Osteoconductive Amine-Functionalized GrapheneâPoly(methyl

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Osteoconductive Amine Functionalized Graphene-Poly(methylmethacrylate) Bone Cement Composite with Controlled Exothermic Polymerization Rakesh Sharma, Govinda Kapusetti, Sayali Y Bhong, Partha Roy, Santosh Kumar Singh, Shikha Singh, Biswajit Ray, Pralay Maiti, and Nira Misra Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00241 • Publication Date (Web): 28 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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Bioconjugate Chemistry

Osteoconductive Amine Functionalized Graphene-Poly(methylmethacrylate) Bone Cement Composite with Controlled Exothermic Polymerization Rakesh Sharma1#, Govinda Kapusetti2#, Sayali Yashwant Bhong2, Partha Roy3, Santosh Kumar Singh4, Shikha Singh5, Biswajit Ray5, Pralay Maiti6 and Nira Misra1*

1

School of Biomedical Engineering, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India 2

Department of Medical Devices

National Institute of Pharmaceutical Education and Research –Ahmedabad 3

Department of Biotechnology, Indian Institute of Technology Roorkee 247667 Roorkee 247667, India 4

Centre of Experimental Medicine and Surgery,

Institute of Medical Sciences, Banaras Hindu University, Varanasi 5

Department of Chemistry,

Banaras Hindu University, Varanasi 221005, India 6

School of Materials Science and Technology, Indian Institute of Technology (Banaras Hindu University), Varanasi 221005, India

# Equal author contribution * To whom correspondence should be addressed Email : [email protected] (Nira Misra) Contact : 0091-9452569747 1 ACS Paragon Plus Environment


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Abstract Bone cement finds its extensive usage in joint arthroplasty since last 50 years, still, the development of bone cement with essential properties like high fatigue resistance, lower exothermic temperature, and bioactivity has been an unsolved problem. In our present work, we have addressed all the mentioned shortcomings of bone cement by reinforcing it with graphene (GR), graphene oxide (GO) and surface modified amino graphene (AG) fillers. These nanocomposites have shown hypsochromic shifts suggesting strong interactions between the filler material and the polymer matrix. AG based nanohybrids have shown greater osteointegration and lower cytotoxicity as compared to other nanohybrids as well as pristine bone cement. They have also reduced the oxidative stress on cells while resulting in calcification within twenty days of implantation of nanohybrids into the rabbits. They have significantly reduced the exothermic curing temperature to body temperature and increased the setting time to facilitate practitioner, suggesting that reaction temperature and settling time can be dynamically controlled by varying the concentration of the filler. Thermal stability and enhanced mechanical properties have been achieved in nanohybrids vis-à-vis pure bone cement. Thus, this newly developed nano-composite can create natural bonding with bone tissues for improved bioactivity, longer sustainability and better strength to the prosthesis.

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Introduction The key success of a joint prosthesis depends on the bonding of the system; fixing of the metal prosthesis onto anatomically uneven bone tissues. A simple approach of filling a grouting material between bone and the implant to make mechanical bonds was initiated by Sir John Charnley in 1960 using non-degradable poly (methyl methacrylate) (PMMA) based material, which is the only successful approach till date.1 The basic function of such grouting materials (commonly known as bone cement, BC) is to establish a bridge between the bone and implant for uniform transfer of pressure as well as shock absorption during physical activities. The use of grouting materials prevents the failure of a joint prosthesis and improves the life span of a joint prosthesis to approximately 15 -18 years.2 Despite the huge success of joint prosthesis, the existing BC is highly prone to mechanical failure. Fatigue generated in the material during stress transfer/ impact loading causes failure of cement mantle leading to osteolysis and eventual aseptic loosening.3-4 However, the major limitation of BC is its inability to share any interactive interface with the bone, thus, lacking any adhesiveness to the bone, resulting in the complete failure of the prosthesis.5-6 The commercially available BC has two components namely; the powder and the liquid material, and specified quantities of each of them are mixed just before its application at the surgical site. The mixture starts hardening as benzoyl peroxide (BPO), present in the liquid component, starts radical polymerization. Exothermic nature of this polymerization reaction increases the temperature of surrounding environment up to ~80-100oC leading to thermal necrosis of the bone tissue and subsequently inducing aseptic loosening.7-9 Enormous research has been done to resolve these issues; however, many of them remain unsolved. Fatigue related failures have been resolved to a great extent by using PMMA copolymer based bone cement.10 The most promising methodology for mechanical property enhancement has been to reinforce the polymer matrix with suitable filler materials.11-14 Interestingly, osseointegration of BC to 3 ACS Paragon Plus Environment


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provide a stable bonding has also been achieved by the similar approach of using specific bioactive additives such as hydroxyapatite and titanium fibers, etc. as fillers.15-17 Though fatigue resistance and bioactivity have been reported in some formulations, no such formulation has been approved by USFDA or any such agency for clinical applications. In previous studies, we have also reported some exciting formulations which offer higher fatigue resistance, reduced exothermic temperature, and improved osteoconductivity.18-19 In the mainstream of materials research, graphene (GR) is a versatile nano-material due to its exceptional characteristics. Researchers have demonstrated outstanding results in disparate fields like electronics, composites, energy, sensors, catalysis and biomedical sensing.20-23 Mono-layered carbon atoms arranged in a planar honeycomb lattice have shown fascinating properties, such as rapid charge carrier mobility (200,000 cm2 V−1 s−1),24-25 high Young’s modulus (1 TPa),26 high thermal conductivity (5000 W/mK), sustainability to high electric current and absolute impermeability to gases. GR consists of a π-conjugated layer of six atom ring as a unit block; these thin sheets are highly capable of interacting with a variety of compounds through π–π interactions in the process of making nanocomposites, biomolecule immobilization and functional groups stacking.27-29 GR provides a higher rate of surface modifications as well as offers huge surface area as compared to other nanomaterials. Due to its sheet-like (2-D) structure, both the planes of GR can be utilized for the addition/adsorption of molecules or functional groups in a controlled manner. Further, its surface modification approach could be used to tune its biocompatibility and colloidal stability attributes. GR and its derivates are largely reported as a paternal contender in biosensors,30 scaffolds,31-32 drug delivery,33-34 antibacterial agents,35 and bioimaging33-36 applications. In this study, we have tried to address various limitations of BC by the synthesis of nanohybrids with GR and its derivatives. The synthesis of GR and its functional derivatives was carried out, followed by the synthesis of their nanohybrids with BC by the addition of a small amount of GR and its 4 ACS Paragon Plus Environment

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derivatives. The synthesized nanohybrids have been evaluated for exothermic temperature, chemical interactions, mechanical properties and finally biocompatibility through in-vitro and in-vivo approaches against pristine BC.

Results and discussion Characterization of the fillers: FTIR has been employed to determine the chemistry of functional groups on graphene. Figure 1a shows the FTIR spectra of GR, GO, and AG. There are no prominent transmission peaks observed in GR since it is IR inactive while a band observed at 3400 cm-1 is due to atmospheric moisture in GR. GO has prominent peaks at 1055, 1390,1623, 1745 and 3400 cm-1 and can be ascribed to alkoxy C–O, C–OH, carboxyl O=C–O, aromatic C=C, C=O (carboxylic acid and carbonyl moieties) and O–H stretching frequency, respectively, confirm the successful oxidation of graphite.38-39 Further, GO was reduced using hydrazine for the synthesis of graphene. The reduction is supported by the loss of peak at 1745cm-1, corresponding to C=O stretching frequency. Solvothermal treatment was done for amine functionalization, and it was observed that oxygen-containing groups have disappeared. The formation of AG was affirmed by the presence of peaks at around 1701 cm-1 for amide carbonyl stretch and a large peak around 3440 cm-1 assigned to the -NH2 stretching frequency. One sharp peak was observed around 1557 cm-1 representing the N-H stretching frequency. Bands at 2916 and 1082 cm-1 were ascribed to the presence of CH2 and C-N stretching confirming the presence of amine groups on the graphene framework.40 The functionalization on graphene has also been studied using Raman spectroscopy. In the case of the graphitic lattice, the ratio of carbon atoms with sp2 and sp3 hybridization is the perfect indication of the degree of oxidation or covalent bond formation. The ratio of the peaks was calculated from Raman spectra as ID/IG, where ID and IG are the intensities of the peaks appeared at around 1345 and 1560 cm−1, respectively, and is presented in Figure 1b. The pristine GR is a 2D carbon sheet with sp2 5 ACS Paragon Plus Environment


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hybridization; while the presence of sp3 hybridized carbons in the lattice are inherently classified as defects, where these defects can be recognized as basal edges or defects in the plane (functional groups). The observed values for GO, reduced GO, and AG were 1.09, 1.17 and 1.31, respectively, representing the successful covalent functionalization. The observed Raman shift was almost 10 cm-1 in the case of reduced GO, while 20 cm-1 shift occurs in AG. The increment of ID/IG ratio from GO to reduced GO confirms the reduction process. The crystallite size (La) has been calculated as follows: La (nm) = 2.4 × (10−10) *λ4*(ID/IG) Where, λ is the laser excitation wavelength, 631 nm. The crystallite sizes of GO, reduced GO, and AG were found to be 15.30, 15.32 and 12.84 nm, respectively. Crystallite size has been reduced in AG by 2.5 nm as compared to GO presumably due to the formation of covalent bonding through the amino group with GR sheet after the reduction of hydrate. However, FTIR and Raman studies indicate functionalization of the graphene sheet. A mixture of wrapped single and stacked multi layered like morphology has been observed in GR (Figure 1c). Exfoliated thin layers of graphene are evident in the SEM image of GR against the stacked graphite while folded semi-transparent layers are noticed in AG. However, morphological changes are evident because of the functionalization on and over the graphene sheet. Figure 1d shows the AFM results of GR, GO and AG and their respective height profiles in nanometers. GO has shown an average thickness of ~8 nm similar to previous report whereas for ethylene diamine grafted graphene oxide (AG), the thickness has been found to be ~4 nm probably because of the functionalization using thionyl chloride and ethylene diamine.41 While in case of GR, in contradiction with GO and AG, the average thickness has been observed to be of ~12 nm probably owing to the fused layers, as aggregation increases by the loss of functional group during reduction process.42


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Figure 1: (a) FTIR spectra of GR, GO and AG showing various functional groups to confirm their synthesis, (b) Respective Raman shifts demonstrate the oxidation of graphite to GO, reduction of GO and solvothermal reduction of AG, (c) SEM images of the pseudo single layered graphene (GR), exfoliated layers of graphite after oxidation in GO and fold or wrapped structured transparent graphene layers in AG, and (d) AFM images of GR, GO and AG and their respective height profile (thickness). 7 ACS Paragon Plus Environment


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Maximum exothermic (Tmax) temperature, setting time (tset) and interactions: The chain growth process of bone cement (BC) is a highly exothermic process, and it creates thermal necrosis at the cement-bone interfaces. BC and its different nanohybrids were prepared through manual mixing method. The temperature of the exothermic reaction was measured as a function of time during the mixing/polymerization process. Maximum temperature (Tmax) of 72 ± 1 °C and setting time of 5.5± 0.6 min was observed for BC, and the similar temperature was measured for BC-GO and BC-GR nanohybrids while tset were found to be slightly higher in nanohybrids (Figure2) as compared to pure BC. Interestingly, BC-AG has shown a drastic decrease of temperature of mixing i.e. the recorded temperature was around 38 ± 1 °C (p ≤ 0.001) and the setting time was found to be 22 ± 2 min (Figure2a). Tmax has significantly reduced in BC-AG presumably because of greater heat diffusion in presence of functionalized graphene with better adhesion and significant retarding effect of the amine groups on polymerization.16, 43 Thus, the observed temperature was constant for a long time improving the setting time to a reasonably higher scale. However, the efficiency of polymerization is not compromised as there is no significant change in molecular weight of BC-AG as compared to pure BC (Supplementary Figure S2). Reduction of Tmax is highly desirable for a joint prosthesis which evades any thermal necrosis and resists the aseptic loosening. Longer setting time of BC-AG is also a big advantage for the surgeon to implant the cement comfortably against quick setting time (~7 min) for commercial bone cement. The structure of the nanohybrids has been examined through XRD analysis. Figure 2b shows the X-ray patterns of GO, AG, and their respective bone cement nanohybrids. One sharp peak has been observed in GO at 2θ=10.5o corresponding to (002) plane whose d-spacing is calculated to be 0.81 nm. The introduction of oxygen and water molecules inside the gallery increases the interlayer distance.44 After the solvothermal process, the diffraction peak at 2θ=10.5o disappeared, and one new peak has been observed at 2θ=22.8o possibly due to reaggregation of GO sheets. One broad diffraction peak appeared 8 ACS Paragon Plus Environment

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in GR at 2θ=24.5o and its basal space is 0.49 nm, indexed to (002) plane. The broadened pattern reflects the lower crystallinity of GR as compare to the GO and GA and the lower d-spacing suggests its pseudo single layered structure. PMMA, being amorphous in nature, shows amorphous halo but the radioopaque agents (barium sulfate) show other diffraction peaks in BC. Diffraction peaks of GR, GO, and AG are not observed in BC-GR, BC-GO, and BC-AG, suggesting partially exfoliated or disordered structure. Thin sheets of GR, GO, and AG are homogeneously dispersed in the polymer matrix with random orientation losing its intercalated structure. Moreover, the content of the fillers is significantly less to obtain sufficient signal. Molecular level interactions were examined through UV-Vis and FTIR spectrum profile of the bone cement and its nanohybrids. BC typically possesses the chromophoric ester group, amenable to n→π* and π→π* transitions at 280 and 238 nm, respectively. GO structure is flanked by multiple auxochromic substituents (conjugated as well as non-conjugated carboxy, hydroxy, phenolic and epoxy bridges). BC-GO has shown a blue and hypsochromic shift as compared to BC because of the interaction of electron rich centers of GO with the electrophilic carbonyl moieties of BC. Similar shifts were also observed when GO (with acidic auxochrome–COOH group) was replaced with AG (with -NH2 as a basic auxochrome). Figure 2d shows the FTIR spectra of BC and its nanohybrids in the range of 2000-750 cm1

. Copolymer based bone cement exhibits several characteristic transmittance peaks such as 1730 cm-1

due to C=O stretching vibrations of ester carbonyl, aromatic C=C stretching vibrations frequency has appeared at 1528 cm-1, two peaks were observed around 1450-1374 cm-1 due to C-H deformation bands, and C-O stretching vibrations are visible at 1225-1077 cm-1.45 In nanohybrids, some new peaks appear corresponding to filler material frequencies demonstrating their presence in the polymer matrix. Interestingly, the peak corresponding to C-O (1077 cm-1) has shifted to lower wavenumber region in nanohybrids as compared to pure BC due to interactions, as demonstrated in UV-Vis spectroscopy and this shifting is relatively large in BC-AG (1058 cm-1) as compared to BC-GO and BC-GR. 9 ACS Paragon Plus Environment


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Figure 2: (a) Maximum (peak of the plot) temperatures evolved from the exothermic polymerization reaction and setting time (point halfway between the ambient temperature and the peak temperature) of BC, BC-GO, BC-GR and BC-AG, (b) X-ray diffraction patterns of GR, GO, AG, BC-GR, BC-GO and BC-AG, (c) Blue shifted UV-Vis spectra of BC-GO, BC-GR and BC-AG nanohybrids against pure BC, and, (d) molecular interaction between filler and bone cement matrix of BC-GO, BC-GR, and BC-AG through FTIR spectroscopy.


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Thermal stability and mechanical behavior: Thermal stability of different nanohybrids and pristine bone cement has been evaluated through thermogravimetric analysis (Figure3a). The degradation temperature (Tdeg) has been taken as the temperature corresponding to the loss of 5 wt.%. Tdeg of pure bone cement, BC-GO, and BC-GR and BC-AG are found to be 222 ± 3, 235 ± 3, 238 ± 3 and 259 ± 3 oC, respectively, indicating large enhancement of Tdeg in nanohybrids with respect to BC and amongst all the nanohybrids BC-AG has shown significant (37 oC) increment of Tdeg. Graphene has higher thermal resistance and homogeneously dispersed amine functionalized graphene is responsible for the large increment of thermal stability of amine functionalized nanohybrid vis-à-vis pure BC or other nanohybrids (BC-GO and BC-GR) presumably due to better interactions. Moreover, the improved thermal stability in nanohybrids is attributed to the formation of high aspect ratio and high thermal resistance between GR (functionalized) networks in the polymer matrix which acts as a thermal barrier to inhibit the emission of their decomposition products during heating.46-47 Further, the usual two-stage degradation of PMMA has been observed for pure bone cement and all the nanohybrids arising from the side chain as well as main chain degradation of polymer molecules. Mechanical properties of bone cement play an important role in the longevity of the joint system when functional stress occurs at the interface of the prosthesis-cement-bone system. Figure 3b shows the stress-strain curves of bone cement and its different nanohybrids. Nanohybrids have shown higher toughness, ultimate tensile strength, and Young’s modulus as compared to pristine bone cement. Moreover, BC-GR has shown higher modulus and strength (p< 0.001), at the same time BC-AG has also shown significant enhancement of strength (~ 44 MPa) (p< 0.001), and toughness (99 kJ.m-3) (p< 0.001), as compared to pure BC (strength 25 MPa and toughness 42 kJ.m-3) (Figure 3c). BC-GR has shown greater mechanical properties over BC-GO and BC-AG, presumably due to deterioration of functionalized graphene sheet during oxidation process and considerable thickness increment from 0.34 11 ACS Paragon Plus Environment


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nm to around 0.8 nm arising from the functionalization. Li et al. reported the Young’s modulus of graphene oxide to be reduced by around 200-300 GPa from graphene (1 TPa).48-49 Even though there is a slight reduction of mechanical properties in functionalized graphene sheets, BC-AG has shown much better mechanical properties than that of pure BC due to stronger interactions in the presence of functional groups and higher reinforcing effect of graphene. Hence, observed modulus and tensile strength are considerably enhanced in BC-AG and BC-GO as compared to pristine bone cement. It is quite evident that higher percentage filler loading usually creates partial agglomeration which limits the efficiency of further improvement.50 Also, weak interfacial interactions between the fillers and polymer matrix obstruct the efficient load transfer.51 Moreover, various functionalization of graphene sheets with different content enhances the mechanical properties of PMMA composite.52-53 BC-AG has improved ultimate tensile strength by 113%, which is quite higher than the literature reported values. Hence, it is evident that nanohybrids particularly BC-AG and BC-GR have shown two-fold increment in mechanical properties as compared to pure BC. Further, nanohybrids have exhibited better fatigue resistance than commercial bone cement. Therefore, nanohybrids are better alternative grouting materials for joint prosthesis surgeries for longer lifetime or durability.


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Figure 3: (a) Thermal stability of nanohybrids (BC-GO, BC-GR, and BC-AG) against pure bone cement by measuring the degradation temperature through thermogravimetric analysis (TGA). The scale has been reduced to 100 to 300 oC for better visualization, (b) Stress-strain curves of BC and its 0.5 wt% nanohybrids showing their tensile strength, and, (c) Bar diagrams show the detailed comparison of tensile modulus and toughness of nanohybrids against BC (Multi-level Y-axis shows the modulus and toughness).



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Biocompatibility and animal studies: Basic information of biocompatibility and bioactivity can be determined through in-vitro cell culture studies. Initially, cytotoxicity of bone cement and its nanohybrids were examined through MTT assay using human osteoblast-like cell line (MG-63). The study was conducted at various time intervals, and the cell seeded on tissue culture plate was taken as the control. The cell viability was found to be ~96 ± 2, 98± 1, 90 ± 0.5 and 118 ± 2% for pristine bone cement, BC-GO, BC-GR, and BC-AG (p ≤ 0.001), respectively, after 1 day of incubation. Over the time, BC-GO and BC-GR have shown a reduction in cell viability (below 80 %) while BC exhibits steady cell viability up to 5 days of incubation (Figure4a). Interestingly, BC-AG attributes enormous cell viability as compared to BC and it steadily increases with time. This is to mention here that GO has shown biocompatibility up to a certain concentration level of 20µl/ml.54-56 In the present study, nanohybrid (BC-AG) does not exhibit any toxicity up to the above threshold level. GO and GR penetrate into the cytoplasm by endocytosis pathway and disturb the metabolic activity, gene transcription and translation process provoking the cell apoptosis. In contrast, the larger size of AG due to functionalization leads to much better stability of the cells. The optical density of cells grown in different systems has been shown in Figure 4b. The qualitative analysis of cell viability was examined by DAPI staining and has been shown in Figure 4d. DAPI stains the live cell by DNA binding and exhibits blue light to the cell nuclei when it binds to the DNA helices. The live cell density is found to be 14 ± 3, 7 ± 1, 9 ± 1,2 ± 1 and13 ± 1 (per µm2) for control, BC, BC-GR, BC-GO, and BC-AG, respectively (Figure4c). Observed results strongly support the MTT assay. Moreover, cell health is quite normal in BC-AG, since the shape of the nuclei is spherical in all cells like in control, whereas BC, BC-GO, and BC-GR show irregular shapes presumably due to the slight toxicity of GO and GR.


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Cell viability (% )

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Figure 4: Cytotoxicity and bioactivity evolution of bone cement and its nanohybrids, (a) the bar diagram show MG-63 cell viability percentages on the specimens of BC and its nanohybrids for the incubation time intervals of 1, 3 and 5 days through MTT assay measurement showing better cell viability in BCAG, (b) MG-63 cell adhesion on BC and its nanohybrids in terms of optical density. The absorption values were taken at 570 nm wavelength, (c) DAPI stained fluorescent images of cells cultured on BC and its nanohybrids after one day of cell proliferation, indicating superior cell growth in BC-AG, and (d) Merged images of viable and non-viable tagged PI (red color) and only live cell permeable AO (green color) images, give the detailed analysis of apoptotic study of BC and its nanohybrids, green colour exhibits the live cells, red color apoptotic cells and yellow color reveals the late apoptotic cells, BC-AG has shown high density of live cells.



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Human osteoblast cells, which produce phenotypic osteoblast markers and growth factors, have been used on the specimens of pure bone cement and its nanohybrids for cell adhesion through crystal violet assay.57 Cell adhesion is determined through optical density and the values of 0.7±0.08, 0.6±0.06, 0.65±0.09, 0.9±0.05 (p < 0.01) and 1.25±0.02 (p < 0.001) have been found in control, BC, BC-GO, BCGR and BC-AG, respectively (Figure4b). BC-AG has shown higher osseointegration over the remaining nanohybrids and BC. Human osteoblast cell membrane is negatively charged primarily due to carboxylates and phosphates ions present in proteins and glycans.58 Therefore, positively charged BCAG (higher surface positivity of AG) strongly attract the osteoblasts.59 Further, the cell adhesion phenomena in BC-AG is favored from its better hydrophobic surfaces (contact angle ~98o) over BC and other nanohybrids (Supplementary figure S3). Cytotoxicity of BC and its nanohybrids were analyzed qualitatively by the acridine orange - ethidium bromide staining assay for three days incubation. The colorigraphic assay is based on the cell membrane integrity resulting from alterations of the cellular permeability to the acridine orange - ethidium bromide dyes. Acridine orange is a non-specific dye, stains the nuclei of cells with green color while ethidium bromide is a specific stain to destabilized membrane (dead cell) and gives red/orange color to the nuclei of the cell. The assay provides the dead cell information (apoptosis or necrosis) by colorigraphic morphology. The study reveals that BC-GO has shown highest cell death (red color or early apoptosis) while BC and BC-GR have also shown early as well as late (light yellow) apoptosis. Interestingly, BC-AG provides a much healthier environment to cells (Green color; Figure4d). Therefore, amine functionalized graphene-based bone cement (BC-AG) has shown insignificant necrosis. There are a couple of reports on the bio-acceptance of amine functionalized carbon nanotubesand graphene.58-61 Our results demonstrate the highly biocompatible and bioactive material properties of BC-AG vis-à-vis commercial bone cement as well as other nanohybrids based on graphene. It appears that functionalization with amine group wraps up the so-called toxic


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graphene to eliminate the toxicity effect. Thus, BC-AG nanohybrid is a suitable material for grouting and to establish osteointegration with bone for long survival time. It is well known that exposure of nanoparticles to a cell greatly increases the reactive oxygen species (ROS) level and induces cytotoxicity by dysfunction of mitochondria.62 The degree of ROS is the indicator for oxidative stress and directly influences the cell health. Cell membrane permeable DCFH-DA diffuses into cells and gets deacetylated by cellular esterase to non-fluorescent 2′,7′dichlorodihydrofluorescein (DCFH). Further, it is rapidly oxidized to highly fluorescent 2′,7′dichlorodihydrofluorescein (DCF) by ROS or free radicals (Figure 5a). The ROS level is directly proportional to fluorescence intensity within the cell cytosol. Figure 5b shows the mean fluorescence intensity (MFI) of BC, BC-GO, BC-GR and BC-AG is about 64x103±0.2x103, 60x103±0.15x103, 65x103±0.15x103, 54x103±0.03x103, 32x103±0.07x103, respectively, and the intensity is quite low in BC-AG (p < 0.001) confirming the free radical scavenging nature of AG. The radicals were produced from cement during polymerization. BC-GR and BC demonstrated significantly higher oxidative stress level and spherical cell morphology suggesting their apoptosis nature while BC-AG has shown very low toxicity promoting routine cellular activities for proper cell growth.



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Figure 5: The cell membrane permeable 2’,7’-dichloroflurescein diacetate (DCF-DA) gets oxidized to green fluorescent dichlorofluorescein (DCF) in the presence of free radicals generated by the bone cements. (a) fluorescence images of MG-63 cells in the presence of BC and its various nanohybrids, (b) quantification of ROS production as determinced by DCF-DA fluorescence, the bar diagram shows the ROS levels in terms of the mean fluorescence intensity (MFI) for BC and its nanohybrids.

The in-vivo examination is the best assessment method of bio-acceptance for a biomaterial. Invitro evaluation has confirmed that BC-AG is the suitable material for implantation over the remaining nanohybrids or pure BC. Hence, BC-AG is employed for in-vivo studies. Figure 6a shows the surgical procedure for implantation of different types of cement in rabbit tibia, here control group represents blank (no filling; Figure 6ai). Post-surgery healing procedure has been examined through X-ray imaging and has been conducted for every five days from the date of surgery. BC-AG implanted bone gets healed in 20 days after surgery (bright shade represents the calcification or healing). In contrast, the cavity filled with BC remain unaffected (size and shade) even after 20 days of post-surgery. Similarly, in control, the cavity is also unaffected i.e. yet to start the calcinationin control rabbit. It is worthy to mention that control represents the natural healing process where calcination is yet to start while the cavity filled with 18 ACS Paragon Plus Environment

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BC-AG has already been healed in 20 days. Hence, in-vivo experiment on rabbit demonstrates the efficacy of amine functionalized graphene-containing nanohybrid of bone cement. The deposition of calcium (Ca) and phosphor (P) elements in the interface of scaffold and tissue is a prime evidence of firm osteogenesis and it highly depends on the micro environment of the grouting material to the surrounding osteogenetic cells.63 Moreover, the amount of elemental deposition is associated with the scaffold biocompatibility and osteoconductivity. Supplementary figure S4 shows the EDX analysis of the extracted BC and BC-AG after 20 days of post-surgery. The deposition of Ca and P minerals are significantly high in BC-AG surface (11.8 ± 0.3 wt % and 24.56 ± 0.4 wt %) as compared to the in BC (7.08 ± 0.5 and 8.1 ± 0.4). The results suggest that the BC-AG has offered very conducive microenvironment to the surrounding cells for proper growth and proliferation to rapid mineral secretion. The EDX analysis is quite supportive for the X-Ray imaging that the BC-AG has offered higher osteoconductivity for rapid healing.



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Figure 6:(a) Surgical procedure for the cavity creation and bone cement filling, (i) cavity of 4 mm dia and 10 mm depth created by drilling apparatus (ii) solidified cement in the cavity (b) X-ray images show BC, BC-AG implanted and control (non-filling) rabbit tibia parts for examination of post-surgery healing mechanism at time interval of 0th and 20th day, red coloured circles show the surgery site, BCAG implanted bone shows higher calcification or proper healing of the cavity within 20 days, while cavity remains same in control and no calcification was observed in BC.


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To sum up, functionalization of graphene particularly with amine group has significant advantages for its preparation of composites with bone cement. The functionalized graphene strongly interacts with the matrix bone cement and, thereby, improves its properties like thermal, mechanical, hydrophobicity and biocompatible nature. The cytotoxicity effect of pure graphene or its composites is overcome by wrapping up of graphene layers with amine functionality which helps to significantly improve the biocompatibility. In-vivo study with rabbit establishes the much-enhanced efficacy of amine functionalized graphene as a bone healing agent. Ample of literature has been published to address the problems associated with the BC by the addition of suitable fillers primarily hydroxyapatite, titanium fibers, glass fibers, different forms of carbons, various nanoparticles, natural and synthetic clays etc. However, the hybrids/composites lag behind one or the other to become an alternative to commercial BC. In the present investigation, amine functionalized graphene based bone cement nanohybrid has demonstrated the reduced exothermic curing by controlled polymerization while exhibiting enhanced mechanical properties. Significantly, the novel formulation has offered proper osteoconductivity for rapid mineralization at the interface between the bone tissues and cement, which will eventually resist the aseptic loosening, and improve the life of the prosthesis. Therefore, the amine functionalized graphene filler puts forth its usefulness in various biomedical applications, particularly to promote the cell conductivity for better growth and proliferation.

Conclusions Graphene has been functionalized with amine containing group, and this functionalized graphene has been used to prepare nanohybrid with bone cement. Functionalization has been verified through FTIR and Raman spectroscopic measurement along with the changes in morphology. The mixing temperature has significantly been reduced to the body temperature in functionalized graphene composite and thereby avoiding any cell necrosis during implantation of bone cement. The nature of the 21 ACS Paragon Plus Environment


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interaction between functionalized graphene and bone cement has been worked out through FTIR and UV-vis studies. The thermal stability has increased by 37 oC as compared to pure bone cement. Both the stiffness and toughness increase significantly for the nanohybrids vis-à-vis pure bone cement. The cell viability through MTT assay and cell adhesion has greatly enhanced in nanohybrids in comparison to pure bone cement which has further been confirmed through cell morphology/imaging. Amine functionalized graphene nanohybrid is found to moderate the rate of polymerization and make this class of material suitable for implant. The in-vivo study has been performed with this material, and it is found to be an excellent healing agent for fractured bone as evident from the calcination in just 20 days against no healing in control or against the use of the usual bone cement as grouting material in the same time frame. Experimental Materials: Commercially available poly (methyl methacrylate-co-styrene) copolymer based bone cement (Simplex-P; Stryker) was used for the study. Graphene oxide was synthesized using graphite (Sigma-Aldrich, USA). Graphene and amine-functionalized graphene were synthesized using graphene oxide. Graphene oxide synthesis: Modified Hummer’s method was used for the synthesis of Graphene oxide (GO) and has been shown in the supplementary Figure S1a.37 Briefly, 1.5 gms of graphite was taken in a round bottom flask. H2SO4 / H3PO4 (180:20 ml) was added to it at room temperature. Subsequently, 9 gms of KMnO4 ware added slowly over the period of 1 hr. This reaction was then conducted at 50 ⁰C overnight (12 hrs). The obtained reaction product was brought to the pH of 7 after washing multiple times with distilled water followed by ethanol and then with 5% HCl. This was washed again with distilled water to remove excess acid.


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Synthesis of graphene: GO was well dispersed in water (500 mg / 300 ml) using ultrasonication for 15 min. After getting a homogeneous suspension, ammonia (0.3 ml) was added, followed by hydrazine hydrate (350 mg). The reaction mixture was purged with nitrogen for 20 min, and then the reaction was performed at 95 oC for 1 hr (the chemical reaction has been shown in Supplementary Figure S1b). The final solution was filtered using nylon-6 filter paper of 2 µm size pores. Collected graphene was further sonicated for 40 min to reduce the number of layers. Henceforth, the reduced graphene will be termed as ‘GR.’ Synthesis of amine functionalized graphene: Initially, 300 mg of GO was dried for 2 hrs under reduced pressure. 5 ml of dry DMF was added to dried GO followed by 57 ml of SOCl2 dropwise. The reaction conditions were then set at 65 ⁰C for 24 hrs and then SOCl2 was removed by distillation. Again, 75 ml of DMF was added into the system followed by 5 ml of ethylene diamine. The reaction was then carried out at 100 ⁰C for four days, and the whole reaction scheme has been presented in Supplementary Figure S1c. Finally, amine functionalized graphene was washed with ethanol to bring it to neutral pH. Henceforth, the amine functionalized graphene will be termed as ‘AG’.

Synthesis of nanohybrids: Bone cement was prepared by mixing its powder and liquid components in 2:1 proportion as per manufacturer, in a petridish using a glass rod for 2 mins. For nanohybrids, fillers (GR or AG) were mixed thoroughly with the powder component of the bone cement pack. Then the liquid component was added in the 2:1 proportion (powder: liquid) and mixed well for 90-120 seconds using a glass rod. Various percentages of nanofillers (each type of filler) (0.1, 0.2, 0.5 and 1.0 wt.%) were added to BC material. Based on the optimized results, we have reported 0.5 wt.% nanohybrids for better comparison and understanding. Henceforth, BC-X composite represents the hybrid using the fillers, X (X = GO, GR and AG) having a filler content of 0.5 wt.%.



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Characterizations: X-ray diffraction: Wide angle X-ray diffractometer was used to determine the exfoliation and/or intercalation of bone cement in the presence of GO/GR/AG using Cu Kα radiation and a graphite monochromator (wavelength, λ= 0.154 nm, Bruker AXS D8, Germany). Very fine sheets of the samples were held at quartz sample holder and scanned for diffraction angle 2θ range of 1° to 50° at a scan rate of 1°/min. Raman Spectroscopy: Raman spectroscopy was performed for the confirmation of the synthesis of the graphene and amine functionalized graphene from graphene oxide. Raman measurements were carried out in backscattering geometry using an Ar+ excitation laser source having a wavelength of 514 nm. Thermal characterization: Thermogravimetric analysis was performed using thermogravimetric analyzer (TGA) (Mettler-Toledo), fitted with a differential thermal analyzer (DTA), for a temperature range of 40 – 550 °C at a heating rate of 20°C min-1 in a nitrogen atmosphere. The maximum temperature during exothermic polymerization (Tmax) and the setting time (tset) were measured following relevant ASTM standards28. Tmax was measured using a thermocouple (Selec, DTC 503, Mumbai, India) and tset is a point halfway between the ambient temperature and the peak temperature. Morphology and EDX analysis: The morphology of the BC-GR, BC-GO, and BC-AG has been examined by scanning electron microscopy (SEM). Prior to SEM examination, a sputtering unit (SPRA 40; Zeiss Instruments) operating at 10 kV was used to coat the specimen with the gold layer and, two specimens were used per cement composition. The extracted cement specimens from the rabbit tibia after 20 days of post-surgery were washed with 0.185 M Na-Cacodylate buffer (pH = 7.4; 346 mOsm). The collected specimens were dehydrated in ascending grades of acetone and, then the specimens were subjected to sputter coating with gold. Energy Dispersive X-Ray Analysis (EDX) (200FEG, FESEM Quanta) was performed at 5 and 10 kV to determine the deposited elements on the specimen surface. AFM analysis was performed using NT-MDT multimode AFM, Russia, controlled by a Solver scanning 24 ACS Paragon Plus Environment

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probe microscope controller. First, 0.5 mg/ml solution of the GO/GR/AG in ethanol was properly dispersed via sonication for 30 mins. Then, a drop (10 microliter) of suspension containing GO/GR/AG solutions was added onto the glass plate [1 x1 cm] and excess of the solution from the edge of the glass plate was soaked carefully with tissue paper followed by drying at room temperature for 12 hrs. Corresponding AFM measurements were performed in semi contact mode. Spectroscopic measurement: UV-Visible (UV-Vis) spectroscopic measurements were performed using Shimadzu spectrophotometer (UV-1700), in the range of 200–1100 nm. Samples were dissolved in Tetrahydrofuran (THF) and then coated on quartz glass to form transparent films of thickness ~30µm. Synthesis of GO, GR and AG was confirmed using Fourier transform infra-red (FTIR). FTIR was also used to understand the functional group’s interactions in the nanohybrids in the spectral range of 600 to 4000 cm-1 (Nicolet 670) and with a resolution of 4 cm-1. Mechanical properties: Samples were molded to dog-bone shaped specimens of thickness 1.3mm using compression molding technique to study their mechanical properties. Universal testing machine (Instron 3369) was used and samples were stretched at a rate of 5 mm/min uniaxially. Specimen preparation for cell adhesion studies: Pristine bone cement and its nanohybrids were molded to pellets of radius 10 mm and height 4 mm and edges were smoothened using emery paper18. All specimens were then washed with distilled water followed by 70% ethanol to remove attached debris. Surface sterilization was performed by washing each specimen thrice with phosphate buffer saline (PBS) and overnight UV exposure. Then these specimens were treated for the preparation of the cement extract (leached medium). Following ASTM standards, 1gm of specimen weight was kept per 5 ml of medium for an incubation period of 72 hrs at 37 °C. Post incubation, extracts from each specimen was collected and stored at -20 °C. Cell culture studies: Cell culture studies were performed on the MG-63 cell line of human osteoblastlike cells (National Centre for Cell Sciences, Pune (Maharashtra), India). These cells were cultured in 25 ACS Paragon Plus Environment


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standard MEM supplemented with 10% heat-inactivated fetal calf, 100 U/ml Penicillin, 100 U/ml Streptomycin, and 20 µg/ml Gentamycin at 37°C in a humidified CO2 incubator maintained at 5% CO218. Cell viability: Standard MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed for cell viability studies. The cell density of 5×106 cm-2 MG-63 was incubated in the specimen extracts for 24 and 72 hrs time intervals at 37 °C. Post incubation, 10 µl of MTT solution (5 mg/ml) was incubated in each well for another 4 hrs at 37 °C to form formazan. The medium was then aspirated and replaced with 100 µL of DMSO (Himedia, Mumbai, India) to dissolve water-insoluble formazan.

Quantitative

analysis

was

performed

by

recording

absorbance

on

UV-Vis

spectrophotometer18. Cell adhesion: Modified crystal violet staining assay was performed to evaluate cell adhesiveness of MG-63 cell line to pristine bone cement and its nanohybrids. The cell density of 1×105 cm-2 was seeded on the cement specimen’s surface and was incubated in a CO2 incubator for 24 hrs. Post incubation, unattached cells were washed off using PBS followed by fixing of attached cells on specimen surface with ice-cold 4% paraformaldehyde for 20 min. 20% (v/v) methanol was used to permeate cells by keeping them in solution for 20 min. These cells were then stained with 0.5% crystal violet aqueous solution for 30 min. Excess stains were washed off using three gentle DI water washes. Elution of the residual crystal violet was performed with 10% acetic acid solution for 30 min on an elliptical shaker. Optical density (OD) of the eluted solution was recorded in UV-Vis spectrophotometer (double beam LI2800, Lasany, India) at a wavelength of 570 nm to correlate with the count of attached cells. Cell proliferation studies: Cell density of 0.5×105 cells per ml were suspended in fresh medium in a 64well plate in triplicate and were incubated for 4 hrs. Then, these cells were incubated in specimen extracts for 24 hrs. These cells were then washed with PBS and stained with DAPI in dark for 30 min at room temperature. Fluorescence images were taken using a Nikon Eclipse 80i fluorescence microscope. 26 ACS Paragon Plus Environment

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Cellular ROS detection: Cells with a density of 1×105 cm-2 were cultured for 6 hrs and then incubated with DPBS containing 10 mM of 2,7-Dichlorodihydrofluorescin diacetate (DCFH-DA) (D399, Invitrogen) for 20 min at 37 °C. Excess DCFH-DA was aspirated and then washed off with PBS. DCFHDA gets oxidized by ROS and this oxidized DCF fluorescence was determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using microplate spectrofluorometer. Morphological assessment of apoptosis: Acridine orange (AO) is a cell-permeable DNA-binding dye (green excitation) while propidium iodide (PI) is also DNA-binding dye (orange excitation) but is impermeable to the plasma membrane. Thus, only AO will be able to permeate plasma membrane and stain entire cell as cells with the intact cell membrane (early apoptotic and viable) will exclude PI to enter. Cells with a density of 1×105 cm-2, after treatment with cement specimen, were stained with 1:1 mixture of 100 mg/ml AO and 100 mg/ml PI for 2 mins. Interpretation of images is as follows: a. Green nucleus – Viable cells b. Dense green areas in nucleus – Early apoptosis c. Orange intact nucleus – Secondary necrosis d. Dense orange areas – late apoptosis In Vivo studies: In Vivo studies were performed on 18 male rabbits weighed approximately 2.5-3.0 kg at the animal care unit, Centre for Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi after taking prior permission of institutional ethical committee vide reference no. Dean/13-14/CAEC/199. Based on the better performance of BC-AG nanohybrids, only these hybrids were selected for in-vivo studies. Three groups of six rabbits each were formed against each material (viz. BC, BC-AG, and control) intended for implantation. During surgery, the rabbit was anesthetized using intraperitoneal injections of 1 mg/kg ketamine hydrochloride, 1 mg/kg of midazolam supplemented with 2% xylocaine with adrenaline 1:1000 under a standard condition of animal care guideline. After making sure that the rabbit is fully anesthetized, the left femur/tibia was washed with iodine tincture (Betadine, 10% aqueous) followed by alcohol (70%). Using a specially designed hand 27 ACS Paragon Plus Environment


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drilling apparatus a cavity of 4 mm diameter and 10 mm depth was created in the bone. Group 1 of rabbits was taken as control (blank) while Rabbits of group 2 and 3 were injected with BC and BC-AG, respectively. The incision was covered with micropore with 3-5 stitches. In every 5-day period, left hind limb of each group of rabbits was processed for digital X-ray radiography (SHIMADZURAD Speed, at 41 KV) to examine for osteological developments over time. These radiographs were mostly focused on three regions of interest; proximal metaphyseal, distal metaphyseal and diaphyseal region. Statistical analysis: The results are expressed as a mean ± standard deviation. In terms of test of significance, 1) results of Tmax, tensile modulus and toughness are analyzed using ANOVA followed by posthoc Dunnett’s multiple comparison tests, 2) results from the cell viability tests are analyzed using ANOVA followed by posthoc Bonferroni test, and 3) results from the cell attachment tests are analyzed using ANOVA followed by posthoc Dunnett’s multiple comparison tests; (GraphPad Prism 5,1). The significance is denoted as p < 0.05while its absence indicates insignificant nature of the result.

Acknowledgements The authors are thankful to Dr. A. Dwivedi, Department of Radiology, IMS, BHU for their help in X-ray imaging. Authors show sincere gratitude to Prof. Ranjan Kumar Singh, Department of Physics, BHU for his help in Raman Spectroscopy. They are also grateful to Mr. Sashank Mishra, lab attendant, Centre for Animal Surgery and Medicine, IMS, BHU for his help in animal surgeries for cement implantation.

Supporting Information The scheme of the synthesis process of graphene derivatives, GPC plots for molecular weights of nano-hybrids, contact angle plots of nanohybrids and EDX data 20 days post surgery.


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Osteoconductive Amine Functionalized Graphene-Poly(methylmethacrylate) Bone Cement Composite with Controlled Exothermic Polymerization Rakesh Sharma1, Govinda Kapusetti2, Sayali Yashwant Bhong, Partha Roy, Santosh Kumar Singh, Sikha Singh, Biswajit Ray, Pralay Maiti and Nira Misra

Amine functionalized graphene as a superb nanomaterial for the bone healing when used as a bone cement nanohybrid.


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