Abstract
The objective of this study is to evaluate the biocompatibility of composite powder consisting of silica and titania (SiO2 –TiO2) for biomedical applications. The advancement of nanoscience and nanotechnology encourages researchers to actively participate in reinvention of existing materials with improved physical, chemical and biological properties. Hence, a composite/hybrid material has given birth of new materials with intriguing properties. In the present investigation, SiO2 –TiO2 composite powder was synthesised by sol‐gel method and the prepared nanocomposite was characterised for its phase purity, functional groups, surface topography by powder X‐ray diffraction (XRD), Fourier transform infrared spectroscopy (FT‐IR) and scanning electron microscopy. Furthermore, to understand the adverse effects of composite, biocompatibility test was analysed by cell culture method using MG63 osteoblast cell lines as a basic screening method. From the results, it was observed that typical Si–O–Ti peaks in FT‐IR confirms the formation of composite and the crystallinity of the composite powder was analysed by XRD analysis. Further in vitro biocompatibility and acridine orange results have indicated better biocompatibility at different concentrations on osteoblast cell lines. On the basis of these observations, we envision that the prepared silica–titania nanocomposite is an intriguing biomaterial for better biomedical applications.
Inspec keywords: bioceramics, nanocomposites, silicon compounds, titanium compounds, nanofabrication, sol‐gel processing, surface topography, X‐ray diffraction, Fourier transform infrared spectra, scanning electron microscopy, X‐ray chemical analysis, cellular biophysics, nanomedicine
Other keywords: MG63 osteoblast cell lines, orthopaedic applications, biomedical applications, nanoscience, nanotechnology, nanotoxicology, physical properties, chemical properties, biological properties, biological applications, biomaterial synthesis, composite‐hybrid materials, intriguing properties, sol‐gel method, surface properties, ceramic nanocomposite, phase purity, functional groups, surface topography, powder X‐ray diffraction, Fourier transform infrared spectroscopy, FT‐IR spectroscopy, scanning electron microscopy, energy dispersive X‐ray analysis, biocompatibility test, cell culture method, screening method, crystallinity, XRD, in vitro biocompatibility, acridine orange, silica‐titania nanocomposite powder, SiO2 ‐TiO2
1 Introduction
In orthopaedic and dental applications, implantable biomaterials including ceramics, metals and polymers have been widely used as structural and functional materials to correct abnormalities of affected bones as temporary devices (bone plates, screws), permanent devices (total hip replacement) due to its inherent physico‐chemical, mechanical and biological properties. Sol–gel synthesis is a process of hydrolysis, polycondensation of metal alkoxides (M(OR) n ) and has been received much attention for the past few decades due to its considerable promising as well as intriguing properties such as synthesis of high purity and high homogeneity at ambient conditions, ability to produce a well‐defined structures with complex shapes. In addition, it is a well‐known technique for the development of hybrid or composite materials with better bioactive, unique particle size distribution [1]. Hence, this technique was adopted for the synthesis of glasses, ceramics and nanocomposites with improved surface properties and pore structures.
Silicon dioxide has been increasingly used as a key material in various fields of biomedical due to its better bioactivity, biocompatibility. Titanium dioxide has good fatigue strength, corrosion resistant, better biocompatibility with osteoconductive and photocatalytic properties. Composite made of SiO2 and TiO2 has broad applications with chemical stability, mechanical and thermal stability with high corrosion resistant. Further sol–gel‐derived TiO2 and SiO2 hybrid coatings can acted as an alternative for the calcium phosphate‐based coatings with enhanced barrier like performance against corrosion [2, 3, 4].
Nanostructured hybrid materials can be prepared by sol–gel method and different physical, chemical and biological properties can be achieved by altering the process parameters with respect to individual components. Hence, suitable in vitro cell culture study such as cytotoxicity, proliferation, should be assessed for the composite materials. The cytotoxic nature of materials has been evaluated by using 3‐[4,5‐dimethylthiazol 2‐yl]‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfonyl)‐2H ‐etrazolium) (MTS), 2,3‐bis‐(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H ‐tetrazolium 5‐carboxanilide) (XTT), water soluble tetrazolium salts (WST‐8 and WST‐1) assays. Of which MTT is a basic technique for analysing both cytotoxicity and cell proliferation nature [5].
Hence, in this paper, we have reported the preparation of hybrid silica–titania composite by sol–gel method. FT‐IR, powder X‐ray diffraction (XRD) and scanning electron microscopy (SEM)‐energy dispersive X‐ray analysis (EDAX) have been used to evaluate the synthesised composite for the presence of functional groups, phase purity and morphological characteristics. The in vitro biocompatibility test (MTT assay) and acridine orange (AO) assay was carried out as a basic screening method to understand the cell proliferation/differentiation of osteoblast cell lines by cell culture method.
2 Materials and methods
2.1 Preparation of Si–Ti composite powder
The SiO2 –TiO2 composite powder was prepared by using tetra ethylortho silicate (TEOS, Aldrich, 98%) and titanium (IV) isopropoxide (Aldrich, 98%), as precursors. 70% silica, 30% titania precursor solutions were mixed to produce composite powder. SiO2 solution was prepared by dissolving TEOS in double‐distilled water. The resultant mixture's pH was adjusted with conc. nitric acid (sd fine‐chem) and stirred for 2 h. TiO2 solution was prepared by mixing titanium (IV) isopropoxide, acetic acid (sd fine‐chem), double‐distilled water at a molar ratio of 1:30:300 under constant stirring conditions for the duration of 2 h. The resultant SiO2, TiO2 solution was transferred to round bottom flask fitted with reflux condenser and stirred vigorously for 2 h. The resultant solution was refluxed in an oil bath at a controlled temperature of 90°C for 12 h with constant stirring. Further, the solution was evaporated in a water bath to obtain solid residue and was dried in an oven for 3 h at 100°C. Finally, the composite was sintered at different temperatures such as 600, 900 and 1000°C for 2 h at the heating rate of 1°C/min.
2.2 Cell viability and proliferation (MTT mitochondrial activity)
The biocompatibility nature of SiO2 –TiO2 composite powder was assessed for its cell viability and proliferation on MG63 cells, an human osteosarcoma cell lines by using 3‐[4,5‐dimethylthiazol 2‐yl]‐2,5 diphenyltetrazolium bromide (MTT) colorimetric assay. The high‐throughput screening MTT colorimetric assay is a simple method which converts soluble tetrazolium ring (yellow) to insoluble blue formazan crystals by mitochondrial succinate dehydrogenase. The resulting blue formazan from the reaction is directly proportional to the number of viable cells participated in the reaction. The colour development produced by viable cells quantified by spectrophotometrically at 570 nm (correction wavelength 630 nm) in an enzyme‐linked immunosorbent assay reader and its relative intensities are plotted to calculate the percentage of cell viability and proliferation.
The prepared composite powder was carefully made into solution without affecting the nature of cell lines (survival) by dissolving in 10% dimethyl sulfoxide (DMSO) and the final concentration of DMSO should not exceed >0.5%. The selected MG63 cell lines plated as monolayer in 96‐well format with a concentration of 1 × 105 cells/well and incubated for 24 h to produce 80% of confluent of cells. After incubation, the media was aspirated twice with 100 µl serum‐free medium for removing non‐adherent cells and starved for 1 h at 37°C. After starvation, the test compound with different concentrations (6, 12, 25, 50 and 100 µg/mL) are added and incubated for 24 h, 48 h and 72 h. At the end of incubation period, the sample was aspirated with the mixture containing MTT (0.5 mg/mL), and further incubated for 4 h at 37°C. The medium and MTT solution was removed by washing with phosphate‐buffered saline at the pH of 7.4. The insoluble formazan crystals were solubilised by adding 100 µL of DMSO solution by pipetting up and down for complete dissolution of particles. Further, the optical density was measured at 570 nm by plate reading spectrophotometer (Bio‐Rad 680) and the cell viability and proliferation was determined by using Graph Pad Prism 5 software. The significant difference between the control and treated cells was statistically determined by calculating its mean and standard deviation with ANOVA analysis.
2.3 AO staining assay
To determine the toxicity nature of prepared composite materials, AO assay was carried out. The typical staining of both viable and dead cells was evaluated by fluorescence emission spectra for three kinds of cells such as normal cells (intact nuclei with green fluorescence), early apoptotic cells (visible with bright green and light orange patches) and late apoptotic cell (orange to red patches). The cell lines were seeded in a 24‐well plate and incubated at 37°C for 24 h. After incubation, the medium was replaced with 100 µL medium containing different doses of test compound (6, 12, 25, 50 and 100 µg/mL) and incubated for 24 h. Cells were washed with PBS buffer (pH 7.4) and treated with 100 μL/mg AO in distilled water, followed by immediate examination and viewed under Olympus inverted fluorescence microscope (Ti‐Eclipse). Untreated cells should be used as a control and a minimum of 100 cells should be counted from each sample.
2.4 Characterisation techniques
The characteristic functional groups formed in synthesised SiO2 –TiO2 composite powder was characterised by Fourier transform infrared spectroscopy (FT‐IR) in the range of 400–4000 cm−1 using an SHIMADZU model 8300 spectrophotometer by using KBr pellet technique (0.1 weight%). The XRD pattern was recorded in an X‐ray diffractometer (D8 model, BRUKER, Germany) with a step size of 0.02° and a scan rate of 1°/min with Cu Kα radiation (λ = 1.54056 Å) to determine the phases. The instrument ZEISS SEM equipped with X‐ray microanalysis was used for the determination of surface topography and elemental analysis of the prepared composite powder.
3 Results and discussions
3.1 FT‐IR spectral analysis
The FT‐IR spectra of synthesised sample before and after heat treatment (Fig. 1) have shown distinguished vibrational bands for Si–O–Ti, Si–O–Si at 948 cm−1 and 1058 cm−1, respectively. The raw sample consists of various bands such as 439, 453, 561, 785 and 767 cm−1, which corresponds to the existence of mixture of SiO2 and TiO2 phases in the composite powder. On sintering, the Si–O–Si bands at 1058 and 785 cm−1 are found to be minimised and it may be due to the well adherence of titania phase on silica and a band at 453 cm−1 corresponds to Si–O–Si bending vibrations. Furthermore, the presence of Si–O–Si indicates the hydrolysis of triethoxy group to Si–OH and condensation of this to Si–O–Si form. The presence of band at 767 cm−1 corresponds to symmetrical stretching vibrations of Ti–O bonds of TiO4 tetrahedral system. The band at 948 cm−1 indicates the possibility of homogeneous network of Si–O–Ti bonds. In addition, the vibration band of Si–O–Ti bond and the condensed TiO4 groups appeared in the typical range of 866–954 cm−1 and the results observed from this study resembles with the previous study [6, 7]. The broad band at 3000–3600 cm−1 reveals the fundamental stretching vibration of hydroxyl groups in the composite powder due to the presence of adsorbed water. After sintering, the composite has sharp changes in peak intensity due to the possible removal of adsorbed water and increase in crystallinity.
Fig. 1.
FT‐IR spectrum of Si–Ti composite powder before and after sintering at different temperatures for 2 h
3.2 Characterisation by XRD analysis
The formation of phases in the raw and sintered SiO2 –TiO2 composite at different temperatures is shown in Figs. 2 A and B. Fig. 2 A represents that the synthesised powder sample (raw) as amorphous with the existence of silica hump between 20° and 25°. Fig. 2 B shows the composite sintered at 600°C with the presence of metastable anatase TiO2 peaks at 25.38°, 37.81°, 47.89°, 54.6° and 62.4°. All the peaks are corresponds to the anatase phase of TiO2 with typical broad peak at 2θ = 25.38°, which indicated the characteristic anatase phase of TiO2 [8, 9]. Furthermore, the presence of narrow high diffraction lines at 25.38° (101) of 900 and 1000°C predicts existence of Si–O–Ti and with increase in sintering temperature, the thermal stability of titania was found to be enhanced with the retention of anatase phase of TiO2 [10, 11, 12]. The crystallite size of the composite raw and 1000°C for 2 h shown nanometer in size such as 5 and 13 nm, respectively.
Fig. 2.
XRD pattern of Si–Ti composite powder
(A) Raw (unsintered), (B) Sintered at different temperatures for 2 h
3.3 SEM‐EDAX
Fig.3 shows the SEM‐EDAX images of Si–Ti composite powder sintered at 1000°C for 2 h.
The SEM images clearly predict the irregular morphology with agglomeration due to mixture of oxides. The elemental analysis confirms the presence of silicon, oxygen and titania in the prepared sample with two peaks around 0.2 and 4.5 keV of Ti, 0.5 keV of ‘O’ radical and distinct Si peak around 1.75 KeV.
3.4 Cell viability and proliferation (MTT mitochondrial activity)
To understand the cytotoxicity nature of SiO2 –TiO2 composite, the cell viability/proliferation study was carried out using MG63 cell lines by MTT assay. Various concentrations of 6, 12, 25, 50 and 100 µg/mL was chosen to study the cytotoxicity nature, for the duration of 24, 48 and 72 h. Mean and standard deviation of the study are tabulated in Tables 1 and 2. Results of the study showed that the mean of the MG63 cell lines at 24 h of exposure having higher concentration have shown vitality as 77% compared with 48 and 72 h. Further, the cell vitality for all the concentration at various time period has shown increase in proliferation of cells with significance value of (p < 0.1). ‘Figs. 4 A, C and E show the cell viability, Figs. 4 B, D and F show the proliferation, with different concentrations of powder sample sintered at 1000°C, for the period of 24, 48 and 72 h’.
Table 1.
Mean and standard deviation of cell viability study for the Si–Ti composite powder sintered at 1000°C for 24, 48 and 72 h
| Concentration(µg/mL) | 24 h | 48 h | 72 h | |||
|---|---|---|---|---|---|---|
| Mean | Standard deviation | Mean | Standard deviation | Mean | Standard deviation | |
| 6 | 100.330 | 2.516 | 101.803 | 5.671 | 96.933 | 11.042 |
| 12 | 92.666 | 1.527 | 101.786 | 16.219 | 89.703 | 4.977 |
| 25 | 92.666 | 3.055 | 98.893 | 5.229 | 84.993 | 7.900 |
| 50 | 92.000 | 6.557 | 94.216 | 7.899 | 80.566 | 4.167 |
| 100 | 77.662 | 4.041 | 90.213 | 6.409 | 74.521 | 14.298 |
Table 2.
Mean and standard deviation of cell proliferation study for the Si–Ti composite powder sintered at 1000°C for 24, 48 and 72 h
| Concentration (µg/mL) | 24 h | 48 h | 72 h | |||
|---|---|---|---|---|---|---|
| Mean | Standard deviation | Mean | Standard deviation | Mean | Standard deviation | |
| 6 | 2.000 | 1.000 | 1.894 | 1.392 | 9.103 | 2.539 |
| 12 | 7.330 | 1.527 | 11.414 | 3.554 | 10.038 | 4.537 |
| 25 | 10.000 | 2.000 | 8.943 | 1.591 | 14.767 | 7.816 |
| 50 | 11.000 | 3.605 | 9.214 | 1.716 | 19.380 | 4.131 |
| 100 | 23.000 | 3.000 | 9.513 | 6.088 | 25.393 | 14.104 |
Fig. 4.
Assessment of biocompatibility evaluation of Si–Ti powder on MG63 osteoblast cell lines
(A, C, E) Cell viability, (B, D, F) Proliferation, with different concentrations of powder sample sintered at 1000°C, for the period of 24, 48 and 72 h. (Data are expressed as mean ± SE from three independent experiments.)
Fig. 3.
SEM‐EDAX images of Si–Ti composite powder sintered at 1000°C for 2 h (picture with different magnification)
The cells are found to be grown on the surface of composite. Further, with the help of phase contrast microscopy, the MTT results are substantiated by observing the morphological characteristics of active and dead cells. The images for cell viability and proliferation have clearly shown that the prepared material does not have any toxicological effect and the obtained images are shown in Fig. 5. Aaritalo et al. [13] have investigated the cell adhesion behaviour of SiO2 –TiO2 composite and found that the adhesion was found to be enhanced with addition of SiO2 in the composite consisting of SiO2 and TiO2. Hence, the SiO2 70% and TiO2 30% is the best composition for prolonged osteoblast activity. Additionally, we have carried out coating on Ti–6Al–4V by electrophoretic deposition method. We found that the composite consists of 70:30 results in the formation of better adherent coating and the composite consists of 50:50 have shown accumulation of particles instead of uniform deposition. Hence, the composite consists of 70:30 have been selected for the biocompatibility study.
Fig. 5.
Phase contrast images of biocompatibility study for the Si–Ti composite powder sintered at 1000°C for 24, 48 and 72 h (20× objective).
Notes: (A) Control, (B) 6 μg/mL, (C) 12 μg/mL, (D) 25 μg/mL, (E) 50 μg/mL, (F) 100 μg/mL
3.5 AO staining assay
The AO florescence microscopy staining assay has been used qualitatively to understand the morphological alterations, cell damage and attachment that could be attributed to apoptotic mechanism. The prepared composite with various concentrations was treated to predict the possible damage on MG63 cell lines and is shown in Fig. 6.
Fig. 6.
Fluorescence microscopic evaluation of Si–Ti composite powder after biocompatibility study (20× objective)
Notes: (A) Control, (B) 6 μg/mL, (C) 12 μg/mL, (D) 25 μg/mL, (E) 50 μg/mL mL, (F) 100 μg/mL
The observed images after treatment have clearly shown the presence of active cells (green dots in Fig. 6) and free from any apoptotic and necrotic cells (dead cells). The results clearly revealed that the prepared composite has better biocompatibility nature on human osteosarcoma (MG63) cell lines. Furthermore, the suppression of phase transformation of Meta stable anatase to rutile at 1000°C indicates the stabilisation of anatase TiO2 and decrease in crystallite particle size. The mesoporous structure of composite effectively allows the cellular mediator of neuroinflammatory microglial cells inside the powder pellet and produces structural integrity with high biocompatibility [14]. In a previous study, poorly crystalline surfaces of CaP releases enhanced Ca than crystallined CaP, which leads to enhanced osteoblast adhesion, proliferation and Alkaline Phosphatase activity. The less crystallised composite easily and slowly releases silica with suitable surface properties than pure silica coating produces enhanced cell viability and proliferation and it is further proof for silica releasing materials enhanced cell growth and proliferation [15]. Bone marrow stem cells cultured on 100% anatase titania of previous study show a promising growth and proliferation on nanoporous titania film justifies the suitability of our composites for better biocompatibility with cell lines [16]. Further, in a previous study the increase in surface area of composite(425 m2 /g) increases the number of active sites and produces more reacting sites than silica surface area (175–225 m2 /g) and titania (45–55 m2 /g), which may be the cause for better cell viability and proliferation for the prepared composite [17].
4 Conclusions
In summary, pure Si–Ti nanocomposite was successfully synthesised by sol–gel technique with the aim to strengthen the life‐time of biomedical devices such as orthopaedic and dental prostheses materials for proper integration with the surrounding tissue. The result from the current study implies that the FT‐IR and powder XRD technique confirms the characteristic functional groups with phase pure anatase in the silica–titania network at the sintering temperature of 1000°C for 2 h. At this sintering temperature, composite has irregular agglomerated particles with existence of two phases which was further confirmed by SEM‐EDAX analysis. The biocompatibility study by MTT assay revealed the pronounced effect of cell viability and proliferation with the existence of more number of active cells and is further proved by AO assay. This study clearly predicts more viability nature of cell lines by producing green fluorescence with none of apoptotic cells.
5 Acknowledgments
The authors thank VIT University, Vellore for providing all required facilities and also one of the author Dr U. Vijayalakshmi highly acknowledge DST, New Delhi, India (SB/FT/CS‐091/2012) for financial support.
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