Article history:Received 20 September 2013Received in revised form 11 January 2014Accepted 7 February 2014Available online 16 February 2014
Keywords:Titania–grapheneCytotoxicityBiocompatibilityOsteocalcinMG-63 cell lineSurface area
Bone defects and damages are common these days, which increases the usage of biomaterial for humans. To pre-pare 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 TGSnanocomposites are an irregular sheetwith spherical TiO2 intercalatedmorphology. The SSA of the nanocompos-ites ranging from 167.98 to 234.56 m2 g−1 with mesoporosity and swelling tendency ranging from 11.55 to26.13% leads to an enhancement in human cell attachment as well as avoids the migration and agglomerationof the nanoparticles in the body. Further, the biological analysis in simulated body fluid and human cell lines(AGS andMG-63) collectively reveals that the TG2 (2:2) and TG4 (2:4) samples are found to bemore favourablematerials for biomimic bone action among the prepared TGS nanocomposites.
The conventional autografting and allografting treatments need toovercome the limitations such as donor site shortage and immunoge-nicity [1]. Thus, tissue engineering offers potential biomaterials to re-store 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 recentyears [2,3]. To overcome the above limitations, researchers are focusedon analysing potential nanocomposite materials rather than a singlenanomaterial [4].
Nano-titania (TiO2) is emerging as potential material for biomedicalapplications because of its unique physicochemical properties. Nano-TiO2 provides the site for deposition of calcium and phosphate, therebyaccelerating formation of bone-like apatite layer [5,6]. In addition, theanatase phase of TiO2 is more efficient in nucleation and growth of hy-droxyapatite (HAp) layer than any other crystalline phase of TiO2 pre-sumably because of better lattice match with HAp phase [7]. However,there are some problems related with migration and accumulation incell organelles [8].
Currently, graphene sheets (GS) are also considered as a new poten-tial material in biology; they have incredible properties such as highsurface area, mechanical stiffness, and bacterial inhibition [9]. Eventhough, the addition of graphene oxides reveals promising propertiesin biomedical applications, the reduced graphene oxide (graphene) isa new potential material with enhancedmechanical property, bioactivenature and cytocompatibility via promoting better cell adherence to the
+91 4288 274880.chalam).
human osteoblasts (bone forming) cells. This unique reinforcing behav-iour with a large surface area of graphene attracts us more towards thebiomedical to produce highly desirable biomaterial [10–12]. Moreover,GS can induce osteocytes on stem cells and have osteoconductive/in-ductive properties, which make them suitable for bone regenerationtherapy [13]. Among different methods to synthesise graphene, for ex-ample electrochemical synthesis [14] and ultrasound-assisted synthesis[15], chemical oxidation continues to be the most popular methodowing to its simplicity, viability, and scalability [16]. Oxidation of graph-ite facilitates more interaction with the tissues.
The growth of nano-Ti on nano–graphene is an important approachto produce nanohybrids because controlled nucleation and growth af-fords optimal chemical reaction and bonding between TiO2 and nano–graphene sheets. It results in a very strong electrical andmechanical cou-pling within the hybrid [17]. Several methods that proposed to form thecomposites 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 asan effective method to grow nanoTiO2 crystals on graphene sheets. Thebiological properties (bioactivity in SBF, cell reactivity) depend on thematerial morphology, size, components, concentration and otherphysico-chemical properties such as surface to volume ratio, particlesize, swelling and degradation [2,3,21–26]. When the nanocomposite isused as biomaterial for surgical and other biomedical applications, it fa-cilitates the enhancement of cell attachment and thereby improves thebioactivity and biocompatibility than that of the bulk.
In this study, an attempt has beenmade to prepare a series of TiO2–GS(TGS) nanocomposites by in situ sol–gel method. Furthermore, theprepared samples are characterised comprehensively to explore theirphysicochemical properties. To search the optimal composite material
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with properties to promote desired bone tissue regeneration, we subject-ed the prepared composites to biological studies such as swelling, bioac-tivity, and biocompatibility, respectively, for phosphate–buffered saline(PBS), simulated body fluid (SBF), and cell lines (human gastric adeno-carcinoma (AGS) and osteoblast-like MG-63). In addition, osteocalcinestimation is carried out to explore the new bone cell synthesis. Further,the properties of TGS nanocomposites are correlated with purenano-Ti structures in light of the biocompatibility and optimisation.
2. Materials and method
2.1. Materials
In this study, titanium isopropoxide (97%, Sigma-Aldrich, USA), iso-propyl alcohol (99%, Merck), acetyl acetone (98%, Merck), graphite(98%, Loba Chemie), sodium nitrate (99%, HiMedia), potassium per-manganate (99%, Merck), sulfuric acid (98.08%, Merck), hydrogen per-oxide (30%, Merck), and hydrazine hydrate (80%, Loba Chemie) wereused to synthesise the nanocomposite. Dulbecco's modified Eagle'smedium/Ham's F12 nutrient mixture (DMEM/F-12HAM, catalogue no.56498), RPMI-1640 medium (catalogue no.:R8758) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, catalogue no.070M61471) kit were used as received from Sigma-Aldrich (USA).
2.2. Nanocomposites preparation
A series of TGS nanocompositeswere synthesised through in situ sol–gel method. The graphene oxide was synthesised using standardHummers method [19,17]. Then it was reduced as graphene by addinghydrazine hydrate [16]. The different concentrations of preparedgraphene, namely, 0.25, 0.50, 1, 2, and 4 g L−1, were dispersed indouble–distilled water under sonication and kept ready to formnanocomposite.
Titanium isopropoxidewas diluted by isopropyl alcohol with hydro-lysis controller (acetyl acetone) in themolar ratio of 1:0.7:4, respective-ly [21]. The prepared graphene dispersion at different concentrationsdescribed previously was drop-wise added under sonication for 1 h.
Fig. 1. The schematic representation of the form
Then, the sonicated solution was stirred continuously for 4 h at 310 K.The obtained precipitatewaswashed individually with double–distilledwater followed by ethanol. The washed precipitates were dried in hot-air oven at 393 K to evaporate the solvents. Then the dried powderswere sintered at 673K 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 dif-ferent concentrations, namely, 0, 0.25, 0.50, 1, 2, and 4 g L−1 (hereaftertermed as TG0, TG.25, TG.50, TG1, TG2, and TG4, respectively).
2.3. Characterisation
2.3.1. X-ray diffractionThe 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 ofλ = 0.15406 Å. The samples were scanned at an angle of 2θ rangingfrom 10° to 80° with an increment of 0.05° at a scanning rate of 5° perminute. The peak positions and the respective intensities of the powderpattern were identified in comparison with the reference powder dif-fraction data. The average crystallite sizewasdetermined using Scherrerformula [24]:
D ¼ k λβ 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 thediffraction angle of the sample.
2.3.2. Fourier transform infrared spectrometerThe characteristic peaks of the synthesised TGS nanocomposites be-
fore and after SBF study were measured with a Fourier transform infra-red spectrometer (FTIR; Spectrum 100; PerkinElmer, USA) in thewavelength range of 4000–400 cm−1. The pellets for the FTIR studywere prepared by mixing nanosamples with potassium bromide (99%,
ation of TiO2–graphene nanocomposites.
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Merck) at a ratio of 2:200 (w/w) and compressing themwith a hydraulicpressure pellet maker.
2.3.3. Energy-dispersive spectroscopyQualitative 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 analysedfrom the observed EDAX pattern. The deposition of calcium and phos-phate on the surface of the samples was analysed both qualitatively andquantitatively before and after the bioactivity study using XRF. The occur-rences of the graphene and TiO2 in the prepared nanocomposites areanalysed through the Raman spectra (RENISHAW-M005-141) with thelaser frequency of 514 nm.
2.3.4. Electron microscopic analysisThe scanning electron microscopy (SEM; JSM-6390LV, JEOL, Japan)
with an accelerating voltage of 25 kV was used to observe the surfacemorphology of TGS nanocomposites. Transmission electronmicroscopy(TEM; CM200, Philips, USA) was used to measure the primary particlesize of the sample. TEM images are obtained using transmitted electrontechnique that produces magnification details up to 1,000,000× with aresolution better than 10 Å. The obtained electron diffraction pattern ofthe selected area of the samples was inserted with the TEM images toexplore the crystalline nature and lattice arrangements.
2.3.5. Specific surface areaThe specific surface area (SSA) of the prepared nanosamples
was measured using the Brunauer–Emmett–Teller analyser (AutosorbAS-1MP; Quantachrome, USA). The pore size distribution, averagepore diameter, and total pore volume of the prepared nanocompositeswere calculated using the Brunauer–Joyner–Halenda method [25]. Thesamples were degassed under vacuum at 290 °C with liquid nitrogen(−196 °C) for 3 h to remove the moisture. Liquid nitrogen was usedto avoid any thermally induced changes on the surface of the particles.
2.4. In vitro analysis
2.4.1. Swelling studyThe 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 hydraulicpressure pelletmaker. The initial weightwasmeasured asW0. Then, thepellet was immersed separately in a 50 mL PBS-containing bottle and
Fig. 2. X-ray diffraction pattern of a series of TGS nanocomposite
ultrapure water. This setup was incubated at 310 ± 1 K for 7 days[23]. After the incubation period, the pellets were carefully taken outfrom both PBS and ultrapure water using filter paper and the wetweight of the pellets was measured as Ww. The swelling ratio of theTGS nanocomposites was calculated using the following formula [23]:
Swellingratio W %ð Þ ¼ Ww−W0
W0� 100 : ðiiÞ
2.4.2. Bioactivity studyThe assessment of in vitro test for the bioactivity of the prepared TGS
nanosamples was carried out in 1.5 SBF (1.5 times higher concentrationthan human plasma). The initial formation of HAp takes place with theSBF environment while the initiation of biomineralisation and nucle-ation aswell as growth of the apatite layer (HAp) is developed in the en-vironment of saturated SBF (1.5 SBF), which mimic the naturalmechanism 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 reportedearlier [28–30]. Prepared samples were immersed in 1.5 SBF and incu-bated for 21 days at 310 ± 1 K in a circulating water bath. pH and con-ductivity probes of 5-Star (Thermo Orion, USA) were used to record theion exchange between the SBF and the prepared sample. After incuba-tion, the weight loss was calculated using dry weight of the pellets.The formation of HAp layer on the surface of the pellets was analysedby XRD, FTIR, and XRF studies. The bioactivity study was carried out astriplicates.
25923) and gram–negative (Escherichia coli, ATCC no. 25922) pathogenswere collected from the Microbial Type Culture Collection and GeneBank (India). The collected pathogenswere used to study antibacterial ef-fect of the prepared nanocomposites by disc diffusion method [31]. Theslants of abovementioned bacteria were inoculated in 2 mL sterileLuria–Bertani (LB) broth overnight, for diluents the bacterial strains.After incubation, a loop full of culture was suspended to 100 mL LBbroth 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, placedon the agar plate, and incubated at 310 K for 24 h to observe the antibac-terial activity.
for bioactivity. a) Before in vitro study; b) after in vitro study.
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2.4.4. Cell line studyThe human AGS cell line is a suitable primary in vitromodel to explore
the toxicity of the nanoparticles. In addition, osteoblast-like MG-63 cellline was used to estimate the biocompatibility and bone-forming abilities[32]. AGS cell line (ATCC-1739) and MG-63 cell lines were routinely cul-tured in, respectively, DMEM/F-12HAM (1:1) and RPMI-1640 mediumcontaining 10% foetal bovine serum, sodium pyruvate, sodium bicarbon-ate, 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 es-timated using MTT assay [21]. Before the use of MTT assay in AGS andMG-63, the cells were passaged freshly with respective medium.When the cells achieved 80%–90% confluency, they were seeded into a96-well microtiter plate at a density of 1 × 103 cells per well. The cellswere then allowed to adhere to the plate for 24 h. Filter-sterilised TGSnanocomposites (TG.25, TG.5, TG1, TG2, and TG4) at three differentconcentrations (20, 5, and 1 μg mL−1) were loaded in different wellsand 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 for4 h. At the end of incubation, 1 mL dimethyl sulfoxide was added to re-duce the formazan crystal into pink. Then the optical density (OD) ofthe pink solution was read at 570 nm. The percentage of cell viabilitywas calculated using the following formula:
2.4.5. Osteocalcin estimationThe production of osteocalcin (OC) was estimated in nanoparticle-
treated bone-forming osteoblast like MG-63 cell line for a period of1–21 days of incubation. Cells grown in the absence of prepared nano-particles served as a control. Highly specific monoclonal antibodiesand peroxidase-labelled osteocalcin containing enzyme immune assay
Fig. 3. Fourier transform infrared spectra of TGS nanocomposites
kit (Biological Technologies Inc., USA) were used to estimate the levelof osteocalcin [33]. The level of OC protein in cell culture supernatantswas collected and was anticipated at the absorbance at 450 nm wave-length. The percentage of osteocalcin production was calculated usingthe following formula:
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; SPSSInc., USA)was used tofindout the statistical significance of the obtainedin vitro results. Biocompatible studies were carried out in triplicate foreach sample and the obtained data were expressed as the arithmeticmean ± standard deviation. The results were assessed statisticallyusing one–way analysis of variance followed by Tukey's least significantdifference and Duncan's post hoc tests. Statistical significance was con-sidered at 5% level (p b 0.05).
3. Result and discussion
3.1. Properties of the nanocomposites
The crystalline phase of the prepared TGS nanocomposites is deter-mined 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 formationof crystalline TGS nanocomposite (JCPDS file no. 21-1276) [15,31, 34,36]. However, a small, low-intensity hump is obtained at 12.7°, whichshows the presence of trace amount of graphite oxide [15,31]. It maybe due to reconversion of graphene oxide from the unfound graphene.Using the Scherrer equation [24] and obtained XRD pattern, the average
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 ofgraphene to TiO2 decreases the crystallite size and crystalline nature.
The formation of TGS nanocomposite is confirmed through the ob-tained infrared transmittance spectra in the range of 2500–400 cm−1
(Fig. 3a). The obtained broad characteristic peak at 900–400 cm−1 is at-tributed to the Ti\O\Ti stretching vibration [21]. The peak centred at1029 cm−1 corresponds to the bending mode of Ti\O\C [15,21,34], in-dicating the formation of strongly bonded composites. Especially com-posite having high graphene content (TG.5, TG1, TG2 and TG4) affordsgood bonding. The through-like absorption peaks are located at 1225and 1363 cm−1, which are associated respectively with Ti\OH/C\OHstretching band and C\C bands [15,34–36]. However, the presenceof carboxyl groups (C\O/COOH) and the formation of TGS complexare confirmed from the peaks observed at 1685, 1701, 1706, and1620 cm−1 [15,20,34–36]. The obtained FTIR results confirm the
Fig. 4. Scanning electron microscopic
formation of well-bonded TGS. An additional interesting phenomenon,similar to XRD results, is observed in Fig. 3a, i.e., TGS nanocomposite at673 K shows the presence of graphene oxide. Generally, grapheneswitches 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 ir-regular spherical shape of TGS nanocomposites is observed throughSEM analysis (Fig. 4). Fig. 4 clearly reveals the spherical morphology ofthe TiO2, which is embedded on the graphene sheet. It is interesting tonote that the sample TG.25 to TG1 shows the domination of TiO2
spherical morphology which is due to low content of graphene in thenanocomposite [36]. However, the agglomeration of the particles isobserved randomly in the nanocomposites due to the absorption ofthe moisture from the environment. In addition, the elemental compo-sitions (EDAX) of the prepared samples are shown in Table 1. The
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gradual increase in carbon content confirms the formation of TGSnanocomposite.
TEM images of all the prepared samples are shown in Fig. 5. Table 1shows the particle size of the nanocompositeswith an average diameterof 20 nm. The TEM images of the lower concentration of graphene in thenanocomposites (TG.25, TG.5 and TG1) show the domination of TiO2
spherical morphology; however, at higher concentration of graphenein the composite (TG2 and TG4) facilitates the formation of nanocom-posites with the good basal spacing. The above observation shows theexistence of TiO2 embedded graphene sheets, and it is also revealedon SEM images (Fig. 4). The obtained selected area electron diffraction(SAED) pattern of the samples is inserted in their respective TEM im-ages. The SAED observations show that the crystalline nature of TiO2 isincreased as a result of high concentration of graphene in the composite,which is well indexed with the XRD pattern.
Fig. 5. Transmission electron microscopic images and correspon
The Raman spectroscopic analysis of the prepared TiO2–graphenenanocomposites is shown in Fig. 6. The presence of TiO2 is revealedfrom 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 ofgraphene are observed at ~1588 and 1355 cm−1 along with the 2Dpeak at 2679 cm−1 [14,37,38]. The intensity of the G and D bands in-creases with the increase in the graphene concentration in the compos-ites. Similarly, the TiO2 bands decrease with the increase in thegraphene concentration. The slight shifts of TiO2 and graphene peaksare observed towards the lowerwave number because of the compositeformation. The obtained Raman spectra confirm the existence grapheneand the formation of TiO2–graphene nanocomposites. This is in linewith 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
ding diffraction pattern of prepared TGS nanocomposites.
Fig. 6. Raman spectra of the prepared TGS nanocomposites.
Fig. 7. BET–isotherm curve of the prepared TGS nanocomposites.
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TG4 samples, respectively (Table 1). In addition, the total pore volumeand average pore diameter of all thepreparednanosamples are calculat-ed according to the method suggested by Barrett et al. [25] and the re-sults are shown in Table 1. The increase in graphene content incomposites is directly proportional to the SSA, total pore volume, andpore diameters. It is interesting to note that the surface area and totalpore volume of the prepared TGS nanocomposites are initially de-creased up to 0.5 g (wt.%) of graphene (TG.5), there after it increaseswith the increase in graphene content. The observed initial decreasein Specific Surface Area (SSA) and total pore volume is due to the totaloccupation of TiO2 at lower content of graphene sheet [36,38]. In addi-tion, the existence of higher TiO2 leads to the collapse of the basal spac-ing 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 presentstudy reveals that the TG1, TG2, and TG4 samples are the optimal ratiofor the improved interactions with the bone-inducing cells. The ob-served higher surface area and pore size of the prepared TG1–TG4 nano-composites allow three dimensional way of cell growth with betterattachment of cells and nutrients from the body as described as earlier[23].
In addition the nitrogen adsorption and desorption isotherm of theprepared nanocomposites are shown in Fig. 7. The obtained hysteresisof the prepared nanocomposites is Type-IV isotherm and containsmesoporosity with high energy of adsorption. Moreover, the obtainedhysteresis loop reveals that the nanocomposites posses the pores withnarrow and wide sections and possible interconnecting channels [39].The obtained mesopores with possible inter connecting channels facili-tate the three dimensional cell attachments as well as enhancement inthe biocompatibility of the prepared nanocomposites [36,37].
3.2. In vitro analysis
3.2.1. Swelling behaviourThe swelling percentage of the nanocomposites are calculated from
the wet weight (310 ± 1 K) of the samples immersed in PBS and ultra-pure water and are given in Table 2. Swelling tendency of the particlesdepends on pH, temperature, and the presence of ions in the solution[34,35]. The above parameters are responsible for the obtained highswelling rate of all prepared nanoparticles in PBS solution than in ultra-pure water. Nevertheless, the rate of swelling in PBS and ultrapurewater is having notable similarity; that is, an increase in the graphenecontent in the composite increases the swelling rate in both PBS and
water, which is due to the availing or holding capability of sheet likestructure of graphene [40]. Further, it reveals that TGS nanocompositewith ratio of 2:2 (TG2) and 2:4 (TG4) attains maximum swelling prop-erty, respectively, in PBS (24.65% and 26.13%) and in water (20.89% and19.84%). This tends to increase the absorption of more nutrients fromthe host medium, thereby increasing the surface area and enhancingcell attachment and growth [23].
3.2.2. Bioactivity studyThe pH and conductivity data of the nanocomposites in 1.5 SBF are
shown in Fig. 8. All the samples show similar pattern of ionic interac-tions, besides a slight increase in pH at the higher concentration ofgraphene due to increased exchange of ions. After soaking the samplesin 1.5 SBF, a sudden decrease in pH and an increase in conductivity areobserved 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 theconductivity is decreased. Thus, it confirms the interconnections be-tween pH and conductivity in SBF. Both pH and conductivity measure-ments are gradually fluctuated up and down and become stable afterday 18, which is due to more absorption of supplementary ions to initi-ate HAp layer formation and saturation of ion exchange after comple-tion of the initiation of HAp during 18 days of incubation, respectively(Fig. 8a and b). The observed pH and conductivity measurements con-firm the existence of ion exchanges between the sample and the SBF.These results show that the prepared samples are favourable for forma-tion of HAp layer in human body [21,29].
After 21 days of incubation in the 1.5 SBF, the nanocomposite sam-ples show distinct diffraction peaks in addition to those shown inFig. 2a. The observed peaks at 25.3° and 31.7° correspond to, respective-ly, (201) and (211) planes, indicating the reflection of HAp layer forma-tion (JCPDS file no. 090432; Fig. 2b). Crystallite size of SBF-incubatednanocomposites is shown in Table 2. The observed dramatic increasein crystallite size after the incubation of nanoparticles in SBF is ascribeddue to the swelling and agglomeration of nanoparticles, as well as theformation of apatite layer on the surface of the nanoparticles [21,41].When graphene concentration is increased from 0% to 4%, an increasein the intensity of HAp crystalline peaks and a gradual diminutivepeak shift of TGS are observed toward the higher diffraction angle.This is because of higher particle and crystallite size due to the swellingbehaviour. Thewell-formed crystalline HAp peak indicates the bioactiv-ity of TG.5, TG1, TG2, and TG4 nanocomposites, which is again con-firmed from the elemental analysis.
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Fig. 3b shows IR spectra of TGS nanocomposites immersed in 1.5 SBFfor 21 days. The observed broad Ti\O\Ti peak is slightly sharpened. Inaddition, new peaks are observed at 958 and 1039 cm−1, whichconfirms the existence of phosphate bands [43]. In addition, the peakobserved at 1084 cm−1 corresponds to C\O stretching [15]. TheTi\O\C band is shifted slightly toward right-hand side whencompared with the one shown in Fig. 3a. The peaks observed at 1468and 1510 cm−1 correspond to C\OH/C_C bands [34,43]. The charac-teristic peak observed at 1417 cm−1 corresponds to CO3
2− due to HAplayer formation [21,41,45]. These results confirm that increasinggraphene content in the composite leads to predominant phosphatebands in bioactivity study. On the other hand, the suppressedphosphatebands are due to the broad absorption peak of titanium bands(400–900 cm−1), which is in accordance with an earlier report [44].
Fig. 8.Measurements of average ionic exchanges between1.5 SBF and TGSnanocomposites.a) pH vs. soaking period; b) conductivity during in vitro bioactivity.
After in vitro SBF study, the weight modulation of the samples is cal-culated with the dry weight of the 21-day old samples (Table 2). PureTiO2 (TG0) and lower content of graphene in the composite (TG.25and TG.5) show weight loss, which implies high degradation ability ofthe samples [23]. In contrast, high graphene content in the compositeshows an increase in weight (TG1–TG4) due to the sheet like structureof graphene. The holding and swelling capacity of graphene facilitatesthe nanocomposite samples to withstand and formulate interactionwith tissue [44]. Moreover, these results collectively reveal that thecomposite can avoid migration and deposition of nano-Ti into the cellsand cell organelles, thereby facilitating less toxicity, especially the com-posite having high graphene content.
Quantity of calciumand phosphate deposition on the sample ismea-sured by XRF before and after bioactivity study. Stoichiometric Ca/Pratio confirms HAp layer formation on the surface of the sample [45].Thus, the Ca/P ratios of all the samples are calculated using the obtainedCa and P percentage from the XRF results and are given in Table 2. Theprepared TGS nanocomposite facilitates better HAp layer formationthan the pure TiO2. Samples having less graphene content and pureTiO2 (TG0 to TG1) reveal the formation of carbonate-substituted HAp(Ca/P ratio = 1.67–1.93), while samples TG2 and TG4 show the forma-tion of oxy-HAp (Ca/P ratio = 1.5–1.67) [45,46]. The observed resultsindicate that the increase in graphene content up to 4 g in TiO2–
graphene nanocomposites intended to alter the calcium and phosphatedepositions on the surface of thematerial, thereby it leads to the forma-tion 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 naturalbone (1.67) [45,46].
3.2.3. Antimicrobial activityAntibacterial 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 onthe reasonable factors such as physicochemical property and quantityof the individual materials [34,47]. Moreover, surface characteristics ofthe bacterial cell wall and the interactions between the cell wall andmaterial surface play a dominant role in determination of antimicrobialproperty [21]. Table 2 shows that the prepared TGS nanocompositeshave no bactericidal and bacteriostatic activities in E. coli and S. aureus.It may be due to the null interaction between the bacterial cell walland material surface. These results are in line with the previous studies[47,48].
3.2.4. Biocompatibility studyAGS cell line is an appropriate primary in vitromodel to explore the
toxicity of the nanoparticles. Further, osteoblast-like MG-63 cell linesare used to ensure biocompatibility and bone cell induction. The MTTassay of AGS andMG-63 cell lines, which are exposed to TGS nanocom-posites for different concentrations (1, 5 and 20 μg mL−1), issummarised respectively, in Fig. 9a and b [16]. Nonsignificant variances(p b 0.05) obtained against AGS and MG-63 cell lines that are exposedto nanocomposites compared with the control show almost non-cyto-toxic effect. The absorption of nutrients is enhanced due to the higher
Fig. 9. Cytotoxicity test of prepared TGS nanocomposites using MTT assay in triplicates. a) AGS cell line; b) MG-63 cell line.
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surface area of TiO2–graphene nanocomposite, which results in strongcell attachment as well as interactions. Thus, it leads to a congenial en-vironment to induce the bone forming cells. However, AGS and MG-63cell lines explore little toxicity at the lower concentration of graphene(TG0–TG1). The obtained result confirms that the cell viability of AGSandMG-63 cell lines depends on the graphene content in the nanocom-posites and administration amount (Fig. 9).
3.2.5. Osteocalcin productionOsteocalcin is a bone-derived multifunctional hormone, primarily
deposited in the extracellular matrix of bone. This non-collagenous pro-tein plays a curial role in bonemass rather than the regulation of energy
Table 3Variation in osteocalcin content at different days in triplicate experimental data of TiO2–graph
a, ab and bc represent homogenous subsets of non-significant difference at p b 0.05.
metabolism, fertility and wound healing. OC production in differentdays during the period of 21 days of incubation was observed and themean and standard deviation of the triplicate values are tabulated inTable 3. It reveals that the increase in graphene content increases theinduction of OC due to the OC induction ability of the graphene andobtained higher surface area to enhance the cell attachment, prolifera-tion, maturation, and finally matrix mineralisation [32,40,50]. Themineralised extracellular matrix is composed of smaller but significantamounts of osteocalcin (OC). The new bone synthesis is directly depen-dent on the secretion of osteocalcin in the cells [49–50].
Eventhough, a non-significance of osteocalcin production vs. varyinggraphene content at p b 0.05 is observed, the three different subsets are
ene nanocomposites treated MG-63 cell line in ng μg−1.
261K. Kandiah et al. / Materials Science and Engineering C 38 (2014) 252–262
existing in the respective concentration. The above colorimatic assaysendorse the presence of minor difference in the osteocalcin productionagainst the graphene content in the composite (Table 3).
4. Conclusions
The prepared nanocomposites are characterised to explore theirphysiochemical properties, such as crystalline nature of well-bondedTGS nanocomposite with spherical embedded sheet-like structures.In addition, nanocomposites with the SSA in the range of167.98–234.56 m2 g−1 and swelling tendency of 11.55–26.13% leadto an enhancement in cell attachment as well as avoid the migrationand agglomeration of the nanoparticles in the body. Moreover, theobtained smaller particle size (4.09–11.47 nm) and crystallite size(3.13–4.18 nm) of nanocomposite further stimulate apatite layer for-mation and hence augment the interaction with tissue based on theconventional principle of surface/volume ratio. These properties ofnanocomposites resolve the adverse effects of TiO2 in the field ofbiomedical sciences. It is clearly evident from the obtained physico-chemical characterisation (SEM, TEM and SSA) that the higher contentof graphene in TiO2 (TG2 and TG4) can form the good nanocompositeformation without any subside of basal spacing and obstruction ofTiO2 domination and total occupation than the low content of graphenein the composites (TG.25–TG1). The same is influenced on the biologicalstudies, like swelling property, cell line studies, and Ca/P ratio. Thepresent study collectively reveal that the samples TG2 (2:2) and TG4(2:4) are optimal composites for biomedical applications. Thus, it ismore suitable to the current requirements of bone reconstruction,regeneration, and tissue engineering.
Acknowledgments
This work was financially supported by UGC-DAE-Consortiumfor Scientific Research, Kalpakkam (CSR/Acctts/2010–11/1136dt.06.01.2011). The authors thank Dr. G. Amarendra (Head, MetalPhysics Section, Indira Gandhi Centre for Atomic Research, Kalpakkamnode) for constructive suggestions. The authors also thank Dr. G.Kumaresan and Mr. P. Jayaprakash (Department of Genetics, School ofBiological Sciences, Madurai Kamaraj University) for their technicalsupport in the toxicity studies.
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