed oxide layer in the first stage from titanium substrate, the second step was conducted in the same electrolyte at 50 V for 1 h, and TiO2 nanopores films were produced. Then, the asprepared films were annealed at 450 ◦ C in atmosphere for 2 h using a Muffle furnace with rates of 1 ◦ C/min and cooled down with the furnace. Si-incorporation TiO2 nanopores films were prepared by PIII using the annealed TiO2 nanopores as substrates. The detailed preparation was similar to that described previously [19]. Briefly, a PIII system was used with a filtered cathodic arc plasma source, and the 99.99% pure silicon rod was adopted as the cathode. During PIII, a pulsed negative voltage applied to the samples was 20 kV, the pulse duration was 450 s, and pulse duration of the cathodic arc current was 800 s. The pulsed negative voltage and cathodic arc current were synchronized at a pulsing frequency of 8 Hz. The working pressure was 4.0 × 10−3 Pa. The as-annealed TiO2 nanopores films were labeled as “TS0”, and the Si-implanted films for 0.5 h and 1.0 h were labeled as “TS0.5” and “TS1.0”, respectively.
215
2.4. Cell morphology Four kinds of samples were placed in 24-well plates. A 1 ml cell suspension with a density of 7 × 103 cells/ml was seeded onto each sample and cultured under standard cell culture condition. After 1, 4 and 7 days of culture, samples were washed with phosphate buffer saline (PBS) twice and fixed with 2.5% glutaraldehyde. Then, the samples were successively dehydrated in gradient ethanol solutions (30, 50, 70, 90 and 100%) and critical point-dried. After sputter-coated with gold, samples were examined using SEM (Hitach S3400, Japan). 2.5. Cytoskeleton staining Cells were seeded on the surfaces of samples at a cell density of 7 × 103 cells/ml. After being cultured for 7 days, the samples with cells were washed twice with the PBS, fixed with 4% paraformaldehyde solution (Sigma, USA) for 10 min at room temperature, and permeabilized with 0.1% Triton X-100 (Sigma, USA) for 2 min. Then, cells were washed with PBS, and stained with FITC-Phalloidin (Sigma, USA) in darkness at room temperature for 1 h and further stained with DAPI (Sigma, USA) for 5 min. The cell cytoskeleton and nuclei were examined by a fluorescence microscopy (Olympus, Japan). 2.6. Cell proliferation Cell proliferation was analyzed using Cell Counting Kit-8 (CCK-8, Dojindo, Japan) with measuring dehydrogenases’ metabolic activity through a tetrazolium reaction [21]. Cells were seeded on samples at a cell density of 7 × 103 cells/ml. After cultured for 1, 4 and 7 days, samples were washed twice with PBS to eliminate non-viable cells. Each well was incubated with 1 ml of medium containing 100 l of CCK-8 solution for 3 h in the incubator. Then the optical density was measured by a microplate reader (Multiskan MK3, Thermo) at excitation wavelength of 450 nm. Cell proliferation was calculated according to the manufacturer’s instruction.
2.2. Surface characterizations
2.7. Alkaline phosphatase (ALP) activity
The surface morphologies of titanium and films were examined by scanning electron microscopy (SEM, Hitach S3400, Japan). The phase compositions of films were analyzed by X-ray diffraction (XRD, D/MAX-2550, Rigaku, Japan) using Cu K␣ radiation at 40 kV and 100 mA. The elemental depth profiles and Si chemical states were determined by X-ray photoelectron spectroscopy (XPS, Physical electronics PHI 5802). The surface wettability of samples was conducted by water contact angle measurements (SL200B, Solon Information Technology Co, Ltd, China) using 2 l of sessile distilled water droplets. The average contact angle value was obtained by measuring the angles of droplets on three samples, and three different positions were tested at each one.
ALP activity was evaluated quantitatively through pnitrophenol formed from the enzymatic hydrolysis of p-nitrophenylphosphate (pNPP) [21]. Cells were seed at 7 × 103 cells/ml on samples and cultured for 1, 4, and 7 days, and then washed twice with PBS. Further, samples were incubated with pNPP (Sigma, USA) at 37 ◦ C for 30 min. The ALP activity was determined by measuring the optical density at a wavelength of 405 nm on a microplate reader (Multiskan MK3, Thermo). The ALP levels were normalized to the total protein content, which was measured by the BCA protein assay, and the experiments were carries out in triplicates. 2.8. Statistical analysis
2.3. Cell culture The osteoblast-like cell line MG63 (Shanghai Institute of Biological Science, Chinese Academy of Sciences, China) was seeded on the Ti control, TS0, TS0.5 and TS1.0 films to evaluate the cytocompatibility. Cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, USA) supplemented with 10% fetal bovine serum (Life Technologies, USA) and 1% penicillin–streptomycin at 37 ◦ C in a moist 5% CO2 atmosphere. The culture medium was replaced every 3 days. After reaching confluence, the cells were subcultured into new dishes using trypsin–ethylenediamine tetra-acetic acid (EDTA, Gibco).
The one-way analysis of variance (ANOVA) and Student–Newman–Keuls post hoc tests were used to determine the level of significance by using GraphPad Instant Software (GraphPad Software, Inc., USA). The statistical analysis significance level was set to p < 0.05. 3. Results and discussion Fig. 1 shows the surface views of the titanium, the as-annealed and Si-implanted TiO2 nanopores films. The surface of the titanium substrate appeared as a flat topography without structures,
216
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
Fig. 1. Surface views of Ti (a), TS0 (b), TS0.5 (c) and TS1.0 (d) samples.
as shown in Fig. 1a. After anodization and heat treatment, the prepared film displayed a clean and uniform surface with the regular nanopores presenting, of which the diameter was about 70 nm (Fig. 1b). The similar surface structures with slight smooth were also observed in both Fig. 1c and d, in which the TiO2 nanopores were treated by Si-PIII with 0.5 h and 1.0 h, respectively. It meant that the surface nanostructures of films were unchanged with the Si implantation. The XRD patterns of the TS0, TS0.5 and TS1.0 films are shown in Fig. 2A. The peaks of films were corresponding to crystalline anatase and titanium substrates. The peaks of silicon were not found from the XRD patterns of Si-implanted samples due to the low content of silicon in the films. Fig. 2B shows the XPS spectra of these films. The feature peaks of Si 2p and 2s were observed in the survey spectra of Si-implanted films, especially in the inset higher magnification, indicating that the Si-incorporated TiO2 nanopores were prepared successfully. Fig. 3a and b shows the high-resolution spectra of Si 2p obtained from the surface of TS0.5 and TS1.0, respectively. The Si 2p spectra of TS0.5 can be deconvoluted into two different chemical bonding that were corresponded to SiOx (100.4 eV) and Si (99.6 eV) [22,23] (Fig. 3a). In Fig. 3b, the peak at 102.35 eV was in agreement with Si4+ in titanium silicon oxide due to the range from 101.85 eV to 103.20 eV ascribed to titanium silicon oxide with different
stoichiometric ratio [24]. The silicon depth profiles are plotted in Fig. 3c. The silicon depth of TS0.5 was about 70 nm and the maximum content was 3.15% at the surface with a decreasing amount toward the interior. For TS1.0, the silicon depth was about 100 nm and the maximum amount was about 4.24% and the peak was located at about 20 nm. Fig. 3d shows the peak positions of Si 2p spectra obtained from each depth scale. The bonding energies of silicon in various depths were nearly constant in both films except the value at the surface, which thought to be resulted from the adsorption of oxygen and the shift of the peak. The results indicated that the silicon formed the same compound in TiO2 nanopores, meaning the formation of titanium silicon oxide in TS1.0 and silicon oxide in TS0.5. In Si-PIII, silicon ions with high energy were generated and accelerated toward to the TiO2 nanopores substrate, which was loaded a negative bias. The incoming silicon ions bombard the substrate and created thermal spike to facilitate the nucleation of crystalline phase [25]. With the PIII time increases, more silicon ions reached on the nanopores films surface, indicating a higher amount of silicon and much more energy supported for the atomic scale diffusion [26] and the nucleation of compounds. Therefore, compared to TS0.5, TS1.0 had a deeper profile and higher concentration of silicon and was composed of titanium silicon oxide, which was formed feasibly.
Fig. 2. XRD patterns (A) and XPS spectra (B) of TS0 (a), TS0.5 (b) and TS1.0 (c) films. The inset in (B) is the enlarged section of the spectra with the range from 0 to 200 eV.
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
217
Fig. 3. High-resolution XPS spectra of Si 2p obtained from TS0.5 (a) and TS1.0 (b) films; silicon depth profiles (c) and Peaks position of Si 2p spectra in different depths (d) of TS0.5 and TS1.0 films.
The wettability of titanium, as-annealed and Si-implanted TiO2 nanopores films were evaluated by water contact angle measurement and the results are presented in Fig. 4. The water contact angle of Ti was 87.14 ± 1.78◦ and those of TS0, TS0.5 and TS1.0 films were 27.89 ± 0.85◦ , 26.88 ± 0.45◦ and 27.45 ± 0.54◦ , respectively, meaning that the as-annealed and Si-implanted TiO2 nanopores were hydrophilic and there was no significant difference among them. It was indicated that the nanopores structures were beneficial for improving the surface wettability. The views of MG63 cells cultured on Ti, TS0, TS0.5 and TS1.0 films for 1, 4, 7 days were observed by SEM and typical images are presented in Fig. 5. After being cultured for 1 day, cells on TS0, TS0.5 and TS1.0 films showed in a larger number, elongated
Fig. 4. Water contact angles of Ti, TS0, TS0.5 and TS1.0 samples, all the data are expressed as means ± SD and n = 9.
morphologies and numerous of filopodia extensions compared to Ti control. At day 4, the cells numbers on all samples increased significantly. It was noticeable that cells on TS1.0 spread completely and attached closely to the substrate surface. After 7 days of incubation, cells covered the entire surface of the all samples. Moreover, larger cell spreading area and prominent filopodia and lamellipodia extension were apparent on the nanopores films (TS0, TS0.5 and TS1.0), indicating that nanostrucured films possessed excellent cytocompatibility compared to Ti control. The proliferation of MG63 cells on Ti, TS0, TS0.5 and TS1.0 films were determined by CCK-8 assay, and Fig. 6 shows the result of the cells cultured for 1, 4 and 7 days. At day 1, the CCK-8 values on all samples appeared similar. After cultured for 4 days, cell proliferation on TS0.5 and TS1.0 films was higher than that on Ti control and TS0 film (p < 0.05). With the culture time prolonged to 7 days, cell numbers on TS0.5 and TS1.0 films were still significantly higher than those on TS0 and Ti (p < 0.05), indicating that the Si-incorporated films were more favorable to the proliferation of MG63 cells. The ALP activity of MG63 cells on samples at various time points were displayed in Fig. 7. After being cultured 1 day, there was no significant difference in ALP activity on four groups of samples (p > 0.05). At day 4, it was clear that the ALP activity was significantly higher on TS0.5 and TS1.0 films than that on Ti (p < 0.05). After 7 days of incubation, the ALP activity was also significantly higher on TS0.5 and TS1.0 films than that on Ti (p < 0.05) and TS1.0 film showed significantly higher ALP activity that those on other groups (p < 0.05). Moreover, it could be seen that the ALP activity was increasing with the increased silicon content at day 4 and 7, although statistically significant difference was not observed among all of them. ALP has been recognized as a marker expressed at the early stage of osteogenic differentiation, and the level of ALP
218
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
Fig. 5. SEM views of MG63 cells cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4, 7 days; insets are the higher magnification images.
activity is an important indicator of osteoblast differentiation [27]. ALP expression in this work was significantly upregulated on TS0.5 and TS1.0 films compared with Ti control, and the ALP activity of cells on the films increased with the increase of Si concentration in the films, indicating the beneficial effects of silicon on the differentiation of MG63 cells. Cytoskeleton staining experiment was also carried out to evaluate the cell adhesion and proliferation activity by staining with FITC and DAPI, respectively, and the results are shown in Fig. 8. After cultured for 7 days, the number of cells was large and the material surface was covered completely by cell cytoskeleton for all samples. The cytoskeletons of cells cultured on Ti and TS0 could be observed clearly; meanwhile those on TS0.5 and TS1.0 were fuzzy, which might be result in an image of multilayer cells. It is in good
agreement with the SEM results in Fig. 5, indicating that the Siincorporated nanopores films could be excellent platforms for cells culture. Surface structure and chemical composition of titanium are the key factors to influence the biological performances. In this work, based on titanium substrates, we prepared TiO2 nanopores films by anodization firstly. This nanostructured surface shows hydrophilicity, which is believed to improve the protein adhesion and facilitate the cell behaviors such as cell adhesion, proliferation and differentiation [13]. Their effects are consistent with some other nanostructures, like TiO2 nanotubes [8], nanorods [11], and nanowires [12]. Then, we modified nanopores films using Si-PIII for the incorporation of silicon, which has been proven to benefit
Fig. 6. Cells proliferation shown by CCK-8 assay of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4 and 7 days, # p < 0.05 vs. Ti and $ p < 0.05 vs. TS0.
Fig. 7. ALP activity of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 1, 4 and 7 days, # p < 0.05 vs. Ti and $ p < 0.05 vs. TS0.
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
219
Fig. 8. Fluorescence images of MG63 cultured on Ti, TS0, TS0.5 and TS1.0 samples for 7 days, scale bar = 200 m.
the growth of bone. The Si-implanted films showed better cytocompatibility than the Ti control and as-annealed nanopores film. Moreover, the TS1.0 exhibited the best cell proliferation and the ALP activity. It could be attributed to the highest silicon concentration of the films and the formation of titanium silicon oxide, which could react with H2 O to form Si OH and Ti OH for good bioactivity [19]. 4. Conclusion Si-incorporated TiO2 nanopores films were prepared by anodization and silicon plasma immersion ion implantation. The concentration and depth of silicon in TiO2 nanopores films increased with the duration time of implantation. Silicon oxide was formed on the surface of TiO2 nanopores films implanted silicon for 0.5 h, while titanium silicon oxide was found when the implantation time up to 1.0 h. Both the as-annealed and Si-incorporated nanopores films exhibited good hydrophilicity and cytocompatibility, while the TiO2 nanopores films implanted silicon for 1.0 h showed higher proliferation rate and vitality of MG63cells than others. Acknowledgements Financial support from the National Basic Research Program of China (973 Program, 2012CB933600), National Natural Science Foundation of China (51401234 and 81271704), Shanghai Committee of Science and Technology, China (13441902400 and 14XD1403900) and Innovation Fund of SICCAS (Y46ZC3130G) are acknowledged.
References [1] K. Anselme, et al., The interaction of cells and bacteria with surfaces structured at the nanometre scale, Acta Biomater. 6 (10) (2010) 3824–3846. [2] S. Qian, et al., Selective biofunctional modification of titanium implants for osteogenic and antibacterial applications, J. Mater. Chem. B 2 (43) (2014) 7475–7487. [3] M. Geetha, et al., Ti based biomaterials, the ultimate choice for orthopaedic implants—a review, Prog. Mater. Sci. 54 (3) (2009) 397–425. [4] Y.T. Sul, et al., The bone response of oxidized bioactive and non-bioactive titanium implants, Biomaterials 26 (33) (2005) 6720–6730. [5] K. Das, et al., TiO2 nanotubes on Ti: influence of nanoscale morphology on bone cell–materials interaction, J. Biomed. Mater. Res. A 90A (1) (2009) 225– 237. [6] K. Das, et al., Titanium surface modification to titania nanotube for next generation orthopedic applications, Adv. Bioceram. Porous Ceram. 29 (7) (2009) 37–41. [7] S. Oh, et al., Significantly accelerated osteoblast cell growth on aligned TiO2 nanotubes, J. Biomed. Mater. Res. A 78A (1) (2006) 97–103. [8] K.C. Popat, et al., Influence of engineered titania nanotubular surfaces on bone cells, Biomaterials 28 (21) (2007) 3188–3197. [9] J. Park, et al., Nanosize and vitality: TiO2 nanotube diameter directs cell fate, Nano Lett. 7 (6) (2007) 1686–1691. [10] S. Oh, et al., Stem cell fate dictated solely by altered nanotube dimension, Proc. Natl. Acad. Sci. U.S.A. 106 (7) (2009) 2130–2135. [11] T. Amna, et al., TiO2 nanorods via one-step electrospinning technique: a novel nanomatrix for mouse myoblasts adhesion and propagation, Colloids Surf. B: Biointerfaces 101 (2013) 424–429. [12] X.L. Ding, et al., Titanate nanowire scaffolds decorated with anatase nanocrystals show good protein adsorption and low cell adhesion capacity, Int. J. Nanomed. 8 (2013) 569–579. [13] L.C. Xu, C.A. Siedlecki, Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces, Biomaterials 28 (22) (2007) 3273– 3283. [14] M.J.P. Biggs, et al., Nanotopographical modification: a regulator of cellular function through focal adhesions, Nanomed.-Nanotechnol. 6 (5) (2010) 619–633. [15] E.M. Carlisle, Silicon: a possible factor in bone calcification, Science 167 (1970) 279–280.
220
S. Qian, X. Liu / Colloids and Surfaces B: Biointerfaces 133 (2015) 214–220
[16] L. Bao, et al., Preparation and characterization of TiO2 and Si-doped octacalcium phosphate composite coatings on zirconia ceramics (Y-TZP) for dental implant applications, Appl. Surf. Sci. 290 (2014) 48–52. [17] X. Qiu, et al., Preliminary research on a novel bioactive silicon doped calcium phosphate coating on AZ31 magnesium alloy via electrodeposition, Mater. Sci. Eng. C 36 (2014) 65–76. [18] X.B. Zhao, et al., Delicate refinement of surface nanotopography by adjusting TiO2 coating chemical composition for enhanced interfacial biocompatibility, ACS Appl. Mater. Inter. 5 (16) (2013) 8203–8209. [19] S. Qian, et al., Effect of Si-incorporation on hydrophilicity and bioactivity of titania film, Surf. Coat. Technol. 229 (2013) 156–161. [20] P.K. Chu, Surface engineering and modification of biomaterials, Thin Solid Films 528 (2013) 93–105. [21] B. Wang, et al., Proliferation and differentiation of osteoblast cells on silicondoped TiO2 film deposited by cathodic arc, Biomed. Pharmacother. 66 (8) (2012) 633–641.
[22] W.A.M. Aarnink, et al., Angle-resolved X-ray photoelectron-spectroscopy (ARXPS) and a modified Levenberg–Marquardt fit procedure—a new combination for modeling thin layers, Appl. Surf. Sci. 45 (1990) 37–48. [23] G.E. Franklin, et al., Interface formation and growth of Insb on Si(1 0 0), Phys. Rev. B: Condens. Matter 45 (7) (1992) 3426–3434. [24] M. Anpo, et al., Photocatalysis over binary metal-oxides-enhancement of the photocatalytic activity of TiO2 in titanium silicon-oxides, J. Phys. Chem. 90 (8) (1986) 1633–1636. [25] Y. Lifshitz, et al., The mechanism of diamond nucleation from energetic species, Science 297 (2002) 1531–1533. [26] A. Anders, Atomic scale heating in cathodic arc plasma deposition, Appl. Phys. Lett. 80 (6) (2002) 1100–1102. [27] L.Z. Zhao, et al., Effects of micropitted/nanotubular titania topographies on bone mesenchymal stem cell osteogenic differentiation, Biomaterials 33 (9) (2012) 2629–2641.
