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Article Cite This: ACS Omega 2019, 4, 7448−7458

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Fabrication of Graphene Nanoplatelet-Incorporated Porous Hydroxyapatite Composites: Improved Mechanical and in Vivo Imaging Performances for Emerging Biomedical Applications Sunil Kumar,† Chandkiram Gautam,*,† Vijay Kumar Mishra,‡ Brijesh Singh Chauhan,§ Saripella Srikrishna,§ Ram Sagar Yadav,∥ Ritu Trivedi,‡ and Shyam Bahadur Rai∥

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†

Advanced Glass and Glass Ceramics Research Laboratory, Department of Physics, University of Lucknow, Lucknow 226027, Uttar Pradesh, India ‡ LSS-101 Laboratory, Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, Uttar Pradesh, India § Cell and Neurobiology Laboratory, Department of Biochemistry, and ∥Department of Physics, Banaras Hindu University, Varanasi 221005, Uttar Pradesh, India S Supporting Information *

ABSTRACT: Three-dimensional nanocomposites exhibit unexpected mechanical and biological properties that are produced from two-dimensional graphene nanoplatelets and oxide materials. In the present study, various composites of microwave-synthesized nanohydroxyapatite (nHAp) and graphene nanoparticles (GNPs), (100 − x)HAp−xGNPs (x = 0, 0.1, 0.2, 0.3, and 0.5 wt %), were successfully synthesized using a scalable bottom-up approach, that is, a solid-state reaction method. The structural, morphological and mechanical properties were studied using various characterization techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and universal testing machine (UTM). XRD studies revealed that the prepared composites have high-order crystallinity. Addition of GNPs into nHAp significantly improved the mechanical properties. Three-dimensional nanocomposite 99.5HAp−0.5GNPs exhibited exceptionally high mechanical properties, for example, a fracture toughness of ∼116 MJ/m3, Young’s modulus of ∼98 GPa, and compressive strength of 96.04 MPa, which were noticed to be much greater than in the pure nHAp. The MTT assay and cell imaging behaviors were carried out on the gut tissues of Drosophila third instars larvae and on primary rat osteoblast cells for the sample 99.5HAp−0.5GNPs that have achieved the highest mechanical properties. The treatment with lower concentrations of 10 μg/mL on the gut tissues of Drosophila and 1 and 5 μg/mL of this composite sample showed favorable cell viability. Therefore, owing to the excellent porous nature, interconnected surface morphology, and mechanical and biological properties, the prepared composite sample 99.5HAp−0.5GNPs stood as a promising biomaterial for bone implant applications. delivery, sensor, and environmental engineering.6 Recently, researchers pay much attention to the fabrication and designing of nHAp nanocomposites with sufficient mechanical properties for biomedical applications. However, the main features of bone implantation materials are 3D porous microstructures with a high amount of nHAp and low secondary phases as an additive.7 It is well reported that the porous scaffolds must have a mechanical strength as close as possible to that of the substituted bone; otherwise, this porous structure is to be destroyed during tissue regeneration and load applications.8 The unadulterated nHAp material cannot fulfill the prerequisites of the embedded bone because of its fragile

1. INTRODUCTION Nanohydroxyapatite (nHAp) is an advanced bioceramic nanocrystalline material suitable for designing artificial bone or implants. Bone engineering is a clinical procedure that replaces missing bone and repairs the bone fracture.1 The living bone has the ability to regenerate the hard and soft tissues but requires a porous scaffold for providing mechanical support to it.2 The biocompatibility, osteoconductivity, high porosity, and mechanical strength are the fundamental criteria for designing a bone substitute material.3 Due to the limited number of donor autograft, allograft does not fulfill the requirement of the implantations; synthetic materials are widely being used as a bone substitute material.4 The synthetic bone material obtained from nHAp and its composite possess a chemical and structural composition similar to the natural bone.5 However, nHAp has been widely used in bone, drug © 2019 American Chemical Society

Received: December 29, 2018 Accepted: April 5, 2019 Published: April 24, 2019 7448

DOI: 10.1021/acsomega.8b03473 ACS Omega 2019, 4, 7448−7458

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imaging of intracellular structure is an important matter in the biomedical field.50 The fluorescent nanoparticles are more stable in the biological and excellent tissue imaging applications. The nHAp is a nontoxic, self-activated fluorescent material and becomes an excellent bioimaging material that retains their fluorescing nature in a biological environment.51 Comparatively, to develop the better nHAp-based composite, the optimized strategy has been adopted using different concentrations of the GNPs to synthesize various nanocomposites with improved performances. Current efforts have been made to synthesize highly porous crystalline nHAp and its composites, which can be used for bone implant applications. Thus, highly efficient biocompatible nHAp has been successfully prepared via a microwave irradiation technique. However, nHAp−GNP composites were synthesized by using a simple solid-state reaction method. In order to improve the mechanical properties by maintaining the biological properties; different concentrations of GNPs were used as an additive. However, GNPs may have a more exploitable porous structure than other additives. In the previously reported studies, most of the researchers have been focused only on the structural properties of such types of composite materials and did not have a detailed study on their biological properties. In the present study, we propose the synthesis of porous nHAp and its composites with improved mechanical and in vivo imaging performances for biomedical applications using a simple and scalable bottom-up approach, that is, a solid-state reaction method. Enhancement in the mechanical strength, in vivo imaging, and biocompatibility without changing the other properties by standard sintering etiquette was for the first time reported.

nature and low mechanical properties which limit their applications.9 Therefore, with the aim of improving the mechanical properties, the various nHAp composites have been developed to enhance the mechanical strength of the pure nHAp, such as HAp−MgO,10 HAp−SrCO3,11 HAp− La2O3,12 HAp−Al2O3,13 and HAp−GO.14 Furthermore, it is also reported that the addition of Fe2O3 into the nHAp considerably improves the mechanical strength.15 The TiO2added nHAp nanocomposites present improved fracture toughness and mechanical strength than the pure HAp.16,17 The 1.0 wt % CNT-added nHAp composite displays a 120% enhancement in toughness.18 However, the glass-added nHAp composite remarkably enhanced the density and compressive strength of the nHAp that was sintered at 1200 °C.19 Although, nHAp composites with metals, ceramic oxides, and polymers showed not only promising results for enhanced mechanical properties but also the reverse effect toward the biological properties.20,21 The incorporation of the second phase of carbon nonmaterials such as CNT and graphene are thought suitable in a ceramic matrix due to excellent mechanical and biological properties such as a high Young’s modulus and compressive strength along with adequate bioactivity.22,23 The graphene has been frequently used in various biological applications such as biosensing,24,25 drug delivery,26 tissue engineering,27,28 and cell modulating interfaces and cell scaffolds to control the cell growth.29 The high surface area and low density of graphene act in applications as a mechanical and biological modifier.30−32 The graphene combined with nHAp makes low-density nanocomposites that possess high mechanical strength, high surface area, biostability, and excellent biocompatibility.33 The addition of graphene into nHAp could be promising for maintaining the nHAp bioactivity.34 The mechanical strength and biological properties of nHAp−GNP composites have already been widely studied and have been found to be mechanically strong compared to pure nHAp.35,36 Various studies have been carried out on graphene nanoplatelets added to composites, silicon nitride,33 and aluminum37 on enhanced mechanical and biological properties. Recently, graphene is added as an effective additive for toughening the nHAp composite. The HAp−GNP composite prepared by an additive manufacturing technique showed excellent improvement in compressive strength.38 It is all around detailed that the graphene-included nHAp composite upgraded a few mechanical and natural parameters, for example, the versatile modulus, crack sturdiness, and osteoblast cell bond and their expansion.39−41 The nanocomposites with high porosity and interconnectivity of porous structure are essential to enhance the restorability and provide mechanical support to tissue growth.42 The mechanical property of the composites strictly depend on microstructure, porosity, pore size, and formation of secondary phases.5 The incorporation of graphene into the pure nHAp enhanced its mechanical properties significantly and showed a reverse effect for their densities.43 Recently, biocompatibility of graphene and its composites were successfully evaluated on mouse embryonic fibroblast cells as well as on human lung cells.44,45 It is previously reported that the addition of GNPs into the nHAp matrix is promising for improving the mechanical strength and cell imaging.46,47 The nHAp composites coadded with graphene and gold nanoparticles showed more favorable differentiation important in bone regeneration and remodeling of the bone tissue.48,49 The

2. RESULTS AND DISCUSSIONS 2.1. X-ray Diffraction. The crystallographic nature, phase formation, and phase stability of the GNP-reinforced nHAp composites sintered at 1200 °C for 3 h were analyzed using a powder XRD method. All the XRD patterns were matched with standard stoichiometric HAp (JCPDS file no. 240033) and were not found of any peak of the impurities and their byproducts. These XRD patterns also indicate the presence of the utmost of high intensity peaks, which are lying between the 2θ ranges of 25°−45°. The XRD pattern of pure GNPs is shown in Figure S1. Three different characteristic peaks were observed at different 2θ angles, 26.66°, 44.64°, and 54.74°, corresponding to their lattice planes, (002), (100), and (004), which confirmed the purity of the utilized GNPs (JCPDS file no. 030401). The X-ray diffraction patterns of all the biocomposite samples are shown in Figure 1a−e. The various diffraction peaks in the XRD patterns of composite samples are well matched with the pure hexagonal HAp phase and the pure GNPs. The characteristic X-ray diffraction peaks corresponding to planes (002), (100), and (004) of GNPs were observed at different 2θ angles, 26.1°, 42.24°, and 53.14°, which clearly showed the presence of GNPs in nHAp.6 The XRD patterns also revealed that the addition of GNPs does not alter the phase stability of nHAp; however, minor peaks due to GNPs themselves have been altered slightly. Thus, XRD patterns of the nHAp−GNP composites are almost similar. The sharp and highly diffracted peaks revealed a good crystalline nature of the nHAp−GNPs composites.9 The average crystallite size of the composite samples was determined from the XRD pattern and was found to be in the range of 411 to 479 nm with ±0.003 7449


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Figure 2. Variations of (a) density with different doping concentrations of GNPs in the composite system (100 − x)HAp− xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %). (b) Variation of percentage of porosity with increasing doping concentrations of GNPs in the composite system (100 − x)HAp−xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %). (c) Variations of density and percentage of porosity in the composite system (100 − x)HAp−xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %).

particle shape, and distribution of grain size, and the amount of additive. Thus, the pore size, fraction, and architectures of the composites were also strongly affected by the mechanical properties of the nHAp. The variations in the percentage of porosity and different concentrations of GNPs are shown in Figure 2b. The increasing concentrations of GNPs for nHAp reduced the densification significantly. Thus; the porosity of the composites is increased by increasing the concentration of GNPs from 8.97 to 20.87%. The variations of density and percentage of porosity with different doping concentrations of GNPs in the composite system (100 − x)nHAp−xGNPs (x = 0.1, 0.2, 0.3, 0.5 wt %) is shown in Figure 2c. The density versus percentage porosity bar graph shows a reverse effect, that is, density is increasing with a decreasing percentage of porosity. Moreover, the addition of GNPs into an nHAp matrix creates a 3D porous structure and exhibits a random grain growth by preventing the grain boundaries.45 Therefore, the high mechanical strength, high fracture toughness, and high 3D porous nature of the composite 99.5HAp−0.5GNPs could suggest the required fluid hauling by this sample and offer superior biological activities. 2.3. Morphological Analysis. The surface morphology and porous structure of nHAp and composites of HAp−GNPs, as well as the fractured samples, were analyzed by field emission scanning electron microscopy (FE-SEM). The SEM

Figure 1. X-ray diffraction patterns of (a) 99.9HAp−0.1GNP, (b) 99.8HAp−0.2GNP, (c) 99.7HAp−0.3GNP, (d) 99.5HAp−0.4GNP and (e) 99.5HAp−0.5GNP composites sintered at 1200 °C for 3 h.

accuracies. Thus the calculated average crystallite sizes of all the samples are enlisted in Table 1. These results were also correlated with the results of the TEM and shown in Figure 2. 2.2. Density and Porosity Analysis. The calculated values of the density of nHAp and its composites 99.9HAp− 0.1GNPs, 99.8HAp−0.2GNPs, 99.7HAp−0.3GNPs, and 99.5HAp−0.5GNPs are listed in Table 1. The variations in the density and percentage of GNPs for all the composite samples are shown in Figure 2a. From a minuscule point of view, a decrease in the density of the fabricated composite samples with increasing concentrations of GNPs may be due to the incorporation of a low density (GNPs, 0.4 g/cm3) by a high density (without sintering nHAp, 2.95 g/cm3). Therefore, the density of the composite sample 99.5HAp−0.5GNPs, x = 0.5 was found to be a minimum of 2.83 g/cm3, while it was found to be a maximum of 3.01 g/cm3 for sintered nHAp, x = 0.0. The porous structure of nHAp samples was affected by a microstructural parameter, such as grain size, grain packing,

Table 1. Mechanical Properties of Crystalline Nano HAp and nHAp−GNP Composite Samples Sintered at 1200 °C for 3h samples nHAp 99.9HAp−0.1GNPs 99.8HAp−0.2GNPs 99.7HAp−0.3GNPs 99.5HAp−0.5GNPs natural bone(human cancellous)57−62

density (g/cm3) 2.87 ± 0.087 2.79 ± 0.085 2.72 ± 0.088 2.65 ± 0.070 2.50 ± 0.075 1.8−2.54

average particle size (nm)

compressive strength (MPa)

Young’s modulus (GPa)

± ± ± ± ±

87 ± 5 87 ± 5 91 ± 7 93 ± 8 97 ± 7 5−10

89 ± 5 89 ± 5 93 ± 6 95 ± 6 98 ± 8 0.05−0.1

411 432 479 431 454

12 9 8 6 5

7450

fracture toughness (MJ/m3) 104 105 110 112 116

± ± ± ± ±

5 6 8 5 9

percentage of porosity 8.97 ± 0.15 11.43 ± 0.17 13.87 ± 0.25 15.98 ± 0.24 20.87 ± 0.41


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images of the pure nHAp and composite samples are shown in Figure 3a−e. According to the microstructures, significant

Figure 4. Energy dispersive X-ray analysis spectrums of (a) pure nHAp and (b) 99.5HAp−0.5GNP composites sintered at 1200 °C for 3 h. The insets along with red arrows reveal the selected area of the SEM at which EDAX spectrum was recorded and also present the weight and atomic percentage of different elements.

Figure 3. Scanning electron microscopy images of (a) nHAp, (b) 99.9HAp−0.1GNP, (c) 99.8HAp−0.2GNP, (d) 99.7HAp−0.3GNP and (e) 99.5HAp−0.5GNP composites sintered at 1200 °C for 3 h. The different pores are presented by yellow arrows while blue arrows present the mixed and large grains of HAp−GNPs.

°C for 3 h are selected for the recording of the EDAX spectrum. The EDAX recorded grains, as well as the elemental, were marked by the red arrow. The EDAX spectra revealed the presence of different elements of HAp as Ca, P, C, and O peaks; however, a low intensity peak of Na impurity was observed, which might be due to the precursor impurity (Figure 4a). Hence, the incorporation of GNPs into the nHAp was observed in the form of carbon (i.e. “C”) for the composite sample 99.5HAp−0.5GNPs. Thus, an EDAX spectrum clearly confirmed the presence of the used additive. 2.4. Fractured SEM Analysis. The fractured SEM images of the samples nHAp, 99.9nHAp−0.1GNPs, 99.8nHAp− 0.2GNPs, 99.7nHAp−0.3GNPs, and 99.5nHAp−0.5GNPs are shown in Figure 5a−e and exhibited similar interconnected porous morphology. Moreover, SEM images of the fractured samples show the irregular shape and size of the pores. The addition of GNP content into the nHAp matrix improved the porous microstructures significantly (Figure 5e).5 When the compressive load is applied to the samples, a random fractured debonding morphology of the composite was observed. Due to the compression load, debonding persuaded the surface roughness of the fractured samples. Thus, there is no straight crack propagation presented in the fractured SEM images. However, the microstructure of these composites showed some randomly oriented grain and grain boundaries, which are found to be prevented by GNP nanosheets that acted as a bridge while initially fine cracks are propagated. It is well reported that the load bearing applications of pure nHAp are limited due to its inherently low mechanical strength and high brittleness as compared to graphene and natural bones.52−54 Thus, the grain boundaries of nHAp are weaker than those of GNPs that randomly overlap

changes were observed in the surface morphology with the addition of GNPs. From the SEM images, a more obvious restricted effect was observed for composite 99.8nHAp− 0.2GNPs as compared to the composite 99.7nHAp−0.3GNPs. It is observed that the nHAp−GNP composite had open pores on the surfaces as well as inside the material (disc) with a high degree of interconnectivity. The porosity and pore diameter increased with the increasing amount of GNPs because GNPs reduce the agglomeration of nHAp particles during sintering. The overlapping, interconnectivity of the pores, enhanced the porosity of the nHAp−GNP composites. Moreover, as the concentration of GNPs was increased, the grain boundaries and their separation with the pores were seen clearly. The SEM images of the nHAp and its composite samples are shown in Figure 3a−e. Figure 3a reveals a dense surface morphology of nHAp in comparison to the nHAp−GNP composite samples. It also exhibits a uniform grain growth of nHAp along with an average pore size of ∼5 μm; the pores are marked by yellow arrows throughout the SEM image. This nHAp−GNP composite is predictable for the grains growing at a high rate along the path where no GNPs are present. It is also observed that the composite surfaces are highly porous in nature with the increased pore size interconnectivity between GNPs and nHAp. The highly porous nature and interconnectivity between GNPs and nHAp are attributed to a homogeneous dispersion of GNPs into the nHAp matrix (Figure 3b−e). The elemental analysis was done using EDAX results and is shown in Figure 4a,b. The tentative chemical compositions of nHAp and the 99.5HAp−0.5GNP composite sintered at 1200 7451


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Figure 5. Scanning electron microscopy images of fractured samples (a) nHAp, (b) 99.9HAp−0.1GNP, (c) 99.8HAp−0.2GNP, (d) 99.7HAp−0.3GNP and (e) 99.5HAp-0.5GNP composites sintered at 1200 °C for 3 h. The red dashed lines show randomly oriented crack lines.

each other and make them stronger.55 As 0.1 and 0.2 wt % of GNPs was added to the nHAp, the grain boundaries appeared blurred as shown in Figure 5b,c. The further the relatively large amount of GNPs was increased, the agglomeration of the GNPs particles was observed and was reducing its density (Figure 5e). The observed rough surfaces of the porous composite bodies may favor the cellular adhesion that induces the new bone formation activity.6,16 2.5. Transmission Electron Microscopic (TEM) Analysis. The structural and morphological analyses were confirmed on the basis of the TEM results. Figure 6a−c shows the low and high magnification TEM images of the composite sample 99.5HAp−0.5GNPs sintered at 1200 °C for 3 h soaking time with heating at 5 °C/min. Figure 6a presents a low magnification image of the composite 99.5HAp− 0.5GNPs and shows a well-interconnected porous network between nHAp and GNPs. The calculated average pore size of ∼4 nm was observed, and the graphene nanoparticles are well dispersed in the nHAp matrix, which is indicated by the dark region specified by yellow arrows. The nanoparticles of the GNPs are uniformly distributed into the porous network of nHAp; the dense nHAp matrix formed a big cluster that enhanced the strength of these composites. The high resolution TEM images are shown in Figure 6b,c. The porous structure of the composite can be seen clearly in these micrographs. However, the agglomeration of the nHAp particles was observed over large nanosheets of GNPs, which makes them very dense in nature (Figure 6b,c).9,10 The highmagnification bright field TEM image shows the large HAp sheets in which GNPs are uniformly embedded with adequate porosity (Figure 6d). The selected area electron diffraction pattern (SAED) is shown in Figure 5e. This pattern clearly

Figure 6. TEM images of the composite sample 99.5HAp−0.5GNPs sintered at 1200 °C for 3 h: (a) low-magnification bright field image, (b) low-magnification bright field image showing the porous interconnected network between HAp and GNPs, (c, d) Highmagnification images containing fine grains of HAp with dispersed GNPs, and (e) SAED pattern of 99.5HAp−0.5GNPs.

exhibits the three different characteristics of diffraction rings corresponding to the lattice planes (000), (002), and (211) of nHAp, which confirmed the highly crystalline nature and are very close to the [11̅00] zone axis of the composite.12 Thus, the obtained results are well consistent with the results of the XRD and SEM. 2.6. Mechanical Behavior. After a structural characterization of the synthesized composites, we performed the mechanical tests. For this purpose, samples were subjected to compression by an Instron universal testing machine. The compressive load−displacement curves are shown in Figure 7a−e. The curves show the load bearing capability and were linearly increased with the increasing concentrations of GNPs.10 The load bearing capability of the 0.5 wt % composite sample 99.5HAp−0.5GNPs was found to be ∼3.41 kN, which is almost 3-fold of the pure nHAp (1.33 kN).11 Moreover, in load versus displacement plots, the displacement decreased with the increase of the amount of GNPs, and it was found to be 0.45 mm for the composite 99.5HAp−0.5GNPs instead of 0.60 mm for pure nHAp.12 Thus, a reduction in displacement shows an improvement in the strength of nHAp as well as an enhancement in the load bearing capability of nHAp that resulted from the addition of GNPs into nHAp. The GNPs enhanced the interlocking and resisted crack propagation for these composites.13 Being 2D in nature, graphene can bear the 7452


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propagation.22 The values of both fracture toughnesses and Young’s modulus increased with the increasing concentrations of GNPs in the nHAp matrix (Table 1). The compressive strength of the prepared nHAp and its composites are shown in Figure 7d. As increasing the weight percentage of GNPs, the compressive strength was also found to have increased from 87 to 96 MPa. Thus, the overall study concluded that the addition of 0.5 wt % GNPs significantly enhanced the mechanical properties of the nHAp.23 The incorporation of GNPs has the ability to tune the mechanical properties of the different composite materials that were demonstrated for useful applications of bone implants. 2.7. Cell Viability Behavior. Qualification of the cell viability examination is necessary for a biomaterial to be considered as an implant/scaffold material. Therefore, among all compositions, the 99.5HAp−0.5GNPs that has achieved the best mechanical properties was examined through this cell viability test. In this test known as the MTT assay, the MTT metabolic activity is measured by using a specific cell type. In the present study, the gut tissues of Drosophila larvae and primary osteoblast cells of a rat are individually used, and cytotoxicity/cell viability results were measured by using MTT [3-(4, 5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide] for both types of cells. In the case of the toxicity measurement on the gut tissues of Drosophila larvae, various increasing concentrations (10, 30, 50, 100, 500, and 1000 μg/ mL) of nHAp and the 99.5HAp−0.5GNP composite were used, as shown in Figure 8a,b. Treatments with 10 μg/mL of

Figure 7. The mechanical characteristics of nHAp and its composites: (a) nHAp, 99.9nHAp−0.1GNPs, 99.8nHAp−0.2GNPs, 99.7nHAp− 0.3GNPs, and 99.5nHAp−0.5GNPs. (b) Variations of Young’s modulus with increasing weight percentage of GNPs. (c) Variations of fracture toughness with increasing weight percentage of GNPs. (d) Bar graph showing the variation of compressive strength with increasing weight percentage of GNPs sintered at 1200 °C for 3 h.

maximum load in both directions, that is, longitudinal and lateral, and therefore, enhanced the load transfer efficiency significantly. The reduced extensibility with improved brittleness may sometime facilitate the material into confined bone defects or enable hard tissue engineering applications.2,5,15,23 The increasing compressive strength of a material revealed that the mechanical strength was significantly enhanced by increasing the concentration of GNPs into the nHAp during sintering.14 The composite 99.5HAp−0.5GNPs achieves the highest value of compressive strength ∼97 MPa, while it is ∼87 MPa for pure nHAp. However, the compressive strength value of the 0.1 wt % GNPs−HAp composite is ∼87 MPa, which is very close to the pure nHAp.15 The compressive strength and elastic modulus of the nHAp−GNPs composites are significantly higher than that of the compressive strength and elastic modulus of the pure nHAp.16 The improvement in mechanical properties suggests the role of the bonding interface between nHAp and GNPs.18 The homogeneous dispersion of GNPs in the HAp matrix improves the mechanical properties of the composites in different ways. Graphene prevented the migration of the grain boundaries of nHAp that ultimately enhance the compressive strength by decreasing the defect size and increasing the interface area between GNPs and nHAp.19 The interaction between the fine grains of HAp and nanosheets of the GNPs improved the binding strength that enhanced mechanical strength, interlocking, and load transfer capability between nHAp and GNPs.20 The variation of Young’s modulus with the increasing concentration of GNPs is shown in Figure 7b. The increasing weight percent of GNPs in the nHAp matrix enhanced the Young’s modulus value up to ∼98 GPa for the composite sample 99.5HAp−0.5GNPs.21 For the fracture toughness as shown in Figure 7c, the highest value of 116 MJ/m3 was observed for the same composite sample. The incorporation of GNP particles are seen inside the intergranular region that provides a high resistance to crack

Figure 8. MTT assay of the (a) nHAp and (b) 99.5HAp−0.5GNP composite in gut tissues of Drosophila larvae for a 1 h incubation period at different concentrations.

nHAp, as well as composite powder samples, exhibited maximum cell viability in their respective groups; however, treatments with the highest concentrations of 500 and 1000 μg/mL of the composite sample and 30 μg/mL of nHAp showed the least cell viability, that is, the highest cytotoxicity. Figure 9a,b presents the rat osteoblast (ROB) cells obtained from calvaria and the cell viability (in terms of optical density, i.e., O.D.) of ROB cells after 24 h of treatment with different concentrations (0, 1, 5, 25, 50, and 100 μg/mL) of the 99.5nHAp−0.5GNP composite. The material’s exposure with lower concentrations of 1 and 5 μg/mL significantly enhanced the cell proliferation as compared to the control. However, higher concentrations 25−100 μg/mL were nonsignificant. It is interesting to note that the exposure with each concentration of the composite 99.5nHAp−0.5GNPs rather than pure nHAp exhibits improved cell viability of the gut tissues of Drosophila larvae. Therefore, the overall cell viability studies demonstrated that there is no cytotoxic effect of the 99.5HAp−0.5GNP 7453


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Figure 9. (a) Rat osteoblast (ROB) cells obtained from calvaria and (b) cell viability (in terms of optical density, O.D.) of ROB cells after 24 h of treatment with different concentrations (0, 1, 5, 25, 50, and 100 μg/mL) of the 99.5HAp−0.5GNP composite. The material’s exposure with lower concentrations 1 and 5 μg/mL significantly enhanced the cell proliferation as compared to the control.

Figure 10. In vivo fluorescence micrographs present the fluorescence activities of nHAp and the 99.5HAp−0.5GNP composite in gut tissues of Drosophila larvae.

no fluorescence as compared to nHAp-treated tissues.49 However, composite 99.5nHAp−0.5GNPs showed a maximum emission spectrum at the green filter (465−495 nm). The gut tissues also exhibited red fluorescence at the red filter (540−625 nm) with a predominant emission in the green range. Further, there was no clear mechanism that existed regarding the fluorescence of newly synthesized composite 99.5HAp−0.5GNPs, because there was neither Ca2+ nor PO43−

composite on any cell type, which may offer the synthesized composite sample for bone implant application. Recently, it has been reported that the nHAp composites are bioactive fluorescent materials that can be used for bioimaging diagnosis.48 The micrographs that present the fluorescence activities of nHAp and the composite 99.5nHAp−0.5GNP sample in the gut tissues of Drosophila larvae are shown in Figure 10. For the control, the untreated gut tissue facilitates 7454


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that exhibited any known fluorescence in the living cells.50 Hence, the nHAp composite could be attributed to selfactivated fluorescing behavior named as “fluorescent HAp” (fHAp).51 In the present study, pure nHAp and composite 99.5HAp−0.5GNPs present improved fluorescence activities in gut tissues of Drosophila larvae as compared to the control. Fluorescence emission in the green channel was found to be stronger in comparison to the red and bright field filters. In the merged channel, the 99.5HAp−0.5GNP composite also shows an improved fluorescence. Moreover, the 99.5nHAp−0.5GNP composite displays a better fluorescence behavior as compared to the pure nHAp. The results demonstrate that the biocompatible HAp−GNPs were performed successfully by in vivo imaging. Thus, nHAp and the HAp−GNP composite have promising fluorescence and wide range applicability for optimization and designing in the research of bioimaging.

the calcium nitrate solution and continuously stirred for 60 min. Further, the pH value of the solution was adjusted to 12 by adding a suitable amount of sodium hydroxide (NaOH) pellets. The suspension with a certain pH value was kept to a household microwave oven of 600 W with a refluxing system for 15 min; the microwave oven followed a working cycle of 5 s on and 10 s off. After microwave irradiation, the sample was cooled to room temperature.9 The obtained material contained a milky suspension, which was centrifuged and washed with deionized water for several times to remove the NH4+, NO3− ions and other volatile impurities. The white precipitate was dried at 100 °C for 20 h in a hot air oven. The dried HAp slurry was crushed by mortar and pestle to convert the crushed material into a fine powder form. Finally, the dried nHAp powder was calcined at 800 °C for 2 h with a heating and cooling rate of 5 °C/min.10 4.2. Synthesis of nHAp−GNP Composites. To synthesize the nHAp−GNP composites, different concentrations of GNP powders (0.1, 0.2, 0.3, and 0.5 wt %) were mixed by dissolving into 200 mL of ethanol. The dissolved mixture was stirred by a magnetic stirrer using zirconia beads for 4 h to produce a homogeneous composite of nHAp−GNPs.11 The obtained slurry was dried in a hot air oven at 80 °C for 6 h. The dried slurry was crushed and sieved, then ball-milled for 10 h to make fine powder for compaction. The nHAp−GNP composite fine powder was then cold-compressed using a hydraulic press machine at 2 ton and was held for 60 s to obtain a cylindrical shape of dimensions (height × diameter) 20 mm × 13 mm.12 The compact green samples were sintered at 1200 °C with a heating and cooling rate of 3 °C/min for 3 h to achieve desired densification. 4.3. Material Characterizations. In order to confirm the phase formation of pure nHAp and nHAp−GNP composite powders, the various X-ray diffraction (XRD) patterns were recorded using a Rigaku Miniflex II X-ray diffractometer equipped with a monochromatic Cu-Kα radiation (λ = 0.15418 nm) operated at 40 kV and 40 mA. Data were recorded in the 2θ range from 20°−60° with fixed scanning at 3°/min. XRD patterns of the sintered nHAp and nHAp−GNP composite samples were then compared with standard JCPDS files no. 24−0033 for the determination of different phases and formation of the reaction product as new phases. The crystallite size (d in nanometers) was calculated with the help of Scherrer’s formula

3. CONCLUSIONS Highly porous nHAp and its nanocomposites have been successfully synthesized via microwave irradiation and a scalable solid-state reaction method. The maintained structure of the nHAp−GNP composite revealed a strong bonding between nHAp and GNPs in the composite. The strong interaction between nHAp and GNPs is due to the increased interfacial area between these nanoparticles. The GNPs are well distributed into the nHAp matrix, which enhanced the mechanical properties of the composite. The addition of a relatively large concentration of GNPs into the nHAp matrix prevents the nHAp grain growth during sintering of the composites at 1200 °C. All the synthesized composites possess a porous 3D interconnected structure with low density. At this high sintering temperature, the mechanical properties of nHAp were significantly improved by the incorporation of GNPs. The enhanced mechanical properties were observed for the composite sample 99.5HAp−0.5GNPs. It is concluded that only a 0.5 wt % amount of GNPs highly modified the surface morphology and mechanical properties. The biological tests of the nHAp and composite 99.5nHAp−0.5GNPs showed good biocompatibility and cell viability with gut tissues of Drosophila third instar larvae as well as with ROB cells. Therefore, the fabricated composite may have great potential for load bearing and bioimaging applications in bone engineering. 4. METHODS AND MATERIALS 4.1. Materials Synthesis. In the synthesis of nHAp, calcium nitrate tetrahydrate (Ca(NO3)2·4H2O), disodium hydrogen phosphate (Na2HPO4), and sodium hydroxide (NaOH) pellets were purchased from Merck (Merck Pvt. Ltd., Mumbai, India) and used as starting raw materials. To adjust the pH value of the solution, sodium hydroxide pellets were used with continuous stirring. The synthesis of nHAp follows the given chemical reaction below:

d=

kλ β cos θ

(1)

where k is a Scherrer constant (k = 0.94), β is the peak width of the diffraction peak profile at full width half maximum (FWHM) in radians, λ is the X-ray wavelength of Cu-Kα radiation (λ = 1.54 nm), and θ denotes the Bragg’s angle in degrees. The densification behavior of pure HAp, as well as nHAp−GNP composite samples, was measured using the Archimedes method. Distilled water was used as a substituting material because it can easily penetrate into the pores of the nHAp−GNP composite samples. The sintered porous pellets of nHAp and nHAp−GNP composite samples were immersed in double-distilled water, and no vapors were seen coming out from them. Then, the dry, soaked, and hanging weights of the pellets were measured using a Shimadzu-made weighing digital balance having readability up to 0.0001 mg. By using the formula below, the density of the sintered samples was calculated

5Ca(NO3)2 ·4H 2O + 3Na 2HPO4 + 4NaOH → Ca5(PO4 )3 (OH) + 10NaNO3 + 23H 2O

To synthesize the nHAp, the amount of reactant in the molar ratio of Ca2+/PO43− was adjusted to 1.67. However, Ca(NO3)2·H2O:Na2HPO4 were taken in the molar ratio of 5:3; Ca(NO3)2·H2O (1.0 M) and Na2HPO4 (0.6 M) were separately dissolved in 100 mL of double-distilled water. The Ca(NO3)2·4H2O solution was stirred by a magnetic stirrer; after 45 min of stirring, the Na2HPO4 solution was added to 7455


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Wt(air) i g y × ρ(water) densityjjj 3 zzz = Wt(air) − Wt(water) k cm {

the tissues were washed twice with 1× PBS and incubated with 0.6 mg/mL MTT for 2 h at 37 °C in the dark. After incubation, the tissue was washed twice with 1× PBS and incubated with 200 μL DMSO for another 1 h at 37 °C to dissolve the purple-colored formazan crystal, and the colored solutions were transferred into the distinct wells of a 96-well culture plate. The absorbance intensity was analyzed by the microplate reader (Auto Reader 4011, SPAN Diagnostics Ltd.) at 492 nm with a reference wavelength of 630 nm. All experiments were performed in triplicate, and the cell viability was expressed in percentage in relation to the control. The cells’ viability was determined by the absorption at 570 nm. The absorption of the wells without the sample was considered as blanks. The adsorption of HAp resulted in the highest absorption, which reflected the best cell viability. The effect of the samples on the cell proliferation can be written as the cell viability using the following formula

(2)

where ρ (water) is the density of distilled water (1 g/cm ), Wt (air) is the weight of the specific gravity bottle with the sample in air, and Wt (water) is the weight of the specific gravity bottle with sample and water. The percentage porosity of the HAp samples was measured using the Archimedes principle according to the equation below 3

g

( )

apparent density P=1−

cm 3

total theoretical density of pure nHAp × 100%

g

( ) cm 3

(3)

where the apparent density was calculated by mass per unit volume of the composite samples in g/cm3. The standard theoretical density of pure nHAp was 3.156 g/cm3.,56,57 The surface morphology of the nHAp and nHAp−GNP composite samples, as well as the fractured samples, were recorded using a scanning electron microscope (SEM) attached with an FE-SEM detector (JEOL JSM-6400, Japan). The energy dispersive X-ray analyses (EDS) were also carried out to investigate the elemental composition of the nHAp and HAp−GNP composites. JEOL 2100 field emission gun transmission electron microscope was used to record the TEM images and diffraction patterns. To record the images, a small amount of the composite particle was diluted with isopropyl alcohol. The diluted particle was dropped onto a carbon-coated copper grid and allowed to dry in vacuum for the duration of 30 min. Then TEM samples were placed in the vacuum for 12 h. TEM images were analyzed through the Image J software to determine the size and shape of the particles. The mechanical behavior of the nHAp and HAp−GNP composites were measured using a universal testing machine (UTM Instron 3639) in compression mode at a crosshead speed of 5 mm/min. The samples’ size of 1.3 cm in diameter and 2.0 cm in length was used for mechanical testing on cylindrical pellet samples. The mechanical characterizations were also carried out at 23 °C temperature and 20% humidity atmospheric conditions. The load bearing behavior of the composites was studied by plots of the load−displacement curve until failure. The Young’s modulus was obtained from the slope of the stress−strain curve for each sample. The fracture toughness of the sintered nHAp−GNP composites was measured as the area under the stress−strain curve within the limit of an initial point to the elastic limit. 4.3.1. MTT Assay of HAp and nHAp−GNP Composites. 4.3.1.1. On Larval Tissues of Drosophila. Drosophila (Oregon R+) obtained from the Bloomington stock center, Indiana, USA, was used in this study. An MTT [3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide] assay was performed to test the cytotoxicity of the nHAp−GNP composite in gut tissues of Drosophila third instar larvae. The nHAp− GNP composites were dissolved in 2% dimethyl sulfa-oxide (DMSO) with increasing concentrations (10, 30, 50, 100, 500, and 1000 μg/mL) in fresh Eppendorf tubes. For cytotoxicity experiment, initially, 10 larvae were teased out in 1× PBS (pH 7.4) and incubated with each composite for 1 h at room temperature. However, for the control, the gut tissues were incubated for 1 h in DMSO only. After the end of incubation,

cell viability =

At × 100% Ac

(4)

where At is the absorbance of the test sample, and Ac is the absorbance of the cells without HAp treatment. 4.3.1.2. On Rat Osteoblast Cells Drawn from Calvaria. The experimental protocol for studying the cell viability of rat osteoblast calvarial cells due to the exposure of prepared materials was adopted exactly from our previously reported studies.12,63 4.3.1.3. Statistical Analysis of the MTT Test. For the analysis of biological data, the following statistics were used. The biological data are represented as mean ± standard error of the mean (mean ± S.E.M). The group differences were determined using a one-way analysis of variance (ANOVA) with a Neuman−Keuls post hoc test by Prism version 5.0 software. Moreover, probability values of p < 0.05 are taken to be statistically significant (*P < 0.05), when compared with the control, that is, without the treated cells. 4.3.2. Treatment of nHAp−GNP Composites in Gut Tissues of Drosophila Larvae. The gut tissues of Drosophila third instar larvae were dissected in 1× PBS (pH 7.4), and for the control, 10 dissected gut tissues were incubated in 2% DMSO for 2 h at room temperature; the same number of gut tissues was also incubated separately with each nHAp composite concentration 100 μg/mL for 2 h in Maximo slides. The 2% DMSO solvent was used for each of the HAp composites. After the end of incubation, the gut tissues were washed twice with 1× PBS solution for 5 min, and we captured the HAp composite treated with gut tissues fluorescence images of Drosophila using a Nikon Niu upright fluorescence microscope.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03473. Synthesis of nanohydroxyapatite (nHAp), details on the synthesis of nanocomposites via a solid-state route, and XRD analysis of pure graphene nanoplatelets (PDF) (PDF) 7456


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(9) Mishra, V. K.; Bhattacharjee, B. N.; Kumar, D.; Rai, S. B.; Parkash, O. Effect of a chelating agent at different pH on the spectroscopic and structural properties of microwave derived hydroxyapatite nanoparticles: a bone mimetic material. New J. Chem. 2016, 40, 5432−5441. (10) Gautam, C. R.; Kumar, S.; Biradar, S.; Jose, S.; Mishra, V. K. Synthesis and enhanced mechanical properties of MgO substituted hydroxyapatite: a bone substitute material. RSC Adv. 2016, 6, 67565. (11) Gautam, C. R.; Tamuk, M.; Manpoong, C. W.; Gautam, S. S.; Kumar, S.; Singh, A. K.; Mishra, V. K. Microwave synthesis of hydroxyapatite bioceramic and tribological studies of its composites with SrCO3 and ZrO2. J. Mater. Sci. 2016, 51, 4973−4983. (12) Gautam, C. R.; Kumar, S.; Mishra, V. K.; Biradar, S. Synthesis, structural and 3-D architecture of lanthanum oxide added hydroxyapatite composites for bone implant applications: Enhanced microstructural and mechanical properties. Ceram. Int. 2017, 43, 14114−14121. (13) Mukherjee, S.; Sen, S.; Chanda, A. Mechanical and In-vitro Performance of CNT Reinforced Hydroxyapatite Prepared by a Shear Mixing Method. J. Nanomed. Nanotechnol. 2013, 04, 169. (14) Baradaran, S.; Moghaddam, E.; Basirun, W. J.; Mehrali, M.; Sookhakian, M.; Hamdi, M.; Nakhaei Moghaddam, M. R.; Alias, Y. Mechanical properties and biomedical applications of a nanotube hydroxyapatite-reduced graphene oxide composite. Carbon 2014, 69, 32−45. (15) Kumar, A.; Biswas, K.; Basu, B. Hydroxyapatite-titanium bulk composites for bone tissue engineering applications. J. Biomed. Mater. Res., Part A 2015, 103, 791−806. (16) Zhao, H.; Dong, W.; Zheng, Y.; Liu, A.; Yao, J.; Li, C.; Tang, W.; Chen, B.; Wang, G.; Shi, Z. The structural and biological properties of hydroxyapatite-modified titanate nanowire scaffolds. Biomaterials 2011, 32, 5837−5846. (17) Rezvani, Z.; Akbari, M. Surface modification of hydroxyapatite crystals by Mg−Al−CO3-layered double hydroxides in HA/Mg−Al− CO3−LDH nanocomposite. New J. Chem. 2015, 39, 189−5196. (18) Stout, D. A.; Webster, T. J. Carbon nanotubes for stem cell control. Mater. Today 2012, 15, 312−318. (19) Nordin, J. A.; Prajitno, D. H.; Saidin, S.; Nur, H.; Hermawan, H. Structure−property relationships of iron−hydroxyapatite ceramic matrix nanocomposite fabricated using mechanosynthesis method. Mater. Sci. Eng. C 2015, 51, 294−299. (20) Azhari, A.; Toyserkani, E.; Villain, C. Additive manufacturing of graphene-hydroxyapatite nanocomposite structures. Int. J. Appl. Ceram. Technol. 2015, 12, 8−17. (21) Zhang, Y.; Pan, C. Measurements of mechanical properties and number of layers of graphene from nano-indentation. Diamond Relat. Mater. 2012, 24, 1−5. (22) Suk, J. W.; Piner, R. D.; An, J.; Ruoff, R. S. Mechanical properties of monolayer graphene oxide. ACS Nano 2010, 4, 6557− 6564. (23) Akhavan, O. Graphene scaffolds in progressive nanotechnology/stem cell-based tissue engineering of the nervous system. J. Mater. Chem. B 2016, 4, 3169−3190. (24) Paul, W.; Sharma, C. P. Blood Compatibility and Biomedical Applications of Graphene. Trends Biomater. Artif. Organs. 2011, 25, 91−94. (25) Zhang, L.; Xia, J.; Zhao, Q.; Liu, L.; Zhang, Z. Functional graphene Oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs. Small 2010, 6, 537−544. (26) Park, S.; Suk, J. W.; An, J.; Oh, J.; Lee, S.; Lee, W.; Potts, J. R.; Byun, J.-H.; Ruoff, R. S. The effect of concentration of graphene nanoplatelets on mechanical and electrical properties of reduced graphene oxide papers. Carbon 2012, 50, 4573−4578. (27) Zhou, K.; Thouas, G. A.; Bernard, C. C.; Nisbet, D. R.; Finkelstein, D. I.; Li, D.; Forsythe, J. S. Method to impart electro- and biofunctionality to neural scaffolds using Graphene−Polyelectrolyte Multilayers. ACS Appl. Mater. Interfaces 2012, 4, 4524−4531.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +918840389015. ORCID

Chandkiram Gautam: 0000-0003-3190-5577 Vijay Kumar Mishra: 0000-0003-1447-4787 Shyam Bahadur Rai: 0000-0002-6321-1038 Author Contributions

The present research work was conceived and supervised by C.R.G., and materials synthesis, fabrication, and manuscript writing were performed by C.R.G., S.K., and V.K.M. Biological testing was performed and analyzed by B.S.C., S.S., R.S.Y., S.B.R., V.K.M. and R.T. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.G. would like to thank the University Grant Commission, New Delhi, India for providing the financial support under Raman Post-Doctoral Research Award Fellowship (Award no. F5-65/2014 (IC)) and also appreciatively acknowledges the Science and Engineering Research Board, Department of Science and Technology (SERB-DST), New Delhi, Government of India for giving the financial support under Empowerment and Equity Opportunities for Excellence in Science (file no. EEO/2018/000647). Authors highly acknowledge Prof. P.M. Ajayan and Prof. Robert Vajtai, Department of Materials Science and NanoEngineering, Rice University, Houston, Texas, U.S.A. for managing the required facilities to carry out this research work. V.K.M. is thankful to SERB, Government of India for providing financial aid in form of NPDF (file no. PDF/2015/000915).

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