Materials Science and Engineering C 38 (2014) 252–262

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TiO2–graphene nanocomposites for enhanced osteocalcin induction Kavitha Kandiah, Prabhu Muthusamy, Selvam Mohan, Rajendran Venkatachalam ⁎ Centre for Nano Science and Technology, K.S. Rangasamy College of Technology, Tiruchengode 637 215, Tamil Nadu, India

a r t i c l e

i n f o

Article history: Received 20 September 2013 Received in revised form 11 January 2014 Accepted 7 February 2014 Available online 16 February 2014 Keywords: Titania–graphene Cytotoxicity Biocompatibility Osteocalcin MG-63 cell line Surface area

a b s t r a c t Bone defects and damages are common these days, which increases the usage of biomaterial for humans. To prepare a potential biomaterial, we synthesised a series of titania–graphene nanocomposites (TGS) (2:x (0.25, 0.5, 1.0, 2.0, and 4.0 g)) using in situ sol–gel method. The obtained structural results show that the prepared TGS nanocomposites are an irregular sheet with spherical TiO2 intercalated morphology. The SSA of the nanocomposites ranging from 167.98 to 234.56 m2 g−1 with mesoporosity and swelling tendency ranging from 11.55 to 26.13% leads to an enhancement in human cell attachment as well as avoids the migration and agglomeration of the nanoparticles in the body. Further, the biological analysis in simulated body fluid and human cell lines (AGS and MG-63) collectively reveals that the TG2 (2:2) and TG4 (2:4) samples are found to be more favourable materials for biomimic bone action among the prepared TGS nanocomposites. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The conventional autografting and allografting treatments need to overcome the limitations such as donor site shortage and immunogenicity [1]. Thus, tissue engineering offers potential biomaterials to restore and retain the natural bone. In the field of biomedicine, biomaterials with combined properties, such as biomimetic, bioactive, and biocompatible properties, have attracted attention in the recent years [2,3]. To overcome the above limitations, researchers are focused on analysing potential nanocomposite materials rather than a single nanomaterial [4]. Nano-titania (TiO2) is emerging as potential material for biomedical applications because of its unique physicochemical properties. NanoTiO2 provides the site for deposition of calcium and phosphate, thereby accelerating formation of bone-like apatite layer [5,6]. In addition, the anatase phase of TiO2 is more efficient in nucleation and growth of hydroxyapatite (HAp) layer than any other crystalline phase of TiO2 presumably because of better lattice match with HAp phase [7]. However, there are some problems related with migration and accumulation in cell organelles [8]. Currently, graphene sheets (GS) are also considered as a new potential material in biology; they have incredible properties such as high surface area, mechanical stiffness, and bacterial inhibition [9]. Even though, the addition of graphene oxides reveals promising properties in biomedical applications, the reduced graphene oxide (graphene) is a new potential material with enhanced mechanical property, bioactive nature and cytocompatibility via promoting better cell adherence to the ⁎ Corresponding author. Tel.: +91 4288 274741 4; fax: +91 4288 274880. E-mail address: [email protected] (R. Venkatachalam).

http://dx.doi.org/10.1016/j.msec.2014.02.010 0928-4931/© 2014 Elsevier B.V. All rights reserved.

human osteoblasts (bone forming) cells. This unique reinforcing behaviour with a large surface area of graphene attracts us more towards the biomedical to produce highly desirable biomaterial [10–12]. Moreover, GS can induce osteocytes on stem cells and have osteoconductive/inductive properties, which make them suitable for bone regeneration therapy [13]. Among different methods to synthesise graphene, for example electrochemical synthesis [14] and ultrasound-assisted synthesis [15], chemical oxidation continues to be the most popular method owing to its simplicity, viability, and scalability [16]. Oxidation of graphite facilitates more interaction with the tissues. The growth of nano-Ti on nano–graphene is an important approach to produce nanohybrids because controlled nucleation and growth affords optimal chemical reaction and bonding between TiO2 and nano– graphene sheets. It results in a very strong electrical and mechanical coupling within the hybrid [17]. Several methods that proposed to form the composites are electrochemical deposition [14], sol–gel process [18], two–step solution phase synthesis [16], hydrothermal preparation [19], and self–assembled composite film [20]. The in situ method is used as an effective method to grow nanoTiO2 crystals on graphene sheets. The biological properties (bioactivity in SBF, cell reactivity) depend on the material morphology, size, components, concentration and other physico-chemical properties such as surface to volume ratio, particle size, swelling and degradation [2,3,21–26]. When the nanocomposite is used as biomaterial for surgical and other biomedical applications, it facilitates the enhancement of cell attachment and thereby improves the bioactivity and biocompatibility than that of the bulk. In this study, an attempt has been made to prepare a series of TiO2–GS (TGS) nanocomposites by in situ sol–gel method. Furthermore, the prepared samples are characterised comprehensively to explore their physicochemical properties. To search the optimal composite material

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with properties to promote desired bone tissue regeneration, we subjected the prepared composites to biological studies such as swelling, bioactivity, and biocompatibility, respectively, for phosphate–buffered saline (PBS), simulated body fluid (SBF), and cell lines (human gastric adenocarcinoma (AGS) and osteoblast-like MG-63). In addition, osteocalcin estimation is carried out to explore the new bone cell synthesis. Further, the properties of TGS nanocomposites are correlated with pure nano-Ti structures in light of the biocompatibility and optimisation.

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Then, the sonicated solution was stirred continuously for 4 h at 310 K. The obtained precipitate was washed individually with double–distilled water followed by ethanol. The washed precipitates were dried in hotair oven at 393 K to evaporate the solvents. Then the dried powders were sintered at 673 K for 1 h and then ground to reduce agglomeration. The schematic representation of TGS nanocomposite is shown in Fig. 1. Using the above procedure, we prepared TGS nanocomposites with different concentrations, namely, 0, 0.25, 0.50, 1, 2, and 4 g L−1 (hereafter termed as TG0, TG.25, TG.50, TG1, TG2, and TG4, respectively).

2. Materials and method 2.3. Characterisation 2.1. Materials In this study, titanium isopropoxide (97%, Sigma-Aldrich, USA), isopropyl alcohol (99%, Merck), acetyl acetone (98%, Merck), graphite (98%, Loba Chemie), sodium nitrate (99%, HiMedia), potassium permanganate (99%, Merck), sulfuric acid (98.08%, Merck), hydrogen peroxide (30%, Merck), and hydrazine hydrate (80%, Loba Chemie) were used to synthesise the nanocomposite. Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture (DMEM/F-12HAM, catalogue no. 56498), RPMI-1640 medium (catalogue no.:R8758) and MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, catalogue no. 070M61471) kit were used as received from Sigma-Aldrich (USA).

2.3.1. X-ray diffraction The prepared TGS nanocomposites were characterised by X-ray powder diffractometer (XRD; X'Pert PRO; PANalytical, Almelo, the Netherlands) using Cu Kα as radiation source with a wavelength of λ = 0.15406 Å. The samples were scanned at an angle of 2θ ranging from 10° to 80° with an increment of 0.05° at a scanning rate of 5° per minute. The peak positions and the respective intensities of the powder pattern were identified in comparison with the reference powder diffraction data. The average crystallite size was determined using Scherrer formula [24]:

D¼

2.2. Nanocomposites preparation A series of TGS nanocomposites were synthesised through in situ sol– gel method. The graphene oxide was synthesised using standard Hummers method [19,17]. Then it was reduced as graphene by adding hydrazine hydrate [16]. The different concentrations of prepared graphene, namely, 0.25, 0.50, 1, 2, and 4 g L−1, were dispersed in double–distilled water under sonication and kept ready to form nanocomposite. Titanium isopropoxide was diluted by isopropyl alcohol with hydrolysis controller (acetyl acetone) in the molar ratio of 1:0.7:4, respectively [21]. The prepared graphene dispersion at different concentrations described previously was drop-wise added under sonication for 1 h.

k λ β cosθ

ðiÞ

where k is the Scherrer constant, λ the wavelength of Cu Kα radiation (1.5406 Å), β the full width at half maximum (FWHM), and θ the diffraction angle of the sample. 2.3.2. Fourier transform infrared spectrometer The characteristic peaks of the synthesised TGS nanocomposites before and after SBF study were measured with a Fourier transform infrared spectrometer (FTIR; Spectrum 100; PerkinElmer, USA) in the wavelength range of 4000–400 cm−1. The pellets for the FTIR study were prepared by mixing nanosamples with potassium bromide (99%,

Fig. 1. The schematic representation of the formation of TiO2–graphene nanocomposites.

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Merck) at a ratio of 2:200 (w/w) and compressing them with a hydraulic pressure pellet maker. 2.3.3. Energy-dispersive spectroscopy Qualitative and quantitative elemental analyses of TGS composites were performed using X-ray fluorescence spectrometry (XRF; EDX-720; Shimadzu, Japan) and an energy-dispersive X-ray spectrometer (EDAX). The elemental composition of synthesised nanocomposites was analysed from the observed EDAX pattern. The deposition of calcium and phosphate on the surface of the samples was analysed both qualitatively and quantitatively before and after the bioactivity study using XRF. The occurrences of the graphene and TiO2 in the prepared nanocomposites are analysed through the Raman spectra (RENISHAW-M005-141) with the laser frequency of 514 nm. 2.3.4. Electron microscopic analysis The scanning electron microscopy (SEM; JSM-6390LV, JEOL, Japan) with an accelerating voltage of 25 kV was used to observe the surface morphology of TGS nanocomposites. Transmission electron microscopy (TEM; CM200, Philips, USA) was used to measure the primary particle size of the sample. TEM images are obtained using transmitted electron technique that produces magnification details up to 1,000,000× with a resolution better than 10 Å. The obtained electron diffraction pattern of the selected area of the samples was inserted with the TEM images to explore the crystalline nature and lattice arrangements. 2.3.5. Specific surface area The specific surface area (SSA) of the prepared nanosamples was measured using the Brunauer–Emmett–Teller analyser (Autosorb AS-1MP; Quantachrome, USA). The pore size distribution, average pore diameter, and total pore volume of the prepared nanocomposites were calculated using the Brunauer–Joyner–Halenda method [25]. The samples were degassed under vacuum at 290 °C with liquid nitrogen (− 196 °C) for 3 h to remove the moisture. Liquid nitrogen was used to avoid any thermally induced changes on the surface of the particles. 2.4. In vitro analysis 2.4.1. Swelling study The swelling behaviour of the prepared TGS nanocomposites was tested experimentally in PBS (pH 7.4) at 310 K and ultrapure water [26]. The prepared samples (150 mg) were pelletised using a hydraulic pressure pellet maker. The initial weight was measured as W0. Then, the pellet was immersed separately in a 50 mL PBS-containing bottle and

ultrapure water. This setup was incubated at 310 ± 1 K for 7 days [23]. After the incubation period, the pellets were carefully taken out from both PBS and ultrapure water using filter paper and the wet weight of the pellets was measured as Ww. The swelling ratio of the TGS nanocomposites was calculated using the following formula [23]: Swelling ratio

W ð% Þ ¼

W w −W 0  100 : W0

ðiiÞ

2.4.2. Bioactivity study The assessment of in vitro test for the bioactivity of the prepared TGS nanosamples was carried out in 1.5 SBF (1.5 times higher concentration than human plasma). The initial formation of HAp takes place with the SBF environment while the initiation of biomineralisation and nucleation as well as growth of the apatite layer (HAp) is developed in the environment of saturated SBF (1.5 SBF), which mimic the natural mechanism of human body [27,28]. In the view of the above reason, the present study uses the 1.5 SBF directly for the bioactivity study [29,30]. The 1.5 SBF was prepared with analytical grade of chemicals (Sigma-Aldrich and HiMedia) using standard procedures as reported earlier [28–30]. Prepared samples were immersed in 1.5 SBF and incubated for 21 days at 310 ± 1 K in a circulating water bath. pH and conductivity probes of 5-Star (Thermo Orion, USA) were used to record the ion exchange between the SBF and the prepared sample. After incubation, the weight loss was calculated using dry weight of the pellets. The formation of HAp layer on the surface of the pellets was analysed by XRD, FTIR, and XRF studies. The bioactivity study was carried out as triplicates. 2.4.3. Antimicrobial activity Bone-infecting gram–positive (Staphylococcus aureus, ATCC no. 25923) and gram–negative (Escherichia coli, ATCC no. 25922) pathogens were collected from the Microbial Type Culture Collection and Gene Bank (India). The collected pathogens were used to study antibacterial effect of the prepared nanocomposites by disc diffusion method [31]. The slants of abovementioned bacteria were inoculated in 2 mL sterile Luria–Bertani (LB) broth overnight, for diluents the bacterial strains. After incubation, a loop full of culture was suspended to 100 mL LB broth and incubated at 310 K for 3–4 h. Freshly grown cell suspension (0.1 mL) was uniformly swabbed onto a nutrient agar plate. Then, 50 mg of each prepared nanosample was loaded on sterile disc, placed on the agar plate, and incubated at 310 K for 24 h to observe the antibacterial activity.

Fig. 2. X-ray diffraction pattern of a series of TGS nanocomposite for bioactivity. a) Before in vitro study; b) after in vitro study.

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Table 1 Composition and physico-chemical properties of prepared TiO2–graphene nanocomposites. Sample name

TG0 TG.25 TG.5 TG1 TG2 TG4

Concentration of TiO2:graphene (g L−1)

Crystallite size (nm)

Particle size (nm)

Surface area (m2 g−1)

Total pore volume (cm3 g−1)

Average pore diameter (nm)

Elemental composition — at.% Ti%

O%

C%

2:0 2:0.25 2:0.5 2:1 2:2 2:4

3.60 4.18 3.52 3.30 3.13 3.68

10.58 11.47 9.21 4.09 8.19 8.86

208.41 167.98 136.30 186.52 216.04 234.56

0.095 0.082 0.069 0.093 0.104 0.117

1.262 1.963 2.026 1.996 1.932 2.000

52.71 43.60 30.00 22.70 15.05 12.87

42.62 36.10 43.50 35.67 31.79 23.62

5.59 19.94 26.50 42.625 52.16 63.51

2.4.4. Cell line study The human AGS cell line is a suitable primary in vitro model to explore the toxicity of the nanoparticles. In addition, osteoblast-like MG-63 cell line was used to estimate the biocompatibility and bone-forming abilities [32]. AGS cell line (ATCC-1739) and MG-63 cell lines were routinely cultured in, respectively, DMEM/F-12HAM (1:1) and RPMI-1640 medium containing 10% foetal bovine serum, sodium pyruvate, sodium bicarbonate, non-essential amino acids, 2 mM glutamine, 100 μg mL−1 penicillin, and 100 μg mL−1 streptomycin at 310 K with 5% CO2. The mitochondrial damage of the nanoparticle-treated cells was estimated using MTT assay [21]. Before the use of MTT assay in AGS and MG-63, the cells were passaged freshly with respective medium. When the cells achieved 80%–90% confluency, they were seeded into a 96-well microtiter plate at a density of 1 × 103 cells per well. The cells were then allowed to adhere to the plate for 24 h. Filter-sterilised TGS nanocomposites (TG.25, TG.5, TG1, TG2, and TG4) at three different concentrations (20, 5, and 1 μg mL−1) were loaded in different wells and then incubated separately in both cell lines for 48 h at 310 K. 80 μg mL−1. MTT solution was added to each well and incubated for 4 h. At the end of incubation, 1 mL dimethyl sulfoxide was added to reduce the formazan crystal into pink. Then the optical density (OD) of the pink solution was read at 570 nm. The percentage of cell viability was calculated using the following formula:

kit (Biological Technologies Inc., USA) were used to estimate the level of osteocalcin [33]. The level of OC protein in cell culture supernatants was collected and was anticipated at the absorbance at 450 nm wavelength. The percentage of osteocalcin production was calculated using the following formula: Percentage of osteocalcin production OD of the nanoparticle−treated cells  100: ¼ OD of the control cells

ðivÞ

The level of osteocalcin was expressed in units of ng μg−1. 2.5. Statistical analysis The Statistical Package for the Social Sciences (version 16.0; SPSS Inc., USA) was used to find out the statistical significance of the obtained in vitro results. Biocompatible studies were carried out in triplicate for each sample and the obtained data were expressed as the arithmetic mean ± standard deviation. The results were assessed statistically using one–way analysis of variance followed by Tukey's least significant difference and Duncan's post hoc tests. Statistical significance was considered at 5% level (p b 0.05). 3. Result and discussion

OD of the nanoparticle−treated cells  100: Percentage of cell viability ¼ OD of the control cells ðiiiÞ

2.4.5. Osteocalcin estimation The production of osteocalcin (OC) was estimated in nanoparticletreated bone-forming osteoblast like MG-63 cell line for a period of 1–21 days of incubation. Cells grown in the absence of prepared nanoparticles served as a control. Highly specific monoclonal antibodies and peroxidase-labelled osteocalcin containing enzyme immune assay

3.1. Properties of the nanocomposites The crystalline phase of the prepared TGS nanocomposites is determined through XRD pattern, as shown in Fig. 2. As indicated in Fig. 2a, the broad diffraction peaks at 25.32° and 26.6° confirm the formation of crystalline TGS nanocomposite (JCPDS file no. 21-1276) [15,31, 34, 36]. However, a small, low-intensity hump is obtained at 12.7°, which shows the presence of trace amount of graphite oxide [15,31]. It may be due to reconversion of graphene oxide from the unfound graphene. Using the Scherrer equation [24] and obtained XRD pattern, the average

Fig. 3. Fourier transform infrared spectra of TGS nanocomposites for bioactivity. a) Before in vitro study; b) after in vitro study.

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crystallite size is calculated and is shown in Table 1. The addition of graphene to TiO2 decreases the crystallite size and crystalline nature. The formation of TGS nanocomposite is confirmed through the obtained infrared transmittance spectra in the range of 2500–400 cm−1 (Fig. 3a). The obtained broad characteristic peak at 900–400 cm−1 is attributed to the Ti\O\Ti stretching vibration [21]. The peak centred at 1029 cm−1 corresponds to the bending mode of Ti\O\C [15,21,34], indicating the formation of strongly bonded composites. Especially composite having high graphene content (TG.5, TG1, TG2 and TG4) affords good bonding. The through-like absorption peaks are located at 1225 and 1363 cm−1, which are associated respectively with Ti\OH/C\OH stretching band and C\C bands [15,34–36]. However, the presence of carboxyl groups (C\O/COOH) and the formation of TGS complex are confirmed from the peaks observed at 1685, 1701, 1706, and 1620 cm−1 [15,20,34–36]. The obtained FTIR results confirm the

formation of well-bonded TGS. An additional interesting phenomenon, similar to XRD results, is observed in Fig. 3a, i.e., TGS nanocomposite at 673 K shows the presence of graphene oxide. Generally, graphene switches to oxide form, that is, graphene oxide, beyond 873 K [15,36]. However, owing to the growth of TiO2 on graphene, it starts at 673 K. A spherical morphology of TiO2 and sheet like structure with an irregular spherical shape of TGS nanocomposites is observed through SEM analysis (Fig. 4). Fig. 4 clearly reveals the spherical morphology of the TiO2, which is embedded on the graphene sheet. It is interesting to note that the sample TG.25 to TG1 shows the domination of TiO2 spherical morphology which is due to low content of graphene in the nanocomposite [36]. However, the agglomeration of the particles is observed randomly in the nanocomposites due to the absorption of the moisture from the environment. In addition, the elemental compositions (EDAX) of the prepared samples are shown in Table 1. The

Fig. 4. Scanning electron microscopic images of TGS nanocomposites.

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gradual increase in carbon content confirms the formation of TGS nanocomposite. TEM images of all the prepared samples are shown in Fig. 5. Table 1 shows the particle size of the nanocomposites with an average diameter of 20 nm. The TEM images of the lower concentration of graphene in the nanocomposites (TG.25, TG.5 and TG1) show the domination of TiO2 spherical morphology; however, at higher concentration of graphene in the composite (TG2 and TG4) facilitates the formation of nanocomposites with the good basal spacing. The above observation shows the existence of TiO2 embedded graphene sheets, and it is also revealed on SEM images (Fig. 4). The obtained selected area electron diffraction (SAED) pattern of the samples is inserted in their respective TEM images. The SAED observations show that the crystalline nature of TiO2 is increased as a result of high concentration of graphene in the composite, which is well indexed with the XRD pattern.

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The Raman spectroscopic analysis of the prepared TiO2–graphene nanocomposites is shown in Fig. 6. The presence of TiO2 is revealed from the observed band at 148, 396,519 and 639 cm−1 [38]. Moreover, this non-destructive analysis reveals that the G band and D band of graphene are observed at ~ 1588 and 1355 cm− 1 along with the 2D peak at 2679 cm−1 [14,37,38]. The intensity of the G and D bands increases with the increase in the graphene concentration in the composites. Similarly, the TiO2 bands decrease with the increase in the graphene concentration. The slight shifts of TiO2 and graphene peaks are observed towards the lower wave number because of the composite formation. The obtained Raman spectra confirm the existence graphene and the formation of TiO2–graphene nanocomposites. This is in line with the obtained FTIR results and previous reports [37–39]. The SSA of the prepared nanoparticles is 208.41, 167.98, 186.52, 212.85, 216.04, and 234.56 m2 g−1 for TG0, TG.25, TG.5, TG1, TG2, and

Fig. 5. Transmission electron microscopic images and corresponding diffraction pattern of prepared TGS nanocomposites.

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Fig. 7. BET–isotherm curve of the prepared TGS nanocomposites. Fig. 6. Raman spectra of the prepared TGS nanocomposites.

TG4 samples, respectively (Table 1). In addition, the total pore volume and average pore diameter of all the prepared nanosamples are calculated according to the method suggested by Barrett et al. [25] and the results are shown in Table 1. The increase in graphene content in composites is directly proportional to the SSA, total pore volume, and pore diameters. It is interesting to note that the surface area and total pore volume of the prepared TGS nanocomposites are initially decreased up to 0.5 g (wt.%) of graphene (TG.5), there after it increases with the increase in graphene content. The observed initial decrease in Specific Surface Area (SSA) and total pore volume is due to the total occupation of TiO2 at lower content of graphene sheet [36,38]. In addition, the existence of higher TiO2 leads to the collapse of the basal spacing of graphene sheet, which is evident from the observed SEM images (Fig. 4) and TEM (Fig. 5). This makes it clear that the SSA of present study reveals that the TG1, TG2, and TG4 samples are the optimal ratio for the improved interactions with the bone-inducing cells. The observed higher surface area and pore size of the prepared TG1–TG4 nanocomposites allow three dimensional way of cell growth with better attachment of cells and nutrients from the body as described as earlier [23]. In addition the nitrogen adsorption and desorption isotherm of the prepared nanocomposites are shown in Fig. 7. The obtained hysteresis of the prepared nanocomposites is Type-IV isotherm and contains mesoporosity with high energy of adsorption. Moreover, the obtained hysteresis loop reveals that the nanocomposites posses the pores with narrow and wide sections and possible interconnecting channels [39]. The obtained mesopores with possible inter connecting channels facilitate the three dimensional cell attachments as well as enhancement in the biocompatibility of the prepared nanocomposites [36,37]. 3.2. In vitro analysis 3.2.1. Swelling behaviour The swelling percentage of the nanocomposites are calculated from the wet weight (310 ± 1 K) of the samples immersed in PBS and ultrapure water and are given in Table 2. Swelling tendency of the particles depends on pH, temperature, and the presence of ions in the solution [34,35]. The above parameters are responsible for the obtained high swelling rate of all prepared nanoparticles in PBS solution than in ultrapure water. Nevertheless, the rate of swelling in PBS and ultrapure water is having notable similarity; that is, an increase in the graphene content in the composite increases the swelling rate in both PBS and

water, which is due to the availing or holding capability of sheet like structure of graphene [40]. Further, it reveals that TGS nanocomposite with ratio of 2:2 (TG2) and 2:4 (TG4) attains maximum swelling property, respectively, in PBS (24.65% and 26.13%) and in water (20.89% and 19.84%). This tends to increase the absorption of more nutrients from the host medium, thereby increasing the surface area and enhancing cell attachment and growth [23].

3.2.2. Bioactivity study The pH and conductivity data of the nanocomposites in 1.5 SBF are shown in Fig. 8. All the samples show similar pattern of ionic interactions, besides a slight increase in pH at the higher concentration of graphene due to increased exchange of ions. After soaking the samples in 1.5 SBF, a sudden decrease in pH and an increase in conductivity are observed on the third and fifth day, due to the solubility of the samples [41,42]. In general, an increase in pH is observed whereas that in the conductivity is decreased. Thus, it confirms the interconnections between pH and conductivity in SBF. Both pH and conductivity measurements are gradually fluctuated up and down and become stable after day 18, which is due to more absorption of supplementary ions to initiate HAp layer formation and saturation of ion exchange after completion of the initiation of HAp during 18 days of incubation, respectively (Fig. 8a and b). The observed pH and conductivity measurements confirm the existence of ion exchanges between the sample and the SBF. These results show that the prepared samples are favourable for formation of HAp layer in human body [21,29]. After 21 days of incubation in the 1.5 SBF, the nanocomposite samples show distinct diffraction peaks in addition to those shown in Fig. 2a. The observed peaks at 25.3° and 31.7° correspond to, respectively, (201) and (211) planes, indicating the reflection of HAp layer formation (JCPDS file no. 090432; Fig. 2b). Crystallite size of SBF-incubated nanocomposites is shown in Table 2. The observed dramatic increase in crystallite size after the incubation of nanoparticles in SBF is ascribed due to the swelling and agglomeration of nanoparticles, as well as the formation of apatite layer on the surface of the nanoparticles [21,41]. When graphene concentration is increased from 0% to 4%, an increase in the intensity of HAp crystalline peaks and a gradual diminutive peak shift of TGS are observed toward the higher diffraction angle. This is because of higher particle and crystallite size due to the swelling behaviour. The well-formed crystalline HAp peak indicates the bioactivity of TG.5, TG1, TG2, and TG4 nanocomposites, which is again confirmed from the elemental analysis.

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Table 2 In vitro biological studies of TiO2–graphene nanocomposites. Sample name

TG0 TG.25 TG.5 TG1 TG2 TG4

After in vitro SBF study

Swelling percentage

Ca/P ratio (%)

Weight modulations (%)

Crystallite size (nm)

PBS (%)

Water (%)

1.45 1.71 1.82 1.75 1.63 1.60

−1.04 −1.07 −0.17 0.93 1.04 1.42

15.92 18.57 26.90 21.54 34.47 37.74

11.00 11.55 13.62 18.05 24.65 26.13

10.80 13.16 15.24 17.80 20.89 19.84

Fig. 3b shows IR spectra of TGS nanocomposites immersed in 1.5 SBF for 21 days. The observed broad Ti\O\Ti peak is slightly sharpened. In addition, new peaks are observed at 958 and 1039 cm− 1, which confirms the existence of phosphate bands [43]. In addition, the peak observed at 1084 cm− 1 corresponds to C\O stretching [15]. The Ti\O\C band is shifted slightly toward right-hand side when compared with the one shown in Fig. 3a. The peaks observed at 1468 and 1510 cm−1 correspond to C\OH/C_C bands [34,43]. The characdue to HAp teristic peak observed at 1417 cm−1 corresponds to CO2− 3 layer formation [21,41,45]. These results confirm that increasing graphene content in the composite leads to predominant phosphate bands in bioactivity study. On the other hand, the suppressed phosphate bands are due to the broad absorption peak of titanium bands (400–900 cm−1), which is in accordance with an earlier report [44].

Zone of inhibition in S. aureus & E. coli (mm) 0 0 0 0 0 0

After in vitro SBF study, the weight modulation of the samples is calculated with the dry weight of the 21-day old samples (Table 2). Pure TiO2 (TG0) and lower content of graphene in the composite (TG.25 and TG.5) show weight loss, which implies high degradation ability of the samples [23]. In contrast, high graphene content in the composite shows an increase in weight (TG1–TG4) due to the sheet like structure of graphene. The holding and swelling capacity of graphene facilitates the nanocomposite samples to withstand and formulate interaction with tissue [44]. Moreover, these results collectively reveal that the composite can avoid migration and deposition of nano-Ti into the cells and cell organelles, thereby facilitating less toxicity, especially the composite having high graphene content. Quantity of calcium and phosphate deposition on the sample is measured by XRF before and after bioactivity study. Stoichiometric Ca/P ratio confirms HAp layer formation on the surface of the sample [45]. Thus, the Ca/P ratios of all the samples are calculated using the obtained Ca and P percentage from the XRF results and are given in Table 2. The prepared TGS nanocomposite facilitates better HAp layer formation than the pure TiO2. Samples having less graphene content and pure TiO2 (TG0 to TG1) reveal the formation of carbonate-substituted HAp (Ca/P ratio = 1.67–1.93), while samples TG2 and TG4 show the formation of oxy-HAp (Ca/P ratio = 1.5–1.67) [45,46]. The observed results indicate that the increase in graphene content up to 4 g in TiO2– graphene nanocomposites intended to alter the calcium and phosphate depositions on the surface of the material, thereby it leads to the formation of the hydroxyapatite layer. Especially, Ca/P ratio of TG2 and TG4 (1.5–1.67) is similar as that of the stoichiometric Ca/P ratio of natural bone (1.67) [45,46]. 3.2.3. Antimicrobial activity Antibacterial activity of prepared TGS nanocomposites is screened against S. aureus and E. coli. The obtained results are given in Table 2. In general, overriding bactericidal and bacteriostatic activities rely on the reasonable factors such as physicochemical property and quantity of the individual materials [34,47]. Moreover, surface characteristics of the bacterial cell wall and the interactions between the cell wall and material surface play a dominant role in determination of antimicrobial property [21]. Table 2 shows that the prepared TGS nanocomposites have no bactericidal and bacteriostatic activities in E. coli and S. aureus. It may be due to the null interaction between the bacterial cell wall and material surface. These results are in line with the previous studies [47,48].

Fig. 8. Measurements of average ionic exchanges between 1.5 SBF and TGS nanocomposites. a) pH vs. soaking period; b) conductivity during in vitro bioactivity.

3.2.4. Biocompatibility study AGS cell line is an appropriate primary in vitro model to explore the toxicity of the nanoparticles. Further, osteoblast-like MG-63 cell lines are used to ensure biocompatibility and bone cell induction. The MTT assay of AGS and MG-63 cell lines, which are exposed to TGS nanocomposites for different concentrations (1, 5 and 20 μg mL− 1), is summarised respectively, in Fig. 9a and b [16]. Nonsignificant variances (p b 0.05) obtained against AGS and MG-63 cell lines that are exposed to nanocomposites compared with the control show almost non-cytotoxic effect. The absorption of nutrients is enhanced due to the higher

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Fig. 9. Cytotoxicity test of prepared TGS nanocomposites using MTT assay in triplicates. a) AGS cell line; b) MG-63 cell line.

surface area of TiO2–graphene nanocomposite, which results in strong cell attachment as well as interactions. Thus, it leads to a congenial environment to induce the bone forming cells. However, AGS and MG-63 cell lines explore little toxicity at the lower concentration of graphene (TG0–TG1). The obtained result confirms that the cell viability of AGS and MG-63 cell lines depends on the graphene content in the nanocomposites and administration amount (Fig. 9). 3.2.5. Osteocalcin production Osteocalcin is a bone-derived multifunctional hormone, primarily deposited in the extracellular matrix of bone. This non-collagenous protein plays a curial role in bone mass rather than the regulation of energy

metabolism, fertility and wound healing. OC production in different days during the period of 21 days of incubation was observed and the mean and standard deviation of the triplicate values are tabulated in Table 3. It reveals that the increase in graphene content increases the induction of OC due to the OC induction ability of the graphene and obtained higher surface area to enhance the cell attachment, proliferation, maturation, and finally matrix mineralisation [32,40,50]. The mineralised extracellular matrix is composed of smaller but significant amounts of osteocalcin (OC). The new bone synthesis is directly dependent on the secretion of osteocalcin in the cells [49–50]. Eventhough, a non-significance of osteocalcin production vs. varying graphene content at p b 0.05 is observed, the three different subsets are

Table 3 Variation in osteocalcin content at different days in triplicate experimental data of TiO2–graphene nanocomposites treated MG-63 cell line in ng μg−1. Samples

Days 1

TG0 TG.25 TG.5 TG1 TG2 TG4

9.25 9.50 10.97 10.27 11.27 12.07

3 ± ± ± ± ± ±

0.4bc 0.3bc 0.8ab 0.3ab 1.3ab 0.3a

8.03 9.55 11.63 13.65 13.07 13.42

7 ± ± ± ± ± ±

0.4bc 0.3bc 0.7ab 1.2ab 0.8ab 1.0a

8.50 9.68 10.93 14.22 14.92 14.55

a, ab and bc represent homogenous subsets of non-significant difference at p b 0.05.

12 ± ± ± ± ± ±

0.3bc 0.5bc 1.0ab 1.3ab 1.0ab 1.3a

8.50 9.17 10.82 13.82 14.82 15.15

14 ± ± ± ± ± ±

0.6bc 0.8bc 0.7ab 1.5ab 1.5ab 0.9a

10 10.17 11.67 14.82 15.43 15.88

21 ± ± ± ± ± ±

0.6bc 0.6bc 1.0bc 1.4ab 1.2ab 1.5a

10.45 10.67 11.48 13.37 15.40 15.32

± ± ± ± ± ±

0.4bc 1.0bc 0.5bc 1.9ab 1.5ab 1.5a

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existing in the respective concentration. The above colorimatic assays endorse the presence of minor difference in the osteocalcin production against the graphene content in the composite (Table 3). 4. Conclusions The prepared nanocomposites are characterised to explore their physiochemical properties, such as crystalline nature of well-bonded TGS nanocomposite with spherical embedded sheet-like structures. In addition, nanocomposites with the SSA in the range of 167.98–234.56 m2 g− 1 and swelling tendency of 11.55–26.13% lead to an enhancement in cell attachment as well as avoid the migration and agglomeration of the nanoparticles in the body. Moreover, the obtained smaller particle size (4.09–11.47 nm) and crystallite size (3.13–4.18 nm) of nanocomposite further stimulate apatite layer formation and hence augment the interaction with tissue based on the conventional principle of surface/volume ratio. These properties of nanocomposites resolve the adverse effects of TiO2 in the field of biomedical sciences. It is clearly evident from the obtained physicochemical characterisation (SEM, TEM and SSA) that the higher content of graphene in TiO2 (TG2 and TG4) can form the good nanocomposite formation without any subside of basal spacing and obstruction of TiO2 domination and total occupation than the low content of graphene in the composites (TG.25–TG1). The same is influenced on the biological studies, like swelling property, cell line studies, and Ca/P ratio. The present study collectively reveal that the samples TG2 (2:2) and TG4 (2:4) are optimal composites for biomedical applications. Thus, it is more suitable to the current requirements of bone reconstruction, regeneration, and tissue engineering. Acknowledgments This work was financially supported by UGC-DAE-Consortium for Scientific Research, Kalpakkam (CSR/Acctts/2010–11/1136 dt.06.01.2011). The authors thank Dr. G. Amarendra (Head, Metal Physics Section, Indira Gandhi Centre for Atomic Research, Kalpakkam node) for constructive suggestions. The authors also thank Dr. G. Kumaresan and Mr. P. Jayaprakash (Department of Genetics, School of Biological Sciences, Madurai Kamaraj University) for their technical support in the toxicity studies. References [1] A. Seidi, M. Ramalingam, I. Elloumi-Hannachi, S. Ostrovidov, A. Khademhosseini, Gradient biomaterials for soft-to-hard interface tissue engineering, Acta Biomater. 7 (2011) 1441–1451. [2] C. Isikli, V. Hasirci, N. Hasirci, Development of porous chitosan–gelatin/ hydroxyapatite composite scaffolds for hard tissue engineering applications, J. Tissue Eng. Regen. Med. 6 (2) (2012) 135–343. [3] H. Zhou, J. Lee, Nanoscale hydroxyapatite particles for bone tissue engineering, Acta Biomater. 7 (2011) 2269–2781. [4] Y.B. Luo, X.L. Wang, D.Y. Xu, Y.Z. Wang, Preparation and characterization of poly(lactic acid)-grafted TiO2 nanoparticles with improved dispersions, Appl. Surf. Sci. 255 (2009) 6795–6801. [5] D. Hua, K. Cheuk, Z. Wei-ning, W. Chen, X. Chang-fa, Low temperature preparation of nano TiO2 and its application as antibacterial agents, Trans. Nonferrous Met. Soc. China 17 (2007) s700–s703. [6] P. Song, X. Zhang, M. Sun, X. Cui, Y. Lin, Graphene oxide modified TiO2 nanotube arrays: enhanced visible light photoelectrochemical properties, Nanoscale 4 (2012) 1800. [7] A.J. Nathanael, N.S. Arul, N. Ponpandian, D. Mangalaraj, P.C. Chen, Nanostructured leaf like hydroxyapatite/TiO2 composite coatings by simple sol–gel method, Thin Solid Films 518 (24) (2010) 7333–7338. [8] H. Chen, P. Zou, J. Connarn, H. Paholak, D. Sun, Intracellular dissociation of a polymer coating from nanoparticles, Nano Res. 5 (2012) 815–825. [9] O.C. Compton, S.W. Cranford, K.W. Putz, Z. An, L.C. Brinson, M.J. Buehler, S.T. Nguyen, Tuning the mechanical properties of graphene oxide paper and its associated polymer nanocomposites by controlling cooperative intersheet hydrogen bonding, ACS Nano 6 (3) (2012) 2008–2019. [10] M. Kalbacova, A. Broz, J. Kong, M. Kalbac, Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells, Carbon 48 (2010) 4323–4329. [11] Y. Yang, A.M. Asiri, Z. Tang, D. Du, Y. Lin, Graphene based materials for biomedical applications, Materials Today 16 (2013) 365–373.

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