Modulation of Protein Adsorption and Cell Proliferation on

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Modulation of Protein Adsorption and Cell Proliferation on Polyethylene Immobilized Graphene Oxide Reinforced HDPE Bionanocomposites Rahul Upadhyay,†,∥ Sharmistha Naskar,§,∥ Nitu Bhaskar,† Suryasarathi Bose,*,‡ and Bikramjit Basu*,†,§ †

Laboratory for Biomaterials, Materials Research Center, Indian Institute of Science,Bangalore 560012, India Department of Materials Engineering, Indian Institute of Science, Bangalore 560012, India § Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore 560012, India ACS Appl. Mater. Interfaces 2016.8:11954-11968. Downloaded from pubs.acs.org by UNIV OF EDINBURGH on 01/28/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: The uniform dispersion of nanoparticles in a polymer matrix, together with an enhancement of interfacial adhesion is indispensable toward achieving better mechanical properties in the nanocomposites. In the context to biomedical applications, the type and amount of nanoparticles can potentially influence the biocompatibility. To address these issues, we prepared high-density polyethylene (HDPE) based composites reinforced with graphene oxide (GO) by melt mixing followed by compression molding. In an attempt to tailor the dispersion and to improve the interfacial adhesion, we immobilized polyethylene (PE) onto GO sheets by nucleophilic addition−elimination reaction. A good combination of yield strength (ca. 20 MPa), elastic modulus (ca. 600 MPa), and an outstanding elongation at failure (ca. 70%) were recorded with 3 wt % polyethylene grafted graphene oxide (PE-g-GO) reinforced HDPE composites. Considering the relevance of protein adsorption as a biophysical precursor to cell adhesion, the protein adsorption isotherms of bovine serum albumin (BSA) were determined to realize three times higher equilibrium constant (Keq) for PE-g-GO-reinforced HDPE composites as compared to GO-reinforced composites. To assess the cytocompatibility, we grew osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) on HDPE/GO and HDPE/PE-g-GO composites, in vitro. The statistically significant increase in metabolically active cell over different time periods in culture for up to 6 days in MC3T3 and 7 days for hMSCs was observed, irrespective of the substrate composition. Such observation indicated that HDPE with GO or PE-g-GO addition (up to 3 wt %) can be used as cell growth substrate. The extensive proliferation of cells with oriented growth pattern also supported the fact that tailored GO addition can support cellular functionality in vitro. Taken together, the experimental results suggest that the PE-gGO in HDPE can effectively be utilized to enhance both mechanical and cytocompatibility properties and can further be explored for potential biomedical applications. KEYWORDS: cytocompatibility, graphene oxide, HDPE, nanocomposites, grafting, toxicity, osteoblast, hMSC

1. INTRODUCTION

In the last two decades, biomaterials research has witnessed impressive progress in the development of joint/bone replacements based on polymeric materials.13−15 Various polymers such as high density polyethylene (HDPE) and ultrahigh molecular weight polyethylene (UHMWPE) are being investigated for biomedical applications.16−20 However, the strength and elastic modulus need to be enhanced without compromising biocompatibility properties. In order to overcome these issues, the hybrid composites of HDPE with graphene oxide (GO) and polyethylene grafted graphene oxide (PE-g-GO) have been developed in the present study. GO is a two-dimensional (2D) nanosheet composed of hydrophobic π domains in the core region and ionized groups around the

Natural bone is a composite of collagen, a polymer matrix, and hydroxyapatite, an inorganic reinforcement.1,2 The load bearing capabilities of the bone gain needs to be critically considered in developing an artificial bone implant.3,4 The mechanical properties of the implant materials must match with natural bone, and this aspect is more significant for hip and knee joint.5 If the implant has higher mechanical properties than natural bone, it can cause stress shielding in patients.6,7 This has been reported as one of the major problems that can lead to bone loss or in revision surgery.8,9 The wear resistance of the implant material has always been important concern as the wear debris generated from the implant can cause osteolysis, which damages the integrity of the implant due to aseptic loosening.10 The biocompatibility of the implant material is therefore very important in order to avoid toxic effects.11,12 © 2016 American Chemical Society

Received: January 24, 2016 Accepted: April 25, 2016 Published: April 25, 2016 11954

DOI: 10.1021/acsami.6b00946 ACS Appl. Mater. Interfaces 2016, 8, 11954−11968

Research Article

ACS Applied Materials & Interfaces edges.21,22 These are the features that enhance its interactions with proteins through hydrophobic and electrostatic interaction.23−25 In the biomedical field, GO has been used for applications such as tissue engineering, drug delivery, gene delivery and bioimaging.26−29 It is envisaged that the dispersion of nanoparticles in a polymer matrix decides the mechanical and cytocompatibility properties of the developed implant.30−32 Therefore, polyethylene is grafted onto GO for enhancing the interfacial adhesion with the matrix and henceforth, the dispersion. As far as the processing aspect is concerned, melt mixing has been reported to be superior than other methods like solution blending,33,34 because it facilitates a better dispersion of nanoparticles in the polymer matrix. During melt mixing, in situ grafting between the reactive functional group present either on the polymer or on the nanoparticle facilitates enhanced interfacial adhesion between the matrix and the nanoparticles. Although, earlier studies have reported the improvement in modulus and strength of UHMWPE and HDPE based composites, but retaining the above-mentioned properties at higher filler content still remains a challenge.35,36 Extensive efforts have been made to improve the dispersion and interfacial adhesion in the biocomposites for their applicability as acetabular cup liner used in total hip replacement.27,37 Significant research has been pursued on joint/bone replacements using HDPE/HA/Al2O3 and HA/UHMPE composites, but the dispersion of ceramic fillers and the interfacial adhesion still remains a key challenge even with the use of coupling agents.38,39 In the present study, PE chain is immobilized onto GO surface to improve structural properties and enhance biocompatibility. The stress transfer from the matrix to the filler is expected to improve, which eventually will result in enhancing the structural properties to a considerable extent. Efforts were therefore made to improve the mechanical properties of HDPE-based composites in which surfacemodified GO nanoparticles were melt-mixed with HDPE. The mechanical and cytocompatibility properties were assessed and compared with the composites filled with unmodified GO sheets. Protein adsorption studies have been performed, as it is precursor to cell growth on any substrate. Cell viability and cell adhesion analysis were evaluated for HDPE/GO and HDPE/ PE-g-GO composites. Mouse osteoblast cell line (MC3T3) and human mesenchymal stem cells (hMSCs) were selected for all the in vitro experiments. It was observed that osteoblast and hMSC growth/adhesion systematically increases with increasing GO content and substantial cell growth was observed for PE-g-GO based composites as compared to unmodified GO based composites. Taken together, this study opens up new avenues in designing unique polymeric substrates with enhanced cell growth.

was added very slowly to oxidize the mixture. This reaction mixture was stirred for 24 h at room temperature. After completion of the reaction, the mixture was poured into deionized water and stirred for 2 h. Then, 5−10 mL of H2O2 (35%) was added dropwise into the solution until the color turned bright yellow. The solution was filtered, and the residue obtained was washed in succession with deionized water, HCl, and ethanol. The final residue obtained was vacuum-dried at room temperature. 2.1.1.2. Synthesis of Amine Functionalized Graphene Oxide (GONH2). Amine functionalized graphene oxide was synthesized through nucleophilic substitution reaction by direct coupling of 4,4′methylenedianiline (MDA) onto GO by a procedure described earlier.41 Briefly, 500 mg of GO was dispersed in 200 mL of THF using a probe sonication method. Then, 500 mg of MDA was later added to it and the resultant mixture was refluxed at 80 °C (oil bath) in N2 atmosphere for 6 h. The mixture obtained was centrifuged and washed thrice with ethanol. After drying overnight under vacuum at 55 °C, the sample of GO-NH2 was obtained. Table 1 shows the elemental analysis of the as synthesized GO-NH2.

Table 1. Compositional Analysis of the Amine Functionalized Graphene Oxide (GO-NH2) element

mass percentage

C H N S O

51.01 3.84 2.93 2.08a 40.14b

a The small percentage of sulfur could be due to contamination. bThe oxygen percentage was calculated by subtracting the sum of percentage of C, H, N, and S from 100.

2.1.1.3. Synthesis of Polyethylene-Grafted Graphene Oxide (PE-gGO). PE-g-MAH and GO-NH2 were blended in HAAKE mini lab melt extruder at a speed of 60 rpm at 100 °C in a weight ratio 3:1 to ensure a complete conversion of amine groups of GO-NH2 to imide groups. Scheme 1 shows different steps involved in the synthesis of PE-g-GO. From FTIR spectra (Figure 1), it is confirmed that there is no free amine group in PE-g-GO suggesting that all amine groups of GO-NH2 are converted to imide groups. To further validate the complete conversion of all free amine to imide group, we performed CHNS (vario MICRO CUBE CHNS analyzer) analysis of GO-NH2, the mass percentage of C, H, N, and O are summarized in Table 1. The source of nitrogen in GO-NH2 was amine group only. Mass percentage of Nitrogen is found to be 2.93%, which implies, 1 g of GO-NH2 contains 29.3 mg of reactive amine Nitrogen in GO-NH2. Saponification value of PE-g-MAH (sigma Aldrich) is 32 mg of KOH/ g, that is, 1 g of PE-g-MAH contains 16 mg reactive anhydride oxygen because 2 mole of KOH react with one mole of di carboxylic acid to form anhydride group. Hence, a ratio of 1:1 (PE-g-MAH and GONH2,) will not completely convert all free amine to imide group due to excess presence of amine group in GO-NH2 compared to the reactive anhydride group in PE-g-MAH. As a result, we used a ratio of 3:1 (PEg-MAH and GO-NH2, respectively) to ensure a complete conversion of all free amine to imide group. The bubble formation in the extruded samples indicated the release of water vapor during the imidization reaction. The blending process was continued for another two cycles, until the bubble formation was ceased. The product (PE-g-GO) was further confirmed by FTIR. 2.1.1.4. Preparation of Nanocomposites. All the nanocomposites (HDPE/GO and HDPE/PE-g-GO) were prepared by using HAAKE minilab melt extruder at rotation speed of 60 rpm maintained at 220 °C for the duration of 20 min. The extruded composites were then subsequently compression molded at 150 °C for 2 min at pressure of 10 Psi. 2.1.2. Mechanical Characterization. Uniaxial tensile testing was carried out using INSTRON 5967 in an effort to measure the yield strength, elastic modulus and % elongation until failure. All the

2. EXPERIMENTAL SECTION 2.1. Materials and Methods. High-density polyethylene (HDPE) of density 0.95 g/cm3 was obtained from Swasan Chemicals (India). Graphite flakes, maleated polyethylene (PE-g-MAH), 4,4-methylene dianiline (MDA), Bovine Serum Albumin (BSA) and WST-1 Assay (Roche) were procured from Sigma-Aldrich (USA).The solvents tetrahydrofuran (THF), xylene, toluene, ethanol, and methanol were obtained from Merck (India). 2.1.1. Biocomposite Processing. 2.1.1.1. Synthesis of Graphene Oxide (GO). GO was synthesized from graphitic flakes by modified Hummers method.21,40 Briefly, the graphite flakes were mixed with concentrated H2SO4 and H3PO4 (9:1 ratio) in a water bath. KMnO4 11955


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ACS Applied Materials & Interfaces Scheme 1. Synthetic Approach Followed in Grafting of Polyethylene on Graphene Oxide (GO)a

a

PE-g-MAH: polyethylene grafted maleic anhydride; MDA: 4,4-methylene dianiline ; PE-g-GO: polyethylene grafted graphene oxide. electron microscopy (TEM) was carried out on a Tecnai G2 Spirit Bio-TWIN TEM instrument (120 kV) to observe the dispersion of the GO and PE-g-GO in the HDPE matrix. 2.1.4. Protein Adsorption Study. The adsorption of proteins from cell culture medium onto any solid surface is critical for cellular adhesion as the cell membrane receptors bind to the adsorbed proteins which further in downstream interacts with cytoskeleton complex.42 Hence, the adsorption of proteins together with other factors dictates the biological response of a material. In the present study, the adsorbed amount of protein was determined using solution depletion method.43,44 The change in the concentration of the protein in bulk solution, prior to and after adsorption, gives an account of amount of protein adsorbed onto the material surface. Bovine serum albumin (BSA, A2058, Sigma-Aldrich) was chosen as the model protein for the adsorption studies as albumin is the most abundant protein in the circulatory system. 2.1.4.1. Determination of the Time Required to Attain Equilibrium. It is necessary to determine the time required for each sample type to attain equilibrium. A known concentration of BSA solution (50 μg/mL) was prepared in which disc shaped substrates, with a surface area of 1 cm2, were soaked for a long period of time. During this period, readings were taken at 280 nm wavelength using a multimode plate reader (Eppendorf AF2200) at every 1 h interval. Then, 200 μL of the solution was taken in each well of a 96-well plate to determine the optical density (OD). The substrates were kept in this manner until the OD was constant as a function of time. 2.1.4.2. Quantitative Estimation of the Adsorbed Protein. BSA solution of 20 graded concentrations (maximum concentration was 1000 μg/mL, minimum concentration was 50 μg/mL, with an increment of 50 μg/mL) was prepared in deionized water. Very thin disc of ca.1 cm2 of surface area, were taken as substrates for the protein adsorption studies. Prior to protein adsorption, the surface of the substrates were cleaned by ultrasonication. These substrates were immersed and kept undisturbed in 1 mL of the prepared protein solution, in order to allow protein adsorption. After 24 h, absorbance of the spent bulk solution was measured at 280 nm of wavelength using the multimode plate reader by taking 200 μL in each well of a 96-well plate. Standard curve was plotted to estimate the amount of protein left in the solution. Subsequently, equilibrium constant (Keq) and surface molecular concentration (Cm) were determined from the obtained data.

Figure 1. FTIR spectra recorded from as synthesized GO and PE-gGO. samples were prepared according to ASTM standard, D-638 type-V for tensile testing. In particular the dumbbell shape specimens were tested under a cross head speed of 5 mm/min and the loading was continued until specimens were fractured. 2.1.3. Structural and Morphological Analysis of GO and PE-gGO. The attenuated total internal reflection Fourier transform infrared spectra (ATR-FTIR, Perkin- Elmer) of GO and PE-g-GO were recorded from 4500 to 500 cm−1. Raman spectroscopy (Horiba LabRAM HR) of GO and PE-g-GO were carried out with a 532 nm monochromatic laser. The percentage grafting of polyethylene was evaluated using Netzsch STA 409 PC Thermogravimetry Analyzer (TGA). Dispersed GO and PE-g-GO nanoparticles were drop casted on a silicon wafer and dried in vacuum for atomic force microscopy analysis (AFM, NanoWizardR 3, JPK Instruments).The SEM micrographs of tensile fractured composites were acquired using FEI, QUANTA (USA) at 10 kV (Figure S2, Supporting Information).The electron transparent thin sections of ca. 80 nm of selected composites were obtained using Ultramicrotome (Leica) in liquid nitrogen atmosphere using antistatic device. Transmission 11956


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the samples (HDPE, HDPE/GO and HDPE/PE-g-GO composites) were prepared with a diameter of ca. 11 mm for the cell culture experiments. The substrate size was taken in the manner that they would fit at the bottom of 48-well plate, leaving no or negligible uncovered area. The substrates were sterilized, exposing them in UV light for 1 h while keeping them dipped in absolute ethanol (Merck, Germany). Gelatin (0.2%) coated glass coverslips had been used as control. Before cell seeding, all the substrates were rinsed properly with phosphate buffer saline (1× PBS; pH 7.2−7.4) so that no trace of ethanol was left. Each well containing the substrate was seeded with 200 μL of the prepared cell suspension and maintained in the culture for 2, 4, and 6 days. 2.1.5.2. Human Mesenchymal Stem Cell (hMSC) Culture. Human mesenchymal stem cells (hMSCs) were cultured as a part of this investigation with respect to the cytocompatibility of the substrates (HDPE, HDPE/GO and HDPE/PE-g-GO composites) and were considered to be justified in reference to the application of the biomaterials.47 hMSC cell line were procured from Institute for Regenerative medicine, Texas A&M HSC COM, and the necessary approval from the Institutional Committee for Stem Cell Research and Therapy (IC-SCRT), IISc, Bangalore, was obtained before carrying out hMSC related experiments. It is important to mention that the rationale of considering hMSC as an experimental cell line was that these cells have been shown to be multipotent, capable of differentiating down to osteogenic lineages along with adipogenic and chondrogenic lineages. The cells were cultured using complete culture medium containing αMEM (alpha modified Eagle’s medium; Invitrogen) supplemented with 20% mesenchymal stem cell−fetal bovine serum (MSC-FBS; Invitrogen), 1% antibiotic antimycotic solution (Sigma) and 2 mM L-glutamine (Invitrogen). Cells were maintained at a temperature of 37 °C in a saturated humidified atmosphere containing 5% CO2. Prior to seeding they were revived in a tissue culture graded T25 flask (Eppendorf, Germany). Upon reaching 70−80% confluency, the cells were harvested using 0.05% Trypsin-EDTA (Invitrogen) and subcultured for further use. Cells were concentrated by centrifuging at 900 rpm for 3 min and then resuspended in the complete growth medium to achieve single cell suspension of cell concentration of ca. 104 cells per milliliter of media. The seeding conditions were kept the same as those for MC3T3. The culture duration for the hMSC was planned for 3, 5, and 7 days. 2.1.6. Cell Viability using WST-1Assay. Cell viability was evaluated biochemically using water-soluble tetrazolium salts (WST-1, 4-[3-(4iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene-disulfonate,Roche).48 As WST-1 assay has no interaction with graphene oxide, hence this assay has been chosen over MTT (3(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The WST-1 assay was performed after the prescribed culture durations of 2, 4, and 6 days for MC3T3 and 3, 5, and 7 days for hMSC. Both cell types were cultured in 48-well plates on the substrates fitted and covering the bottom of each well. Approximately 2 × 103 cells/well were seeded on the sterilized samples in 48-well plates and subsequently placed in 5% CO2 incubator at 37 °C and 95% humidified atmosphere. The substrates were transferred to a new plate after being washed thrice with 1X PBS. To perform the colorimetric WST-1 (Roche) assay, we prepared 200 μL of 10% v/v solution of WST-1 and complete culture medium and added them to each of the wells. After the samples were incubated for 4 h at 37 °C in 5% of CO2 atmosphere along with saturated humidity, the optical density (OD) of the developed color was measured by a microplate reader (iMark, Biorad laboratories, India) at a wavelength of 450 nm. The absorbance value provides a direct correlation to the number of viable cells in each well. The measurements were obtained by averaging the data from three wells repeated thrice. Cell viability (%) was calculated by using the following equation:

The amount of adsorbed protein onto the surface of the substrates was quantitatively determined as the difference ΔCb, of the protein prior to and after adsorption, ΔC b = Cs = C bi − C bf

(1) 2

where, Cs is the surface concentration (μg/cm ), Cbi is the initial bulk concentration (μg/mL) and Cbf is the final bulk concentration (μg/ mL) after the allowed duration of adsorption. A standard curve with optical density (OD) versus protein concentration (C; Figure S4, Supporting Information) was plotted from which the following equation was obtained after fitting with Origin Pro 9.1 software.

OD = 0.254 ln(C) + 0.001

(2)

where the coefficient of determination (R2) is 0.9912. This equation was used to calculate the protein concentration from the measured OD as well as the amount of protein adsorbed onto the substrate. 2.1.4.3. Determination of Keq. The Keq values for different sample composites were evaluated. It is envisaged that the phenomenon of adsorption of albumin onto hydrophobic surfaces follow Langmuir’s isotherm,45 which is described as

Cs =

M ⎛⎜ KeqC b ⎞⎟ ANA ⎝⎜ 1 + KeqC b ⎠⎟

(3)

where Cs is the weight of protein adsorbed per unit area of surface, M is the molecular weight of the adsorbing protein molecule, Keq is an equilibrium adsorption constant, Cb is the bulk protein concentration in solution, A is the surface area per site, and NA is Avogadro’s number.45 A linear form of the equation can be derived as Cb C 1 = + b Cs KeqCm Cm

(4)

A linear relation between Cb/Cs versus Cb can be clearly expected. The equilibrium constant (Keq) and monolayer concentration (Cm) are obtained from the slope divided by the intercept and inverse of slope, respectively. 2.1.5. Cell Culture. Two cell lines, mouse osteoblast precursor cells (MC3T3) and human mesenchymal stem cell (hMSC), were selected to observe their responses toward all the substrates (HDPE, HDPE/ GO, and HDPE/PE-g-GO composites). Both the studies were planned, in respect to their culture duration, in such a way that they would be considered as two independent studies. Three observations were planned at every alternate day. Each study was repeated thrice with triplicates. 2.1.5.1. Mouse Osteoblast Precursor Cell (MC3T3) Culture. MC3T3 is an osteoblast precursor cell line derived from Mus musculus (mouse) calvaria.46 Our reasons behind choosing MC3T3 cell line are (1) they have behavior similar to primary calvarial osteoblasts; (2) osteoblast cell line is ideal in context of the use of a biomaterial as a bone implant; and (3) they have good adherence on both lower and higher stiffness surfaces without compromising the reproducibility of the experiments. All the cell culture experiments were conducted with cells at passage of 2−7. Prior to seeding, cells were revived from cryopreserved stock and expanded in tissue culture graded T25 flask (Eppendorf, Germany) containing complete culture media, which was αMEM (Alpha Minimum Essential Medium; Invitrogen), supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotic antimycotic solution (Sigma-Aldrich). Cells were maintained in an environment of 5% CO2 incubator (Sanyo, MCO-18AC, USA) with 37 °C temperature and 95% humidification. Revival took 3 days, during which, after 2 days, the media had been changed once. As the cells were 70−80% confluent, they were enzymatically lifted from the T-25 flask by using 2 mL of 0.05% Trypsin-EDTA (Invitrogen) and subcultured for further use. The cells of passage 2−4 were concentrated by centrifugation at 1100 rpm for 3 min, resuspended in known amount of media, counted with a Neubauer chamber, and then diluted in complete culture media to a cell density of ∼104 cells per ml of media. The single cell suspension thus prepared has been used for seeding onto the samples. Disc shaped, thin substrates of all

cell viability =

mean absorbance of sample − absorbance of blank × 100 mean absorbance of control − absorbance of blank

(5) 2.1.7. DAPI Staining to Visualize Cell Viability. To observe the nuclei of proliferated active cells, we placed the sterilized samples of all 11957


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ACS Applied Materials & Interfaces the substrate composite and gelatin coated coverslips (control) in 48well plate. MC3T3 osteoblast cells were seeded approximately at a density of 2 × 103 cells/well. The cells were cultured for 2 days, 4 days and 6 days. Then the samples were washed twice with 1× PBS followed by fixation of samples in 4% paraformaldehyde (PFA; SD Fine-Chem Lit) solution for 30 min. After being washed three times with PBS, 0.1% of triton X was added and kept for 10 min for permealization. Blocking with 1% bovine serum albumin (BSA) for 1 h was done to prevent nonspecific binding of the dye, and washing with 1× PBS was done afterward. Hoechst stain 33342 DAPI (Invitrogen) was added and kept for 15 min to visualize the active nuclei. After the sample was washed with 1× PBS to remove the excess stain, the cells were observed under fluorescence microscope (Nikon LV 100D, Japan).49 2.1.8. SEM Analysis of Cell Morphology. Scanning electron microscopy (SEM) analysis was performed to evaluate the morphological appearance of the cells. The cellular morphology was investigated after being cultured for the prescribed duration, in respect to each cell type. In the course of preparing the samples for SEM imaging all the samples were washed twice with 1× PBS. Fixation was carried out for 30 min in 2% glutaraldehyde. Then the substrates containing the cells were washed and dehydrated in a graded series of ethanol treatment. The specimens were sputter-coated with gold and photographed with FEI, QUANTA, at an acceleration voltage of 10 kV. 2.1.9. Statistical Analysis. All the results were expressed as mean ± standard error (SE). Student’s t test and one-way ANOVA with posthoc Tukey’s test were performed to reveal the statistical differences regarding the results of the protein adsorption and cytocompatibility studies among the substrates of different samples (HDPE, HDPE/GO, and HDPE/PE-g-GO composites). All the statistical analysis had been performed using SPSS-16.0 software (SPSS Inc.@2010). The cutoff p-values were set at 0.05 and 0.01, lower than those considered to be too low to accept the null hypothesis.

Figure 2. Raman spectra acquired from as synthesized (a) GO and (b) PE-g-GO.

observations suggest that the defect and disorder in PE-g-GO are minimum than that of GO. Atomic force microscopy (AFM) is a powerful surface analytical technique used to acquire high-resolution topographic images of a surface down to molecular/atomic resolution. Figure 3 shows AFM images of GO and PE-g-GO nanosheet in tapping mode. The thickness of the GO sheets was ca. 1 nm, whereas the thickness of PE-g-GO sheets was ca. 10 nm. The increase in the thickness for PE-g-GO sheets was due to grafting polymer chains onto the GO surface. Thermogravimetric analysis (TGA) is a technique in which the mass of a compound or a material is dynamically monitored as a function of temperature or time. Figure 4 shows the TGA profile for GO and PE-g-GO. The experiments were performed at a heating rate of 10 °C/min in nitrogen atmosphere. In Figure 4, Ti (420 °C) and Tf (490 °C) are, respectively, the initial and final degradation temperatures, whereas Wi and Wf are respective weights at Ti and Tf. Wi and Wf are wt % at temperature Ti and Tf, respectively. (Wi − Wf) is the weight loss due to polyethylene grafting. From the measured weight loss, it can be suggested that the percentage of grafted polyethylene in PE-g-GO was approximately 65%, which is independent of GO content. TEM micrographs have been taken to observe the dispersion of GO and PE-g-GO in HDPE matrix. For TEM analysis, ca. 80 nm thin sections were cryo-sectioned using a Leica Ultramicrotome at −120 °C. Figure 5 shows the representative bright field TEM micrographs of modified and unmodified GO reinforced HDPE composites. In Figure 5a, thin hairy lines and dark spots indicate the intersection and agglomeration of graphene oxide sheets in HDPE matrix, respectively. It can be inferred from Figure 5a that GO is poorly dispersed in HDPE matrix primarily due to lack of interfacial adhesion. The van der Waals interactions and restacking of GO sheets in HDPE matrix further facilitate their agglomeration in the host matrix. The grafting of PE chains on to GO enhances the state of dispersion in HDPE matrix by improving the interfacial adhesion between GO and HDPE which has further led to their better dispersion, as seen in Figure 5b. These micrographs suggest that PE-g-GO can facilitate better interaction with the host polymer compared to unmodified GO, thereby imparting

3. RESULTS AND DISCUSSION In this section, structural, physical, and cytocompatibility characterization are described and analyzed in the context of potential biomaterial application. 3.1. Synthesis and Characterization of Polyethylene Grafted GO (PE-g-GO). The synthesized PE-g-GO was further characterized with the spectroscopic techniques. FTIR spectra of GO and PE-g-GO are shown in Figure 1. The absorption band of GO at 1730, 1623, 1045, and 3407 cm−1 are ascribed to stretching frequency of CO (carboxylic groups), −CC−, epoxy ring, and hydroxyl group, respectively.50 The characteristic peaks of PE-g-GO around 2920 and 2850 cm−1 were assigned to stretching frequency of −CH2, while peaks at 1376 and 1640 cm−1 were assigned to stretching frequency of C−N (imide) and stretching frequency of imide CO (Figure 1), respectively. Raman spectroscopy provides signatures of molecular vibrations of Raman-active compounds. Figure 2 shows two signature bands in the Raman spectrum of GO and PE-g-GO. In Raman spectrum of GO, the G band was observed at 1585 cm−1 which is attributed to the first order scattering of the E2g mode of the sp2 domain of graphite (C−C scattering).51 In contrast, the D band at 1351 cm−1 corresponds to the defects or breakdown of translational symmetry arising at the edges.52 The relative intensity ratio of the disorder induced D band to G band (ID/IG) of GO was 0.97. In the Raman spectrum of PE-gGO, the position of D and G band were approximately same as in GO but the relative intensity ratio of the disorder induced D band to G band (ID/IG) was relatively lower at 0.89. The above 11958


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Figure 3. AFM images of (a) GO and (b) PE-g-GO and height profiles taken along the line indicated on respective AFM images of (c) GO and (d) PE-g-GO.

Figure 4. TGA profile recording weight loss for GO and PE-g-GO.

superior mechanical properties at low loadings as will be discussed in the subsequent section. 3.2. Mechanical Properties of HDPE/GO and HDPE/PEg-GO bionanocomposites. The uniaxial tensile deformation properties of all the composites were evaluated using ASTM standards at a constant cross head speed of 5 mm/min. The representative engineering stress−strain response of the composites is shown in Figures 6 and 7. Irrespective of the filler type and amount, the initial elastic response was recorded in the entire composite. The nonlinear deformation behavior started prior to reaching the yield strength and continued. The extent of nonlinear deformation below the yield strength however varied with the type and amount of the filler. The deformation characteristics were qualitatively same in case of GO and PE-g-GO based composites. The magnitude of the yield strength, elastic modulus and failure strain varied significantly, depending on the presence of GO content (Table 2 and Figures 6 and 7). The yield strength and the elastic modulus of pure HDPE were found to be 16.4 and 490 MPa respectively, at a cross head speed of 5 mm/min. A significant

Figure 5. Bright-field TEM images of (a) HDPE/GO 3% and (b) HDPE/PE-g-GO 3%.

improvement could be observed in the yield strength (17.4 MPa) and elastic modulus (586 MPa) with the addition of 1 wt % GO (Table 2). The yield strength and elastic modulus were, however, decreased with the addition of 2 wt % GO and 3 wt % GO, possibly because of the agglomeration or restacking of GO 11959


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Figure 6. (i) Engineering stress−strain plot for HDPE, HDPE/GO 1%, HDPE/GO 2%, and HDPE/GO 3%. (ii) Plot showing the variation of elastic modulus and yield strength of HDPE composites with different amount of fillers.

Figure 7. (i) Engineering stress- strain plot for HDPE, HDPE/PE-g-GO 1%, HDPE/PE-g-GO 2% and HDPE/PE-g-GO 3% (ii) Plot showing the variation of Elastic Modulus and yield Strength of HDPE composites with different amount of fillers.

respectively, in contrast to unmodified GO filled HDPE. More importantly, the extent of nonlinear deformation considerably increases in the reinforced HDPE composites (Figure 7). A critical look at Figure 7 reveals that the PE-g-GO filled HDPE composite exhibited a constant deformation region at a stress level below the yield strength. For example, a constant deformation of 12.5 MPa was recorded for 1 wt % PE-g-GO addition. However, such deformation with much lower extent took place at 15 MPa for 3 wt % PE-g-GO addition. Table 3 summarizes the mechanical properties of various PEbased composites. Though different research groups have used different grades of HDPE, however this table reports the properties of HDPE with lower reinforcement content of less than 5% in an effort to establish a fair comparison with previous research. Since HDPE, depending on the source or molecular weight or processing route may exhibit a variation in the mechanical properties therefore, a comparison is being made with respect to percentage change in elastic modulus or yield strength due to specific reinforcement addition. For instance, Wan et al. added 0.3% of GO in HDPE/MAPE (90/10, wt/wt %) through solution casting and then prepared the composite film for mechanical studies by compression molding.53 The incorporation of GO nanoplatelets of 0.3% increased the

Table 2. Summary of Tensile Properties Obtained for HDPE and Its Composites, Designed Using Melt Mixing Followed by Compression Molding matrix

filler (wt %)

HDPE HDPE HDPE HDPE HDPE HDPE HDPE

neat GO 1% GO 2% GO 3% PE-g-GO 1% PE-g-GO 2% PE-g-GO 3%

yield strength (MPa) 16.4 17.4 14.0 15.3 17.0 18.7 20.0

± ± ± ± ± ± ±

0.8 0.6 0.2 0.5 0.7 0.3 0.4

elastic modulus (MPa) 490 586 412 418 522 564 595

± ± ± ± ± ± ±

16 10 6 13 18 3 14

elongation at failure (%) 56 41 36 48 70 92 71

± ± ± ± ± ± ±

10 3 5 4 9 6 15

nanoparticles due to hydrogen bonding or electrostatic interaction. In contrast, a different scenario was noted in the presence of PE-g-GO-based composites. As compared to pure HDPE, both elastic modulus and yield strength scaled up with PE-g-GO (Table 2 and Figure 7), possibly because of uniform dispersion of PE-g-GO in HDPE matrix. From Table 2, we find that the addition of 2 and 3 wt % PE-g-GO in HDPE matrix enhanced the yield strength and elastic modulus by 30% and 40% 11960


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Table 3. Comparison of HDPE-based Composites with Different Carbonaceous Reinforcement in Reference to Mechanical Property with Respect to the Present Work system

filler (wt %)

Young’s modulus (MPa)

tensile strength (MPa)

% increase in modulus w.r.t. neat HDPE (MPa)

% increase in strength w.r.t. neat HDPE (MPa)

ref

HDPE/MAPE (90/10, wt/wt %) HDPE/5% CPE25

GO 0.3%

580 ± 40

13.0 ± 4.2

30

12

Wan et al.53

GO 0.5%

1228 ± 20

75 ± 1.5

16

21

HDPE

MWCNTs 2.5%

796 ± 42

19.5 ± 2.9

30

no increment

HDPE

expanded graphite 3% PE-g-GO 3%

1460 ± 20

18.7 ± 0.4

17

4

Chaudhary et al.54 Chrissafis et al.55 Zheng et al.56

595 ± 14

20 ± 0.4

22

20

present study

HDPE

ultimate tensile strength up to 12% and young modulus up to 30% with significant decrease in ductility. They reported aggregation of GO beyond 0.3 wt %. Chaudhary et al. prepared graphene based nanocomposites by solution mixing with chlorinated polyethylene as a compatibilizer followed by melt mixing with high density polyethylene.54 An increase of 16% in young’s modulus and 21% in tensile strength at 0.5 wt % graphene oxide incorporation was reported. In another work, Chrissafis et al. prepared HDPE based nanocomposites by melt mixing on a Haake-Buchler Reomixer, containing 2.5 wt % of MWCNTs. They reported aggregation of MWCNTs as observed from transmission electron micrographs.55 As a result, there was no increment in tensile strength however; ca. 30% increment was found in Young’s modulus. Zheng et al. prepared HDPE reinforced graphite composites by melt compounding.56 Although mechanical properties improved as a function of filler content, the overall enhancement was not impressive due to lack of good interfacial adhesion between filler and matrix. An increase of 17% in Young’s modulus and 4% in tensile strength at 3 wt % of expanded graphite was observed. In the present study, we have developed hybrid composites of HDPE and polyethylene grafted graphene oxide which showed improved structural properties as compared to GO filled composites. PE was grafted on to GO to enhance the interfacial adhesion between matrix and filler. Also, well dispersed graphene oxide sheets were observed to adhere in HDPE matrix (Figure 5) from TEM micrographs. Here, we have achieved ca. 22% increment in Young’s modulus and ca. 20% increment in yield strength for HDPE with 3 wt % PE-gGO as compared to neat HDPE. Therefore, the improvement in tensile properties, as compared to the previous work can be attributed to the recirculation channel coupled to the melt extruder, which facilitated better dispersion of fillers in the polymer matrix when compared to solution blending technique.36,57 It has also resulted in improved interfacial adhesion, which improved the stress transfer from matrix to the fillers. 3.3. Protein Adsorption Analysis. Protein adsorption study was performed on different substrates (HDPE, HDPE/ GO and HDPE/PE-g-GO composites) as per the methodology described in section 2.1.4 There is a marked decrease in the absorbance of the bulk solution for HDPE with 3 wt % PE-gGO (Figure 8). The depletion of protein infers increased adsorption of protein on the substrate surface. With respect to protein adsorption, the difference between HDPE reinforced with 3% GO and 3% PE-g-GO is ca. 13.8% at 1000 μg/mL bulk concentration. HDPE without any reinforcement shows 29.1%

Figure 8. Adsorption of BSA form the bulk solution onto the substrate surfaces.

lower adsorption compared to HDPE reinforced polyethylene grafted 3 wt % GO at the same initial bulk concentration. Evaluation of the plot of Cs versus Cbi qualitatively followed the Langmuir isotherm (Figure 8) and therefore more quantitative analysis was performed as per eq 4. In particular Cbf/Cs versus Cbf plot was analyzed to obtain equilibrium constant (Keq) and monolayer concentration (Cm), as shown in Figure 9. The fact that Langmuir isotherm can be accepted as the most appropriate model of adsorption in the current study because (Cbf/Cs) versus Cbf has shown linear relationship. HDPE/PE-g-GO 3% has exhibited the highest Keq value (0.58 cm3/μg) among all the test samples. The lower Keq value of HDPE suggests the possibility of obtaining fast equilibrium of the reversible adsorption−desorption process (Figure 10). It is worthwhile to note that protein adsorption is an irreversible phenomenon which can be very well explained thermodynamically.58,45 Monolayer concentration is low for the HDPE (4.8 μg/cm2) and HDPE/PE-g-GO 3% (6.0 μg/cm2). Overall ca. 24% increase in Cm and more than one order increase in Keq value are recorded for HDPE/PE-g-GO 3% composite compared to pure HDPE. Such increase remains statistically significant between HDPE/GO 3% and HDPE/PE-g-GO 3% composite. The adsorption mechanism is interplay of hydrophobic interaction and electrostatic interaction with the substrate. It has been reported that albumin has hydrophobic patches on the surface suggesting irreversible hydrophobic interaction with hydrophobic biomaterials, in contrast the albumin weakly 11961


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for two different cell lines, MC3T3 and hMSC. The WST-1 assay was performed to quantify the cell viability throughout this study. The assay is based on the conversion of the tetrazolium salt of WST-1 to produce a colored product by the mitochondrial dehydrogenase enzymes.61 The intensity of the color gives a relative count of viable or metabolically active cells. Cell growth was quantitatively analyzed in the terms of cell number over different time point in culture. In addition, cell adhesion, spreading, and cytoplasmic protrusions were analyzed in a qualitative manner to assess the cytocompatibility. 3.4.1. MC3T3 Osteoblast Cells. As previously mentioned, osteoblast cells were considered because of the potential clinical application of such materials as orthopedic implants. 3.4.1.1. Cell Viability Analysis Using WST-1 Assay. Cell proliferation is significantly enhanced for the specified duration of the in vitro culture (2, 4, and 6 days) as reflected from the increased OD values (Figure 11). The enhanced cell viability Figure 9. Plot of Cbf/Cs versus Cbf to determine the adsorption parameters for BSA adsorption onto the samples of different compositions (HDPE, HDPE/GO, and HDPE/PE-g-GO composites).

Figure 10. Graphical representation of Keq and Cm values for the samples of different compositions (HDPE, HDPE/GO, and HDPE/ PE-g-GO composites). Statistical analysis is performed using One-way ANOVA and post hoc Tukey’s test; # denotes difference is significant at p ≤ 0.05 compared to control; ** denotes difference is significant at p ≤ 0.01 compared control; † denotes difference is significant at p ≤ 0.05 compared to control.

Figure 11. Bar graph for WST-1 assay for MC3T3 shows increased optical density for after modification of the graphene oxide. One-way ANOVA performed for the groups shows significant increase of the optical density as a representative parameter of the cell viability. Post hoc Tukey’s test shows distinguishable differences between the samples of days 4 and 6; * significant at p ≤ 0.05 and ** significant at p ≤ 0.01 for day 4 w.r.t. control; † significant at p ≤ 0.05 and †† significant at p ≤ 0.01 for day 6 w.r.t. control; • and # significant at p ≤ 0.05 w.r.t. HDPE/GO 3%

interacts with hydrophilic surface.59 According to the values of the contact angles (Figure S3, Supporting Information), the hydrophobicity of HDPE remains unaltered even after reinforcement with nanofillers like GO and PE-g-GO. Hence, it can be assumed that BSA binds to the HDPE based composites by hydrophobic interaction. Moreover, addition of GO can promote electrostatic interactions with the zwitterions of the BSA molecule, having the isoelectric pH 4.7 due to the presence of hydrophilic groups.25 Albumin follows a timedependent spreading on the surface reflecting in a lower value of the monolayer concentration (Cm) for hydrophobic surfaces than hydrophilic ones.60 In addition to the above-mentioned factors, other parameters like molecular weight, isoelectric pH of the protein and surface topography also play an important role to determine the mode of protein−substrate interaction. 3.4. In Vitro Cytocompatibility Analysis. Cytocompatibility studies were performed by considering the cell viability and in vitro cellular proliferation as two quantitative parameters

can be explained in relation to the substrate composition. The substrate containing 3% modified graphene oxide in HDPE matrix has been found to have an impressive 150% enhancement in cell viability at the first observation (i.e., after 2 days of culture), which at the end of sixth day resulted in ca. 300% increase, with respect to the control (Figure 12). As far as the growth rate was concerned, HDPE/PE-g-GO 3% has supported the highest cell growth. Moreover, surprisingly pure HDPE has also supported osteoblast growth. 3.4.1.2. DAPI Staining of Osteoblast Cell. DAPI staining was performed for the osteoblast cells and significant increase in the cell count was observed. DAPI binds to the double stranded Deoxyribonucleic acid (DNA) and gives an emission at blue wavelength when excited at 340 nm. The result corroborates with the viability pattern observed for WST-1 assay. As can be seen in Figure 13, the nuclei are deeply stained with DAPI which can be counted easily to estimate the number of cells grown per unit area of the substrates. For all the samples, the cells have grown significantly, compared to growth 11962


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Figure 14. Graphical representation of the cell count on the materials after day 2 and day 6 culture. One-way ANOVA had been performed showed significant differences between the groups. Post hoc Tukey’s test also showed statistical significant differences within the specific groups. The plotted data can be converted into the rate of cellular growth which can be estimated as ca.23 cells/hour on average; •• significant at p ≤ 0.01 for HDPE; ** significant at p ≤ 0.01 for HDPE/ GO 3%; †† significant at p ≤ 0.01 for HDPE/PE-g-GO 3%; □ significant at p ≤ 0.05 for control.

Figure 12. Cell viability graph shows increase of cell viability for MC3T3 at day 6 cultured on the modified graphene oxide reinforced samples. Student’s t test had been performed to reveal statistically significant differences with mean; ** significant at p ≤ 0.01; * significant at p ≤ 0.05

3.4.2. Human Mesenchymal Stem Cells. Human mesenchymal stem cells (hMSCs) are the pluripotent cells which can uptake the path of osteogenic differentiation if suitable cues are available. Other than the biological cues, material properties can also influence the decision taken by the cells. Before the onset of differentiation, the cells need to get proliferated initially and also should show biological activities like high values of viability, good adhesion to the material, increased cytoplasmic appendages, and so on. These later aspects were investigated as part of the present study. 3.4.2.1. Cell Viability Analysis Using WST-1 Assay. Human Mesenchymal stem cell (hMSC) has expressed slightly altered pattern of cell viability in respect to the samples. On day 3, the amount of viable cells for HDPE is very low as compared to other samples, though modified and unmodified GO reinforced HDPE composites samples have been noticed with a significant difference between them (Figure 16). A significant increase of 120% of the cell viability is observed as compared to day 3. In addition, a marked increase is also found when day 7 results are compared (Figure 17). 3.4.2.2. Cell Adhesion and Morphological Analysis. As can be observed distinctly from the SEM micrographs (Figure 18), hMSCs have proliferated significantly on the substrates which contain GO, both polyethylene grafted and nongrafted. As a signatory marker of cell proliferation, the cells are seen to have well established substrate adherence, spreading and cell−cell communication through filopodial extensions.62 The extensive growth of filopodia and lamellipodia is observed in a mesh like appearance for the samples containing modified and unmodified graphene oxide. It is evident that cells are able to communicate better and subsequently are able to proliferate more on both modified and unmodified graphene oxide reinforced HDPE composites than control. The spreading of the cells also is a reliable parameter to describe cellular fate on the biomaterial. The cells are observed with good spreading on both modified and unmodified graphene oxide reinforced HDPE composites, though on the modified GO containing composite has resulted with maximum spreading, as observed from the day 3 results. The amount of

Figure 13. Florescence microscopy images of MC3T3 cell nuclei, after staining using DAPI (scale bar, 100 μm).

after 2 days of culture. HDPE/PE-g-GO 3% has shown ca. 200% increase in cell count at day 6, which is lowered in case of HDPE/GO 3%. The percentage difference among modified and unmodified GO reinforced HDPE composite is ca. 46% on day 6 (Figure 14). 3.4.1.3. Cell Adhesion and Morphology Analysis. Scanning electron microscopy (SEM) was performed to get qualitative analysis of cell morphology. Our study on the cell morphology showed distinguishable alteration of morphology due to the difference in the composition of various sample substrates. As the signature criteria for the cells to exhibit proliferation on a biomaterial surface (e.g.- cell adhesion, cell spreading, cell cell communication), we have emphasized on these parameters to analyze the cell growth on respective substrates. After 6 days, the osteoblast cells poorly adhere in the case of control and HDPE substrates (Figure 15). If we compare the growth pattern, as a representative of the cellular functionality, it is fair to comment that both 3% of modified and unmodified GO reinforced HDPE composites have increased the cellular adhesion, cell−cell communication and cell spreading than control. There is a slightly sluggish growth pattern found for HDPE/GO 3%, when compared to the HDPE/PE-g-GO 3% composite and this is evident from the spreading of the cells. Somehow, cells appear to be more shrunk on the HDPE/GO 3% composite surface while cultured cells are more spread out on HDPE/PE-g-GO 3% composite. 11963


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Figure 15. Representative SEM images of MC3T3 cells proliferating on the surface of various sample substrates after 6 days of culture (a) control (b) HDPE (c) HDPE/GO 3% (d) HDPE/PE-g-GO 3%.

inclusion into HDPE renders polarity which is enough to alter the pattern of cellular responses. It is well-known that every cell type has its own sets of genes at the level of repression versus expression through which the cell type manifests different adaptations in different microenvironments and hence, two different cell types were studied here. As reported by several researchers that osteoblast cells are very sensitive to surface wettability and topography. As investigated by Zhang et al., cell number on hydrophilic modified surfaces were significantly lower than on hydrophobic surfaces with the same topography.64 Our study also shows a similar tendency of growing more osteoblast cells on HDPE and HDPE/PE-g-GO composites. Unlike the osteoblast cells, hMSC has strict repulsion toward hydrophobic surfaces and hence more favorably has grown on GO reinforced composites. Also important to mention that upon achieving confluency, they show aligned pattern which was a good indication of cell differentiation. Confirmation of such prediction needs further investigations. In literature, the biocompatible nature of GO based polymer substrates has been studied and constantly been debated by several investigators.65,66 Our present results provide strong evidence toward noncytotoxic nature of HDPE/PE-g-GO 3%. Interestingly, an addition of PE-g-GO up to the level of 3 wt % has been observed to be beneficial for the cell proliferation on HDPE/PE-g-GO composite compared to neat HDPE. A better proliferation of the cells on HDPE/PE-g-GO substrate could be attributed to the following aspects, (1) hydrophobic interactions of PE-g-GO with proliferating cells, (2) good dispersion of PE-g-GO in HDPE matrix possibly has caused less cellular stress, subsequently less chance of reactive oxygen species (ROS) generation which has reduced toxicity to the cells. Taken together all the results presented above, confirm the positive impact of PE-g-GO in HDPE matrix on osteoblast and

Figure 16. WST-1 assay plot shows the cell viability of hMSC on various composites. One-way ANOVA together with post hoc Tukey’s test conducted for statistical analysis; * significant at p ≤ 0.05 and **significant at p ≤ 0.01 for day 4 w.r.t. control; † significant at p ≤ 0.05 and ††significant at p ≤ 0.01 for day 6 w.r.t. control; # and □ significant at p ≤ 0.05 w.r.t. HDPE/GO 3%.

spreading is diminished with the number of days in the culture once the cells are confluent. The cells, growing on HDPE/PEg-GO 3%, are also characterized with atypical aligned morphology with increasing cell density. In contrast, HDPE substrates have failed to support the growth of hMSC, manifesting poor adhesion and spreading with a low cell count. Cytocompatibility is the critical phenomenon which is sensitive to different stages starting with the adsorption of protein to adhesion/functionality of various cell types. These parameters help to interpret the cellular response toward a material in context of cell−substrate interaction. GO sheets behave as soft membranes with high in-plane stiffness.63 GO 11964


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Figure 17. Cell viability graph shows increase of cell viability for hMSCs at day 7 cultured on the modified graphene oxide reinforced samples. Student’s t test had been performed to reveal statistically significant differences with mean of the control. **significant at p ≤ 0.01

Figure 18. Representative SEM images of hMSC proliferating on the surface of various sample substrates after 5 days of culture (a) control (b) HDPE (c) HDPE/3 wt % GO (d) HDPE/ 3 wt % PE-g-GO.

behavior of these new composites and is subjected to future investigations.

mesenchymal stem cell fate. An interesting observation is that the cell growth and attachment is more favored in the presence of PE-g-GO in HDPE matrix as compared to only GO addition. Therefore, on the basis of our in vitro experiments, it can be concluded that PE-g-GO modulate protein adsorption and cell proliferation synergistically coupled to its improved dispersion and enhanced structural properties. As a concluding note, chemical functionalization of nanofillers are some routes that allow improvement of filler dispersion in matrix and better adhesion between filler and matrix. Fatigue and impact toughness should be carried out in order to obtain a better understanding of the mechanical

4. CONCLUSIONS On the basis of experimental results obtained with HDPE/GO and HDPE/PE-g-GO composites with varying concentration of GO and PE-g-GO up to 3 wt %, we can arrive at the following conclusions: (1) The melt-mixing technique has been successfully adapted to graft polymer chain on to the GO sheets and synthesize HDPE based composites reinforced with GO and PE-g-GO up to 3 wt %. 11965



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(2) In HDPE reinforced with 2 and 3 wt % PE-g-GO, the yield strength and elastic modulus showed an increment of 30 and 40% respectively, as compared to their unmodified counterparts. Maximum yield strength of 20 MPa and elastic modulus of 600 MPa was achieved with 3 wt % PE-g-GO reinforced HDPE composites. (3) Following the protein adsorption kinetics best-fits with langmuir adsorption model, it is clearly discernible that polyethylene grafting on GO modulates the BSA adsorption in terms of 3 times higher equilibrium constant and an order of magnitude higher monolayer concentration. (4) Cytocompatibility assessment using osteoblast and mesenchymal stem cells corroborated good cell attachment and growth with an increase in the number of mitochondrically active cells with time in the culture. WST-1 assay revealed that cell viability and proliferation on HDPE reinforced with 3 wt % PE-g-GO was maximum among the investigated composites. (5) The cell morphological analysis reveals the signatures of cell proliferation with extensive filopodial extensions for both MC3T3 and hMSC. Also, a better cell-material interaction is qualitatively established on PE-g-GO reinforced HDPE composite compared to unmodified GO reinforced HDPE composite. (6) Taken together, the present study demonstrated the modulation of protein adsorption and cell proliferation on 3% PE-g-GO reinforced HDPE compared to unmodified 3% GO reinforced HDPE composite together with better comparison of elastic modulus and yield strength. Therefore, it is believed that this developed material could be a promising candidate for moderate load bearing orthopedic applications.

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ASSOCIATED CONTENT

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00946. Optical images, SEM micrographs, water contact angle data and standard plot for protein adsorption. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. Author Contributions ∥

REFERENCES

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These authors made equal contributions.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for financial support from Department of Science and Technology (Govt. of India), Department of Biotechnology (Govt. of India), and the Centres of Excellence and Innovation in Biotechnology scheme through the Center of excellence project Translational Center on Biomaterials for Orthopedic and Dental Applications. The authors gratefully acknowledge support from Mr. Ravikumar Krishnamurthy for SEM images, Ms. Maya Sharma for Raman spectra, Mr. Prasanna Mural for cryo-sectioning using a Leica Ultramicrotome, Ms. Jini (NCBS, Bangalore), and Dr. Renu Pasricha (NCBS, Bangalore) for TEM. 11966


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