3D-nanoflowers of rutile TiO₂ as a film grown on conducting and non-conducting glass substrates for in vitro biocompatibility studies with mouse MC3T3 osteoblast and human HS-5 cells

Abstract

Thin films of 3D-nanoflowers of rutile TiO₂ on conducting (FTO and ITO) and non-conducting (glass) substrates were grown using a surfactant free one-step hydrothermal process. Field emission scanning electron microscopy (FE-SEM) observations confirmed the transformation of TiO₂ nanostructures from mesh-like to 3D-nanoflowers with an increase in hydrolysis rate during the growth of the TiO₂ films. The X-ray diffraction (XRD) pattern of the TiO₂ nanostructures as films grown on different substrates showed that under various conditions, they have a phase pure rutile crystallite structure. The high resolution transmission electron microscopy (HR-TEM) diffraction pattern of the TiO₂ nanostructures showed tightly packed assemblies of titanium atoms and a lattice spacing of 0.23 nm along the longitudinal axis direction of rutile TiO₂. X-ray photoelectron spectroscopic (XPS) analysis of the TiO₂ nanostructures grown as films on glass substrates showed a spectral shift of 0.53 eV in binding energy, which confirms the charge accumulation on the non-conducting substrate, whereas there was no spectral shift observed for TiO₂ films with similar structures grown on conducting substrates. The accumulated charge on the conducting surfaces can be easily neutralized, whereas the non-conducting surfaces may retain these accumulated charges. The adhesion, viability and proliferation response of mouse osteoblast (MC3T3) and human stromal (HS-5) cells on the 3D-nanoflowers of TiO₂ as films grown on conducting and non-conducting substrates were assessed. The adhesion and proliferation of both the type of cells showed a better response on non-conducting surfaces as compared to conducting surfaces, despite the similar crystallite structures and nanomorphology of TiO₂. Stromal cells had potential to prepare extra-cellular matrix scaffolds for the ex vivo expansion/differentiation of stem cells. Therefore, the current findings can be used to prepare 3D TiO₂ nanostructure supported cellular scaffolds for regenerative medicine in the future.

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1. Introduction

During the past two decades, substantial research work has been carried out to understand the correlation between the different process parameters of various synthesis methods and their effects on tailoring the structural and functional properties of metal and metal oxides.^3–8 In the past, the use of metal oxides such as titanium dioxides with tunable nanostructural and chemical properties has been extensively explored for catalytic, energy and biomedical applications.^9–13 Different synthesis methods were employed to tailor the structural and functional properties of metal oxides by manipulating the processing conditions to obtain the desired arrangements of the nanocrystallites.^13–18 For each synthesis method, detailed studies were carried out to understand the effects of the processing parameters on the modification of basic structural properties; however, to obtain materials with a higher degree of phase purity and tunable surface morphologies with the desired surface chemical functionalities is still a challenge.^19–21

A number of researchers have addressed the challenges associated with the tunable surface morphology and synthesis of micro/nanostructured titanium oxides with highly oriented rod-like crystals.^22–27 The control over the crystallite phase and morphologies were achieved either by surface treatment or metal ion doping.^28–31 Thin films of titanium oxides were deposited with controllable crystallite phases by choosing appropriate metal ion doping and were used for different applications.^32,33 Well ordered 1D TiO₂ as nanorods, nanowires, nanocorals, hierarchical microspheres and nanotubes were synthesized and extensive studies were performed to use these materials for energy and biosensor applications.^23–26 Previously, the biocompatibility of titanium oxides as thin film coatings deposited by plasma sputtering and dip coating was studied with different types of cells.^9,12,28 The simultaneous growth of 1D/3D nanorods/nanoflowers is advantageous for better adhesion at the interface of the 1D/3D nanostructures.³⁴ However, the use of 1D and 3D nano-assemblies of TiO₂ in biomedical fields has not been fully explored to date.

Herein, we report a surfactant free one-step hydrothermal process for the growth of 1D/3D nano-morphologies of TiO₂ under various conditions. The simultaneous growth of 3D nanoflower-like structures on 1D nanorods provides better adhesion at the interface of 1D/3D nanorods. The other advantage of the method used in this study was to achieve uniform coatings on all the sides of the substrates in a single step. The physicochemical properties of the nanostructures of TiO₂ grown on different substrates (non-conducting glass and FTO/ITO coated conducting glass) were investigated. The molar ratio of precursor material and reaction time were optimized to grow 1D/3D nanostructures of rutile TiO₂ as films on different types of substrates. In this study, we have reported, for the first time, the cellular response of osteoblast and HS-5 cells with TiO₂ by changing the conducting and non-conducting nature of the substrates without altering the 3D nanostructures. The use of osteoblast cells for assessing the biocompatibility of metal oxide and polymeric materials is a well-accepted in vitro model system.^35–38 To further assess the applicability of the 1D/3D nanostructures, human stromal cells, which have potential for preparing an extra-cellular matrix scaffold were cultured on different types of nano-assemblies of TiO₂. In vitro cell adhesion, viability and proliferation assays using human HS-5 cells were performed. The outcomes of this study may have potential in the future use of these substrates to prepare 3D nanostructure supported cellular scaffolds^1,2 for application in regenerative medicine.

2. Experimental

2.1 Materials

Titanium isopropoxide (Ti[OCH(CH₃)₂]₄, 97%), hydrochloric acid (HCl, 37 wt%) and isopropyl alcohol were purchased from Sigma-Aldrich (Sigma-Aldrich) and used as received. The plastic wares used for cell culture in this study were obtained from Nunc, USA. Culture media (α-MEM) was obtained from Invitrogen. All other tissue culture reagents and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium were purchased from Sigma. All other analytical grade chemicals and solvents were purchased from local companies.

2.2 Hydrothermal growth of TiO₂ nanostructures as films

For the growth of nanostructures of TiO₂ as films, a solution of titanium isopropoxide was prepared in HCl. Six different dilution ratios of HCl with water, namely, (i) 20 : 80, (ii) 30 : 70, (iii) 40 : 60, (iv) 50 : 50, (v) 60 : 40 and (vi) 70 : 30, were prepared and used to control the hydrolysis rate of titanium isopropoxide. In the final step, 1 ml of titanium isopropoxide was added to 40 ml of HCl solutions mentioned above ((i)–(vi)). The mixed solutions of titanium isopropoxide and HCl solutions were placed in a Teflon-lined stainless steel autoclave. The temperature of the autoclave was set at 180 °C and the growths of the nanostructures were observed after 3 h on FTO substrates for the conditions (i) to (vi). After the reaction was completed, the autoclave was allowed to cool to room temperature and the TiO₂ nanostructures grown on FTO substrates under the different conditions described above were collected and dried in vacuum at 70 °C for 12 h before use.

Fig. 1 shows the FE-SEM images of the TiO₂ nanostructures grown on FTO at various dilution conditions of HCl ((i)–(vi)). The TiO₂ nanostructures obtained under conditions ((i)–(iii)) with a higher water content in HCl showed round ball-like 3D TiO₂ nanostructures (Fig. 1a–c). A very significant change in the surface structure morphology was observed with an increase in the dilution of HCl. The FE-SEM images of the TiO₂ nanostructures obtained at higher concentrations of HCl (condition vi) show a mesh-like nanostructure. The mesh-like nanostructures of TiO₂ were changed to 3D-nanoflower-like structures with the use of an equal volume to volume ratio of water and HCl. TiO₂ nanostructures obtained using an equal volume to volume ratio of water and HCl (condition iv) showed a uniform distribution of 3D-nanoflowers of TiO₂ on FTO. Therefore, this optimum condition (iv) was used for the further investigation of the effects of reaction time on TiO₂ nanostructure growth as films on FTO. In this study, three different types of substrates (i) non-conducting glass (glass), (ii) ITO coated conducting glass (ITO) and (iii) FTO coated conducting glass (FTO) were used.

Fig. 1 FE-SEM images of rutile TiO₂ nanostructures grown on FTO substrates. (a)–(f) represent the nanostructures grown with different dilutions of HCl. HCl to water ratio: (a) 20 : 80; (b) 30 : 70; (c) 40 : 60; (d) 50 : 50; (e) 60 : 40; (f) 70 : 30 at 180 °C.

TiO₂ films were grown under four different types of conditions: (a) 1 h on FTO, (b) 3 h on FTO, (c) 3 h on ITO and (d) 3 h on glass substrates for the quantification of the detailed crystallite structure using X-ray diffraction (XRD), surface chemical analysis by X-ray photoelectron spectroscopy (XPS) and cell culture experiments to assess their biocompatibility.

2.3 Cell culture

MC3T3 and HS-5 cells were maintained in α-MEM with 10% fetal bovine serum (FBS) and 100 U per mL penicillin, and incubated at 37 °C in an atmosphere of 5% CO₂. Trypsin–EDTA was used to detach the cells. Three samples were used for in vitro cell adhesion, viability and proliferation assays and all the experiments were repeated three times. For statistical analysis, a pairwise comparison of inter-experiment variation was carried out. MS Excel software was used for statistical analysis. Standard deviation values were calculated separately for all the assays. The significance of the statistical analysis values was determined using the Student's t-test, where p-values <0.05 were considered significant. The cell culture experiments were performed on TiO₂ 3D-nanoflower structures grown on FTO, ITO and glass substrates under condition (iv) for 3 h. In addition, TiO₂ nanostructures grown at an early stage (1 h) on the FTO substrate under condition (iv) were selected to perform cell culture experiments and tissue culture plastic surface was used as the control.

2.4 Characterization

The crystallite structure of 1D- and 3D-TiO₂ nanostructures grown on different substrates was characterized from the XRD pattern obtained using a XRD-6000 (Japan) X-ray diffractometer in the diffraction angle range of 5°–80° with Cu-Kα radiation (λ = 1.54060 Å). Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) were performed using a JEM 2010F microscope. TEM images, selected area electron diffraction (SAED) patterns and HRTEM images were obtained using a TECNAI F20 Philips operated at 200 kV. The surface morphology of the samples was studied using field emission scanning electron microscopy (FESEM; S-4700, Hitachi). XPS spectra were obtained on a MultiLab200 with standard MgKα radiation to quantify the elemental composition and surface states of the TiO₂ nanostructures. All the spectra were obtained at a working pressure of 10⁻⁹ mbar. An XPS survey and high-resolution spectra of C1s, O1s, and Ti2p were collected. The different surface states were obtained in the high resolution Ti2p, O1s and C1s spectra of the samples by specifying line shape, relative sensitivity factor (RSF), peak position, full width at half maxima (FWHM), and area constraints.

2.5 In vitro assays

Cell adhesion and viability assays. These assays were performed by plating 3 × 10⁴ cells in an area of about 1 cm² TiO₂ nanostructures grown substrates placed in tissue culture grade plates. After 4 h, non-adherent cells were washed off and 100 μL of 2 mg mL⁻¹ MTT was added to the remaining cells in each condition, followed by incubation at 37 °C under 5% CO₂ for 4 h. After removing the culture media, the reaction was stopped by adding dimethylsulfoxide, and measurements were carried out at 595 nm using an ELISA reader. In the cell viability assay, an MTT assay was performed after 24 h of incubation, as described above, to calculate the percentage of cell viability.

Cell proliferation assay. The assay was performed by plating a similar number of cells for all conditions in triplicate followed by incubation for three different time periods, namely, 24, 48 and 72 h. After each time period, the cell number was calculated using the MTT assay described above.

3. Results and discussion

3.1 Surface morphology and crystallite structure characterization

Fig. 2 shows the FE-SEM images of the time dependent growth of TiO₂ nanostructures on FTO grown after 1 h and 3 h. The high resolution FE-SEM images of the TiO₂ nanostructures grown after 1 h showed two distinct regions, namely, 1D-nanorod and 3D-nanoflower nanostructures. The top view of the FE-SEM image of the TiO₂ nanostructures grown after 3 h showed a uniform coverage of 3D-nanoflowers. From these observations, it appears that 1D-nanorods of TiO₂ grow on the substrates as films on which the simultaneous growth of 3D-nanoflowers were initiated. The cross-sectional view of the FE-SEM images of nanostructures obtained after 1 h and 3 h of TiO₂ growth demonstrates the formation of 3D-nanoflowers on the vertically aligned 1D-nanorods of TiO₂. The surface of the TiO₂ nanostructures obtained after 1 h of growth had both 1D-nanorods and 3D-nanoflowers, whereas after 3 h of growth, the surfaces were completely covered by 3D-nanoflowers.

Fig. 2 FE-SEM images of rutile TiO₂ nanostructures grown on FTO substrates for two different times (namely, 1 h and 3 h) with a HCl to water ratio of 50 : 50 at 180 °C.

The effect of different types of substrates on the growth of the TiO₂ nanostructures as films was quantified by the FE-SEM images of the nanostructures grown on glass, ITO and FTO substrates after 3 h. In the FE-SEM images, a uniform distribution of 3D-nanoflowers of TiO₂ as films were seen for all the three different types of substrates, as shown in Fig. 3a–c. The higher resolution FE-SEM images of the 3D nano-flower structures show tips of nano-rods of about 300–500 nm on the surface, which are very tightly packed, as shown in Fig. 3d–f. The cross-sectional view of the FE-SEM image shows the formation of a stable interface of 3D nanoflower-like structures on these substrates.

Fig. 3 FE-SEM images of rutile TiO₂ nanostructures grown on glass (a), FTO (b) and ITO (c) substrates with a HCl to water ratio of 50 : 50 and reaction time of 3 h at 180 °C.

A TEM image of a tip of the TiO₂ 3D-nanoflowers on FTO substrates was obtained at different magnification, and the results are shown in Fig. 4. The presence of densely packed TiO₂ as nanorods was seen in TEM images. The diffraction pattern of TiO₂ was obtained and the lattice spacing (d) of TiO₂ was calculated. The results showed d₀₀₁ = 0.23 nm along the longitudinal axis direction pertaining to the d-spacing of the rutile TiO₂ (001) crystal planes.

Fig. 4 (A) FE-SEM image of TiO₂ nanostructures grown on FTO substrates with a HCl to water ratio of 50 : 50 at 180 °C for 3 h. TEM images (B and C) of the top-view of the TiO₂ nanostructures, as seen in the high resolution FE-SEM image (A). (D) Diffraction pattern of TiO₂ nanostructures grown under similar conditions.

Fig. 5A shows the XRD pattern of the TiO₂ nanostructures grown on FTO substrates after 1 h and 3 h. The FTO substrate shows several strong peaks for Sn at the (110), (002), (112), (202) and (113) planes in the XRD spectra (Fig. 5A(a)), which is in accordance with previously obtained spectra.³⁹ The XRD pattern of the FTO substrate and TiO₂ nanostructures as films grown on FTO after 1 h was similar. This may be observed due to the fact that the TiO₂ film does not grow considerably; thus, the TiO₂ content was not detected by XRD in the presence of a strong background of FTO substrate. TiO₂ nanostructures obtained at higher time points (at 3 h) showed XRD peaks of TiO₂ at 2 theta values of 27.62, 36.28, 41.42, 44.16, 54.62, 56.72, 63.02, and 69.28. These peaks were assigned the (hkl) values of (110), (101), (111), (210), (211), (220), (002) and (301), respectively. All the diffraction peaks were marked to the pure tetragonal rutile phase of TiO₂ (JCPDS no. 21-1276). The XRD patterns of the TiO₂ nanostructures grown on FTO, ITO and glass substrates are shown in Fig. 5B. The XRD peaks appear at 2 theta values of 27.62, 36.28, 41.42, 44.16, 54.62, 56.72, 63.02, and 69.28. The XRD pattern of TiO₂ nanostructures as films grown on the three substrates after 3 h seem to be identical. These peaks were assigned the (hkl) values of (110), (101), (111), (210), (211), (220), (002) and (301), respectively.

Fig. 5 (A) XRD pattern of TiO₂ crystallite growth on FTO with a HCl to water ratio of 50 : 50 at 180 °C for two different times (1 h and 3 h). Prominent diffraction peaks in the XRD pattern of FTO substrate are marked with ‘*’. (B) XRD pattern of TiO₂ crystallite growth on glass (a), FTO (b) and ITO (c) substrates with a HCl to water ratio of 50 : 50 at 180 °C for 3 h.

3.2 Characterization of the surface chemical functionalities

The XPS spectra of the TiO₂ nanostructure surfaces were obtained after 1 h and 3 h on FTO for the quantification of the surface elemental analysis. The %proportions of oxygen (O) to titanium (Ti) atoms were obtained from the wide scan XPS spectra, and the results are shown in Fig. 6. The O/Ti value was 3.1 for the TiO₂ nanostructure surfaces obtained after 1 h, which was reduced to 2.7 for the nanostructure obtained after 3 h. The reduction in O/Ti values after a longer reaction time may correspond to a decrease in the %proportion of oxygen atom on the surface. This observation also indicates that titania atoms are tightly packed on the surface, which was also confirmed by FE-SEM and TEM observations.

Fig. 6 Wide scan XPS spectra of TiO₂ nanostructure growth on FTO substrates with a HCl to water ratio of 50 : 50 at 180 °C for two different reaction times of (a) 1 h and (b) 3 h.

High resolution Ti2p and O1s XPS spectra are shown in Fig. 7A and C. The details for the peak fitting parameters for Ti2p and O1s are given in Fig. 7B and D.¹⁶ A small increase in the Ti⁴⁺ surface states of Ti2p was observed on the surface of TiO₂ obtained after 3 h. The relative increase in the proportion of Ti2p surface states indicates the relative decrease in the number of oxygen atoms surrounding the Ti atoms in these nanostructure assemblies. The relative proportion of the different oxidation states of oxygen in the O1s XPS spectra at the surface of TiO₂ were obtained after 1 h and 3 h, and the results are shown in Fig. 7. Oxygen atoms as O = (O²⁻) surface states were increased with an increasing reaction time. These observations are consistent with the atomic %proportion obtained from wide scan XPS spectra.

Fig. 7 (A) High resolution peak fitted Ti2p XPS spectra and (C) high resolution peak fitted O1s XPS spectra for TiO₂ nanosurfaces prepared with a HCl to water ratio of 50 : 50 at 180 °C for two different reaction times. (B) Peak fitting parameter for Ti2p high resolution XPS spectra and (D) peak fitting parameter for O1s high resolution XPS spectra. (a) and (b) represent 1 h and 3 h of TiO₂ nanostructure growth on FTO substrates, respectively.

The XPS spectra of the nanostructures grown as films on ITO, FTO and glass substrates were obtained to further quantify the chemical nature of the nanostructures. The wide scan XPS spectra of TiO₂ on non-conducting (glass) and conducting (ITO and FTO) substrates are shown in Fig. 8. Detailed elemental analysis and peak fitting for surface state quantification from C1s and Ti2p were carried out as described in our previous studies,²⁷ and the results are shown in Fig. 9 and 10. The higher resolution C1s XPS spectra of the TiO₂ film surface prepared on glass and ITO substrates was fitted with six peaks corresponding to different carbon environments including hydrocarbons (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 283 ± 0.2 eV. The TiO₂ film surface prepared on FTO coated glass substrates was fitted with five peaks corresponding to different carbon environments including hydrocarbons (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 and (C(=O)OX) at 289.2 ± 0.2 eV. The TiO₂ films on glass showed a spectra shift of 0.53 eV towards the lower binding energy side, which was adjusted to its original position. The bombardment of X-ray photons during XPS analysis caused a positive charge to accumulate on the non-conducting surface. This charge accumulation corresponds to an energy shift of 0.53 eV in the XPS spectra for the TiO₂ films grown on non-conducting substrates. There was no spectral shift observed for TiO₂ nanostructures grown on conducting (ITO and FTO) glass substrates.

Fig. 8 Wide scan XPS spectra of TiO₂ nanosurfaces prepared with a HCl to water ratio of 50 : 50 at 180 °C for 3 h on glass (a), FTO (b) and ITO (c) substrates.

Fig. 9 High resolution C1s peak fitted XPS spectra of TiO₂ nanosurfaces prepared with a HCl to water ratio of 50 : 50 at 180 °C for 3 h on glass (a), FTO (b) and ITO (c) substrates.

Fig. 10 High resolution Ti2p XPS spectra of the TiO₂ nanosurfaces prepared with a HCl to water ratio of 50 : 50 at 180 °C for 3 h on glass (a), FTO (b) and ITO (c) substrates.

Different titanium oxidation states were quantified in the high resolution Ti2p XPS spectra of the TiO₂ nanostructures grown on ITO, FTO and glass substrates. The spectra were fitted with four peaks including Ti³⁺2p_3/2 at 453.2 ± 0.4 eV, Ti⁴⁺2p_3/2 at 454.6 ± 0.2 eV, Ti³⁺2p_1/2 at 455.8 ± 0.4 eV and Ti⁴⁺2p_1/2 at 460.3 ± 0.2 eV. There was not much change in the surface states of Ti2p obtained for the TiO₂ nanostructures grown on glass and FTO substrates. TiO₂ nanostructures grown on an ITO substrate showed a significant increase in the Ti³⁺ surface state of Ti2p.

The relative variation in the %proportion of the different surface states of C1s and Ti2p with the different types of nanostructured film prepared on ITO, FTO and glass substrates are shown in Fig. 11. Mainly, five different types of carbon surface states, namely, C/C–H, C–C(=O)OX, C–OX, C=O and C(=O)OX, were observed in the TiO₂ films on all the three substrates. Herein, we are interested to see the relative variation for three active functional groups, namely, C–OX, C=O and C(=O)OX, present on the nanosurface obtained on ITO, FTO and glass substrates. The %proportion of hydroxyl functional groups on the surface of the TiO₂ nanostructures was significantly increased when the substrates were changed to glass, FTO and ITO for the growth of TiO₂. There was no significant change in the %proportion of carbon atoms as C=O in C1s at the surface of the TiO₂ nanostructures grown on the different types of substrates. The TiO₂ nanostructures grown on glass substrates showed the highest levels of carboxylic functional groups. There was not much difference in the %proportion of carboxylic functionality on the TiO₂ nanostructured surfaces on ITO and FTO substrates. An increase in the Ti³⁺ surface states of Ti2p was obtained from the TiO₂ nanostructures grown on ITO substrates. Moreover, there was not much difference in the Ti³⁺ surface states of Ti2p of TiO₂ nanostructures obtained on glass substrates and FTO coated conducting glass substrates.

Fig. 11 Relative variation in the %proportion of (A) COX in C1s, (B) CO in C1s, (C) C(=O)OX in C1s and (D) Ti³⁺ in the Ti2p XPS spectra of the TiO₂ nanosurfaces prepared with a HCl to water ratio of 50 : 50 at 180 °C for 3 h on glass (a), FTO (b) and ITO (c) substrates.

3.3 Biocompatibility of TiO₂ nanostructures

Cell adhesion, viability and proliferation assays using MC3T3 osteoblast and HS-5 cells were carried out on the TiO₂ nanostructure surfaces obtained on FTO after 1 h and 3 h, and the results are shown in Fig. 12. An increase in %cell adhesion on both the types of nanostructures was obtained as compared to the control (tissue culture plastic surfaces). A combination of 1D-nanorod and 3D-nanoflower-like structures of TiO₂ provide better entrapping of cells, and thus the highest percentage of cell adhesion was obtained. The cell viability did not show a significant variation on the two different types of nanostructures. The cell proliferation was also a little less on these structures as compared to the control. The percentage of cell adhesion of HS-5 cells was similar on both the types of TiO₂ nanostructures. The viability of HS-5 cells was increased on the TiO₂ nanostructure with 1D-nanorod/3D-nanoflowers as compared to the control condition. However, the cell proliferation was reduced on these two types of nanostructures.

Fig. 12 Cell adhesion assay carried out using MTT on TiO₂ nanostructures grown at (a) 1 h and (b) 3 h on FTO substrates and (c) tissue culture plastic surfaces. (A) The percentage of osteoblast cell adhesion. The significance values between the groups were determined: ◆ represents p < 0.05 for conditions (a) and (b) with respect to condition (c); ◇ represents p < 0.05 for condition (a) with respect to condition (b). (B) The percentage of osteoblast cell viability. ◆ represents p < 0.05 for condition (a) with respect to condition (c); ◇ represents p < 0.05 for condition (a) with respect to condition (b). (C) Osteoblast cell proliferation. ◆ represents p < 0.05 for conditions (a), (b) and (c) at different times of cell proliferation. ◇ represents p < 0.05 for condition (a) and condition (b) with respect to condition (c) at the same cell proliferation time points. (D) The percentage of HS-5 cell adhesion. ◆ represents p < 0.05 for condition (a) and condition (b) with respect to condition (c); ◇ represents p < 0.05 for condition (a) with respect to condition (b). (E) The percentage of HS-5 viability. ◆ represents p < 0.05 for condition (a) with respect to condition (c); ◇ represents p < 0.05 for condition (a) with respect to condition (b). (F) HS-5 cell proliferation. ◆ represents p < 0.05 for conditions (a), (b) and (c) at different cell proliferation times. ◇ represents p < 0.05 for conditions (a) and (b) with respect to condition (c) at the same cell proliferation time.

MC3T3 cell adhesion, viability and proliferation assays were carried out on TiO₂ nanostructure films grown on FTO, ITO and glass substrates, and the results are shown in Fig. 13. An overall increase in %cell adhesion on the TiO₂ nanostructures grown on all three different types of substrates was observed as compared to the tissue culture plastic surface. The highest %cell adhesion was obtained on 3D nanoflower structures of TiO₂ grown on a glass surface. There was a relative decrease in the %cell adhesion on TiO₂ 3D-nanoflowers grown on ITO and FTO as compared to the response observed on nanostructures grown on glass. A similar response was observed for %cell viability. Osteoblast cell proliferation on TiO₂ 3D nanoflowers grown on ITO and FTO was less as compared to the control, despite having a higher %cell adhesion. The XPS analysis of TiO₂ films on non-conducting glass substrates showed a spectral shift towards the lower binding energy side. This observation indicates that the TiO₂ films grown on non-conducting glass surfaces can retain the charge, whereas the TiO₂ films grown on conducting (ITO and FTO) substrates do not show charge accumulation. TiO₂ 3D-nanoflowers grown on the non-conducting glass substrate showed a higher cell proliferation rate as compared to the control conditions, whereas a decrease in cell proliferation was observed on TiO₂ 3D nanoflowers grown on ITO and FTO. Therefore, the charge nature of substrate plays an important role in determining the cellular response, despite the similar nanostructure of the same material.

Fig. 13 Osteoblast cell response were observed on (a) tissue culture plastic surfaces and 3D nanoflower-like TiO₂ nanostructures grown on (b) FTO, (c) ITO and (d) glass substrates at 3 h. (A) Adhesion%, the significance values between the groups were determined: ◆ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a); ◇ represents p < 0.05 for conditions (c) and (d) with respect to condition (b); ● represents p < 0.05 for condition (d) with respect to condition (c). (B) Viability%, ◆ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a); ◇ represents p < 0.05 for conditions (c) and (d) with respect to condition (b); ● represents p < 0.05 for condition (d) with respect to condition (c). (C) Cell proliferation, ◆ represents p < 0.05 for conditions (a), (b), (c) and (d) with respect to the change in cell proliferation time. ◇ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a) at the same cell proliferation time.

HS-5 cell adhesion, viability and proliferation assays were carried out on TiO₂ nanostructured film grown on FTO, ITO and glass substrates, and the results are shown in Fig. 14. The HS-5 cell adhesion assay showed a lower %cell adhesion on the TiO₂ nanostructures grown on FTO substrates as compared to nanostructures grown on ITO and glass substrates. TiO₂ nanostructures on non-conducting glass substrates showed the highest %cell adhesion and proliferation for HS-5 cells.

Fig. 14 HS-5 cell response were observed on (a) tissue culture plastic surfaces and 3D nanoflower-like TiO₂ nanostructures grown on (b) FTO, (c) ITO and (d) glass substrates at 3 h. (A) Adhesion%, the significance values between the groups were determined: ◆ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a); ◇ represents p < 0.05 for conditions (c) and (d) with respect to condition (b); ● represents p < 0.05 for condition (d) with respect to condition (c). (B) Viability%, ◆ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a); ◇ represents p < 0.05 for conditions (c) and (d) with respect to condition (b); ● represents p < 0.05 for condition (d) with respect to condition (c). (C) Cell proliferation, ◆ represents p < 0.05 for conditions (a), (b), (c) and (d) with respect to the change in cell proliferation time. ◇ represents p < 0.05 for conditions (b), (c) and (d) with respect to condition (a) at the same cell proliferation time.

Despite the fact that the similar nanostructure and crystallite structures of TiO₂ were grown on the conducting and non-conducting surfaces, the improved adhesion and proliferation response of both the cell types on the non-conducting surfaces confirms that the cells prefer a non-conducting surface. This may enable the cells to create and sustain a local surface charge environment, which is induced during the cell–surface interactions, for better cell growth on non-conducting surfaces; this is not possible on conducting surfaces.

4. Conclusions

The ordered growth of nanorod (1D)/nanoflower (3D) structures of TiO₂ was achieved on non-conducting and conducting (FTO and ITO coated) glass substrates. We used a surfactant-free method for the simultaneous growth of 1D/3D nanorod and nanoflower structures with better adhesion at the interface. TiO₂ nanostructures obtained at higher time points (after 3 h) on FTO, ITO and glass substrates exhibit a rutile phase. The findings show that the mixed 1D-nanorod/3D-nanoflower-like structures show better cell adhesion as compared to only 3D-nanoflower-like structures. Cell viability and proliferation rate was higher on TiO₂ 3D-nanoflowers grown on a non-conducting surface as compared to that grown on a conducting substrate.

Acknowledgements

KHP thanks the Basic Science Research Program for supporting this research through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT and Future Planning (2012R1A1A3010655). MD is thankful to the network project (Bioceramics) of Council of Scientific and Industrial Research (CSIR), India for financial support. We are thankful to Dr Vijay Kumar (NIMS, Hyderabad) for permitting the use of an ELISA reader.

References

1. A. Tiwari, M. L. Tursky, D. Mushahary, S. Wasnik, F. M. Collier, K. Suma, M. A. Kirkland and G. Pande, Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line, J. Tissue Eng. Regener. Med., 2013, 7(11), 871–883.
2. M. Goerner, B. Roecklein, B. Torok-Storb, S. Heimfeld and H. P. Kiem, Expansion and transduction of nonenriched human cord blood cells using HS-5 conditioned medium and FLT3-L, J. Hematother. Stem Cell Res., 2000, 9(5), 759–765.
3. C. N. R. Rao and A. K. Cheetham, Science and technology of nanomaterials: current status and future prospects, J. Mater. Chem., 2001, 11, 2887–2894.
4. S. T. Aruna and A. S. Mukasyan, Combustion synthesis and nanomaterials, Curr. Opin. Solid State Mater. Sci., 2008, 12(3–4), 44–50.
5. P. Yang, D. Zhao, D. I. Margolese, B. F. Chmelka and G. D. Stucky, Generalized syntheses of large-pore mesoporous metal oxides with semicrystalline frameworks, Nature, 1998, 396, 152–155.
6. J. Li, S. Tang, L. Lu and H. C. Zeng, Preparation of Nanocomposites of Metals, Metal Oxides, and Carbon Nanotubes via Self-Assembly, J. Am. Chem. Soc., 2007, 129(30), 9401–9409.
7. S. V. Agrawal, S. S. Reddy and M. Dhayal, Ultra small gold nanoparticles synthesis in aqueous solution and their application in fluorometric collagen estimation using bi-ligand functionalisation, RSC Adv., 2014, 4(35), 18250–18256.
8. H. S. Nalwa, Encyclopedia of nanoscience and nanotechnology, American Scientific Publisher, USA, 2011.
9. M. Dhayal, R. Kapoor, P. G. Sistla, C. Kant, R. R. Pandey, Govind, K. K. Saini and G. Pande, Ni doped TiO₂ thin films on borosilicate glass enhance in vitro growth and differentiation of osteoblasts, J. Biomed. Mater. Res., Part A, 2012, 100(5), 1168–1178.
10. A. J. Maira, K. L. Yeung, C. Y. Lee, P. L. Yue and C. K. Chan, Size effects in gas-phase photo-oxidation of trichloroethylene using nanometer-sized TiO₂ catalysts, J. Catal., 2000, 192, 185–196.
11. R. Carbone, I. Marangi, A. Zanardi, L. A. Giorgetti, E. Chierici, G. Berlanda, A. Podesta, F. Fiorentini, G. Bongiorno, P. Piseri, P. G. Pelicci and P. Milani, Biocompatibility of cluster-assembled nanostructured TiO₂ with primary and cancer cells, Biomaterials, 2006, 27, 3221–3229.
12. M. Dhayal, R. Kapoor, P. G. Sistla, R. R. Pandey, S. Kar, K. K. Saini and G. Pande, Strategies to prepare TiO₂ thin films, doped with transition metal ions, that exhibit specific physicochemical properties to support osteoblast cell adhesion and proliferation, Mater. Sci. Eng., C, 2014, 37, 99–107.
13. W. Zhao, C. Chen, W. Ma, J. Zhao and Z. Shuai, Efficient degradation of toxic organic pollutants with Ni₂O₃/TiO_(2−x)B_x under visible irradiation, J. Am. Chem. Soc., 2004, 126, 4782–4783.
14. M. Mazaheri, A. M. Zahedi, M. Haghighatzadeh and S. K. Sadrnezhaad, Sintering of titania nanoceramic: Densification and grain growth, Ceram. Int., 2009, 35, 685–691.
15. M. Dhayal, S. D. Sharma, C. Kant, K. K. Saini and S. C. Jain, Role of Ni doping in surface carbon removal and photo catalytic activity of nano-structured TiO₂ film, Surf. Sci., 2008, 602, 1149–1154.
16. J. Jun, J. H. Shin and M. Dhayal, Surface state of TiO₂ treated with low ion energy plasma, Appl. Surf. Sci., 2005, 252(10), 3871–3877.
17. S. I. Shah, W. Li, C. P. Huang, O. Jung and C. Ni, Study of Nd³⁺, Pd²⁺, Pt⁴⁺, and Fe³⁺ dopant effect on photoreactivity of TiO₂ nanoparticles, Proc. Natl. Acad. Sci. U. S. A., 2002, 99(2), 6482–6486.
18. Y. Cheng, H. Sun, W. Jin and N. Xu, Photocatalytic degradation of 4-chlorophenol with combustion synthesized TiO₂ under visible light irradiation, J. Chem. Eng., 2007, 128, 127–133.
19. P. X. Ma, Biomimetic materials for tissue engineering, Adv. Drug Delivery Rev., 2008, 60, 184–198.
20. X. Liu and C. Ding, Plasma sprayed wollastonite/TiO₂ composite coatings on titanium alloys, Biomaterials, 2002, 23, 4065–4077.
21. D. S. Kommireddy, S. M. Sriram, Y. M. Lvov and D. K. Mills, Stem cell attachment to layer-by-layer assembled TiO₂ nanoparticle thin films, Biomaterials, 2006, 27, 4296–4303.
22. H. Zhang, Y. Han, X. Liu, P. Liu, H. Yu, S. Zhang, X. Yao and H. Zhao, Anatase TiO₂ microspheres with exposed mirror-like plane {001} facets for high performance dye-sensitized solar cells (DSSCs), Chem. Commun., 2010, 46, 8395–8397.
23. R. R. Pandey, K. K. Saini and M. Dhayal, Using Nano-arrayed structures in sol–gel derived Mn²⁺ doped TiO₂ for high sensitivity urea biosensor, J. Biosens. Bioelectron., 2010, 1, 101.
24. Y. Li, Q. Sun, X. Sun and L. Dong, Synthesis of nanocoral structured TiO₂ and its photoelectrical performance in dye sensitized solar cells, ECS Trans., 2013, 53, 57–63.
25. L. L. Li, Y. J. Chen, H. P. Wu, N. S. Wang and E. W. G. Diau, Detachment and transfer of ordered TiO₂ nanotube arrays for front-illuminated dye-sensitized solar cells, Energy Environ. Sci., 2011, 4, 3420–3425.
26. J. Qu and C. Lai, One-dimensional nanostructures as photoanodes for dye-sensitized solar cells, J. Nanomater., 2013, 2013, 762730.
27. M. Adachi, Y. Murata, J. Takao, J. Jiu, M. Sakamoto and F. Wang, Highly efficient dye-densitized solar cells with a titania thin-film electrode composed of a network structure of single-crystal-like TiO₂ nanowires made by the “oriented attachment” mechanism, J. Am. Chem. Soc., 2004, 126, 14943–14949.
28. M. Dhayal, S. I. Cho, J. Y. Moon, S. J. Cho and A. Zykova, S180 cell growth on low ion energy plasma treated TiO₂ thin films, Appl. Surf. Sci., 2008, 254, 3331–3338.
29. K. N. Pandiyaraj, V. Selvarajan, M. Pavese, P. Falaras and D. Tsoukleris, Investigation on surface properties of TiO₂ films modified by DC glow discharge plasma, Curr. Appl. Phys., 2009, 9, 1032–1037.
30. I. Ichinose, H. Senzu and T. Kunitake, Stepwise adsorption of metal alkoxides on hydrolyzed surfaces: A surface sol–gel process, Chem. Lett., 1996, 25, 831–832.
31. B. D. Boyan, T. W. Hummert, D. D. Dean and Z. Schwartz, Role of material surfaces in regulating bone and cartilage cell response, Biomaterials, 1996, 17, 137–146.
32. R. R. Randey, M. Dhayal and K. K. Saini, Engineering electrochemical response of TiO₂-based enzymatic biosensors by aliovalent cation doping, Adv. Electrochem. Sci. Eng., 2013, 1(1), 62–66.
33. R. R. Randey, M. Dhayal and K. K. Saini, Modification of titanium surface states by ruthenium to enhance electrochemical response of TiO₂ thin films for biosensor application, Adv. Electrochem. Sci. Eng., 2013, 1(2), 55–61.
34. K. H. Park and M. Dhayal, Simultaneous growth of rutile TiO₂ as 1D/3D nanorod/nanoflower on FTO in one-step process enhances electrochemical response of photoanode in DSSC, Electrochem. Commun., 2014, 49, 47–50.
35. B. Vagaska, L. Bacakova, E. Filova and K. Balik, Physiol. Res., 2010, 59, 309–322.
36. W. H. Chen, Y. Tabata and Y. W. Tong, Curr. Pharm. Des., 2010, 16, 2388–2394.
37. C. J. Wilson, R. E. Clegg, D. I. Leavesley and M. J. Pearcy, Tissue Eng., Part A, 2005, 11, 1–18.
38. M. Trinadh, G. Kannan, T. Rajasekhar, A. V. S. Sainath and M. Dhayal, Synthesis of glycopolymers at various pendant spacer lengths of glucose moiety and their effects on adhesion, viability and proliferation of osteoblast cells, RSC Adv., 2014, 4(70), 37400–37410.
39. K. H. Park, R. R. Pandey, C. K. Hong, K. K. Saini and M. Dhayal, Electrochemical characterization of enzymatic organo-metallic coating of TiO₂ nanoparticles, Sens. Actuators, B, 2014, 196, 589–595.

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