Preparation, mechanical properties and biocompatibility of graphene ...

European Polymer Journal 48 (2012) 1026–1033

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

Preparation, mechanical properties and biocompatibility of graphene oxide/ultrahigh molecular weight polyethylene composites Yuanfeng Chen a,b, Yuanyuan Qi c, Zhixin Tai a, Xingbin Yan a,⇑, Fuliang Zhu b, Qunji Xue a a

State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics, Chinese Academy of Science, Lanzhou 730000, China Department of Material Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China c School of Stomatology, Lanzhou University, Lanzhou 730000, China

MACROMOLECULAR NANOTECHNOLOGY

b

a r t i c l e

i n f o

Article history: Received 22 November 2011 Received in revised form 9 March 2012 Accepted 15 March 2012 Available online 27 March 2012 Keywords: GO/UHMWPE composites Fabrication Mechanical properties Biocompatibility

a b s t r a c t Graphene oxide (GO)/ultrahigh molecular weight polyethylene (UHMWPE) composites were prepared by liquid-phase ultrasonication dispersion followed by hot-pressing. The microstructure features and mechanical properties of the composites were investigated by scanning electron microscope (SEM) and universal testing machine, respectively. Moreover, the attachment and proliferation of the MC3T3-E1 osteoblasts on the composites’ surfaces were investigated by methyl thiazolyl tetrazolium assay, SEM and fluorescence staining observations to evaluate the biocompatibility of the GO/UHMWPE composites. As shown in the cross-section SEM images, GO sheets were well dispersed within the UHMWPE matrix. The addition of GO sheets up to 1.0 wt.% not only increased the hardness of the pure UHMWPE gradually, but also improved its yield strength slightly. The MC3T3E1 cells well attached and grew on the surfaces of the composites, and the adding of GO did not affect the cells’ morphology and viability. The GO/UHMWPE composites displayed a remarkable combination of enhanced mechanical properties and good biocompatibility, making the composites attractive for potential candidate as artificial joints in the human body. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Ultrahigh molecular weight polyethylene (UHMWPE), which has excellent mechanical properties and stability, is widely used as the material for artificial joints in the human body [1]. However, owing to the low surface hardness and poor wear resistance of polymer, wear particles lead to osteolysis [2–4], which will cause bone loss, joint loosening, discomfort, and ultimately limits the lifespan of the joints [5,6]. Therefore, much effort has been carried out to improve the mechanical and tribological properties of the UHMWPE materials. Up to now, it has been revealed that crosslinked UHMWPE (generated either by gamma irradiation, or by peroxide treatment or thermal treatment) can dramatically increase the wear resistance compared with ⇑ Corresponding author. Tel./fax: +86 931 4968055. E-mail address: [email protected] (X. Yan). 0014-3057/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2012.03.011

non-crosslinked UHMWPE [7]. Unfortunately, some studies have also shown that some mechanical properties, for example, ductility, modulus and fatigue strength would be diminished in crosslinked UHMWPE [8–12]. Another way to improve the wear and the mechanical properties of UHMWPE is the use of inorganic fillers. Much work has been done in this field. Quartz [13], kaolin [14], zirconium particles [15] and carbon nanotubes [16–18] have been chosen as the fillers in the composites. However, the use of these inorganic fillers/UHMWPE composites in highperformance artificial joints is still limited due to their drawbacks, such as relatively high additive amounts for traditional inorganic particles, high cost of carbon nanotubes and unsatisfactory performance of composites. Since graphene was first isolated in 2004 with the help of scotch tape, researchers have excitedly turned to the material to discover its potential applications. Graphene with superior mechanical [19], electrical [20,21] and

Y. Chen et al. / European Polymer Journal 48 (2012) 1026–1033

2. Experimental 2.1. Materials Granulated UHMWPE was supplied from Beijing Oriental Petrochemical Co., Ltd., China. Graphite powder (325 mesh) was purchased from Qingdao Huatai Tech. Co., Ltd., China. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Bio. Engineer. Mater. Co., Ltd., China. MC3T3-E1 cells and RPMI 1640 medium were purchased from Lanzhou Shenggong Biomedical Co., Ltd., China. Trypsin–EDTA solution and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were purchased from Sigma. Other reagents were commercially available and were of analytical reagent grade. All chemicals and solvents were used as received. 2.2. Preparation of the GO/UHMWPE composites High-purity graphite was used to prepare graphene oxide according to a modified Hummers’ method [32]. A series of GO/UHMWPE composites were prepared by liquid-phase ultrasonication dispersion followed by hotpressing. In brief, as-prepared powdery GO was subjected to disperse in 50 ml alcohol with the aid of ultrasonication for 30 min, to form a well dispersed suspension (Fig. 1a). Subsequently, UHMWPE powders were added into the suspension and the mixture was stirred for 30 min and then ultrasonicated for 1 h. Then the alcohol was removed at 60 °C in an oil bath and the solid product was completely dried in an oven at 60 °C (Fig. 1b). Finally, the obtained solid

was molded into a 70 70 1 mm3 shape by hot-pressing at 195 °C under a 10 MPa pressure and holding at this pressure for 20 min. To investigate the effects of GO on the mechanical properties and biocompatibility of UHMWPE, composites with the addition of 0, 0.1, 0.3, 0.5 and 1.0 wt.% GO were prepared. 2.3. Characterization of the composites Transmission electron microscopy (TEM, JEOL, JEM2010) was employed to investigate the morphology of asprepared GO, using an accelerating voltage of 200 kV. Xray photoelectron spectroscopy (XPS) measurement of the GO was performed on a Perkin–Elmer PHI-5702 multi-functional X-ray photoelectron spectroscope (Physical Electronics, USA), using Al-Ka radiation (photon energy 1476.6 eV) as the excitation source and the binding energy of Au (Au 4f7/2: 84.00 eV) as the reference. The fracture morphology of the GO/UHMWPE composites was observed using a field emission scanning electron microscope (SEM, JEOL, JSM 6701F). For SEM observations, the obtained sheets were first frozen in liquid nitrogen, and then quickly fractured. Crystallinity and melt temperature of the composites were determined by differential scanning calorimetry (DSC) (METTLER TOLEDO DSC822e) at a scanning rate of 10 °C/min from 20 to 200 °C under nitrogen atmosphere. The hardness was measured by a microhardness tester with a load of 0.1 N and the sample size was 10 10 mm2. The mechanical properties were measured by a universal testing machine (AGS-X5kN, Shimadzu Corporation). Tensile specimens were cut from the compression-molded panels with size of 70 mm in length and 4 mm in width (Fig. 1c). The tensile tests were performed at a rate of 20 mm/min. 2.4. Biocompatibility test 2.4.1. MC3T3-E1 cell culture and seeding MC3T3-E1 cells were cultured in RPMI 1640 containing 10% FBS, 50 U/ml penicillin and 50 U/ml streptomycin. The medium was refreshed every 3 days and the cells were incubated in a tissue culture incubator at 37 °C with 5% CO2. After reaching about 80% confluence, the cells were detached by 0.05% trypsin. The sample size for cell culture and seeding was 10 10 mm2. A density of 104 cells/ml was seeded in 24-well plates for methyl thiazolyl tetrazolium (MTT) assay and SEM observation after the composite samples were sterilized by 75% ethyl alcohol for 2 h, and a density of 105 cells/ml was seeded for fluorescence staining observation. 2.4.2. MTT assay The MTT assay is one of the chromatic assays that have been used to test the cytotoxicity and cell viability. Cells were seeded as previously described and the cell viability was evaluated after 1, 2 and 4 days by MTT assay, which was indicated by the reduction of MTT into a formazan dye by living cells. MTT solution (100 ll) at 5 mg/ml in phosphate buffered saline (PBS) was added to each well and incubated for 4 h under the same conditions described. After removal of the medium, the converted dye was dis-


optical properties [22], high specific surface area and low cost compared to carbon nanotubes, has attracted intense attention for various applications. Graphene oxide (GO) could be regarded as graphene functionalized by carboxylic acid, hydroxyl, and epoxide groups [23]. It makes itself relatively easy to be dispersed into some polar solvents and to form intercalated composites with polar molecules through the strong interaction. Recently, incorporation of GO or graphene nanofillers into a variety of polymers has given rise to exceptional enhancements in conductivity and mechanical properties [24–26]. More recently, GO and graphene have been proved to exhibit excellent biocompatibility [27–31]. These have motivated us to explore the possibility of GO as a reinforcement in UHMWPE matrix for a new kind of artificial joints. However, to our knowledge, GO/UHMWPE composite materials are rarely reported. In this study, we report for the first time on the synthesis of GO/UHMWPE composites by using a simple combination of liquid-phase ultrasonication dispersion followed by hotpressing. As-prepared GO/UHMWPE composites showed remarkably enhanced hardness and slightly improved yield strength compared with pure UHMWPE. The addition of small amounts of GO did not affect the attachment and proliferation of the MC3T3-E1 osteoblasts cultured on GO/ UHMWPE composite surfaces, indicating its excellent biocompatibility. We expect such GO/UHMWPE composites with good mechanical properties and biocompatibility may find important applications in artificial joints.

1027


1028


Fig. 1. Photographs of (a) GO suspensions in alcohol, (b) UHMWPE powders and GO/UHMWPE mixed powders with 1 wt.% GO and (c) tensile specimens of GO/UHMWPE composites.

solved in 750 ll/well dimethyl sulfoxide. Solution (150 ll) of each sample was transferred to a 96-well plate. Absorbance of converted dye was measured at a wavelength of 490 nm using an ELISA plate reader. 2.4.3. SEM and fluorescence staining observations After 1, 2 and 4 days of culture, samples were rinsed twice with PBS to remove non-adherent cells and subsequently fixed with 3% glutaraldehyde at 4 °C for 4 h. Thereafter, the samples were dehydrated through a series of

(a)

graded ethanol solutions and air-dried overnight. Dry cellular constructs were sputtered with gold and observed under SEM to examine morphology of the cells. In the same way, cells were dehydrated through absolute ethyl alcohol, and the samples were stained with acridine orange, which was cleaved to yield a green fluorescent product by metabolically active cells. The density of the cells which adhered on each sample was measured from randomly selected views of observed at 100-fold magnification with a microscopy (Olympus BX51).

(b)

C=C

C-O

C=O O-C=O

100 nm 280

282

284

286

288

Binding energy (eV) Fig. 2. Characterization of GO sheets: (a) TEM image and (b) C1s XPS spectrum.

290

292

294

3. Results and discussion 3.1. Morphology and crystalline structure Fig. 2 showed the TEM image and XPS spectrum of GO sheets. It was evident that the GO exhibited a typically wrinkled, thin and sheet-like structure (Fig. 2a). Furthermore, the C1s XPS spectrum of GO after the Gaussian fitting indicated the presence of four types of carbon bonds (Fig. 2b): C@C (284.5 eV), CAO (286.6 eV), C@O (287.8 eV) and OAC@O (289.0 eV). It indicates the considerable degree of the oxidation existing in GO material, which results in the hydrophilic nature of GO. The DSC curves and crystallinity of GO/UHMWPE composites were shown in Fig. 3. The peak temperature of crystallization melting occurred at about 137 °C for all the GO/ UHMWPE composites, indicating that the addition of GO has little effect on the melting point of the composites. The enthalpy of crystallization is 125.16, 145.45, 134.68, 136.29 and 130.75 J/g for 0%, 0.1%, 0.3%, 0.5% and 1% weight fraction of GO, respectively. The crystalline fraction of GO/UHMWPE composites was calculated as [33]:

xc ¼

DH

ð1Þ

ð1 /ÞDH0

3.2. Microhardness

where DH is the apparent enthalpy of fusion per gram of composite, DH0 is the heat of fusion of a 100% crystalline PE taken as 293 J/g [34], and / is the weight fraction of the filler in the composites. According to formula (1), the crystallinity of the composites increased from 42.7% to 49.7% with the addition of 0.1 wt.% of GO, then the values reduced slightly (Fig. 3b). When appropriate amount of GO sheets were added into the polymer, the GO surface could act as nucleation sites for the crystallization. Therefore, the nucleation and growth of individual crystallites were so rapid that a very large number of crystallites were formed together. Fig. 4 showed the SEM images of the fracture surfaces of unfilled UHMWPE and GO/UHMWPE composites. It could be seen that the fracture surface of unfilled UHMWPE

(a)

was relatively flat. When the GO content was 0.1 or 0.3 wt.%, the corresponding fracture surface was uneven and the GO sheets were randomly distributed within the polymer matrix. Furthermore, as the content of GO increased to 0.5 wt.%, the morphology of the fracture surface was totally different. GO sheets were distributed homogeneously within the UHMWPE matrix without obvious agglomeration. The magnified image (Fig. 4f) showed GO sheets were embedded into the polymer so that GO and polymer could combine tightly to each other to form a layered structure. As the content of GO further increased to 1.0 wt.%, the layers stacked in a more compact manner. As a result, the extension of the polymer chain was hindered by the closely compacted GO sheets. Our fabrication processing was presented to explain the combination of GO and polymer matrix. After they were mixed by ultrasonication dispersion and high speed stirring, UHMWPE powders were homogenously covered by GO sheets. To some extent, ultrasonication dispersion was an effective way to disperse GO sheets into UHMWPE. Subsequently, these composite powders were hot-compressed at a high temperature to stick together tightly and form a continuous mixed phase.

Fig. 5 showed the variation of microhardness values with the increase of GO into UHMWPE. It could be noticed that the microhardness of the GO/UHMWPE composites increased gradually with the increase of GO. The addition of 1.0 wt.% GO increased the microhardness of unfilled UHMWPE from 5.18 to 5.97, corresponding to an increasing amount of about 15%. It was indicated that low additions of GO sheets could obviously enhance the hardness of UHMWPE. This was due to the excellent mechanical properties of GO sheets, which could bear partial load and be very essential for load transfer. According to Archard’s prediction [35], the increase of hardness would reduce the plastic contact areas for UHMWPE or its composite to metal surface. Therefore, we believe GO/

(b) 55

0 -5

50

-15

Crystallinity (%)

Heat flow (mW)

-10

-20 -25 0% 0.1% 0.3% 0.5% 1%

-30 -35 -40 40

60

80

100

120

140

160

180

200

45

40

35

0.0

0.2

0.4

Temperature ( C) O

Fig. 3. DSC curves (a) and crystallinity (b) of GO/UHMWPE composites.

0.6

GO wt.%

0.8

1.0


1029



1030


Fig. 4. SEM images of the fractured surfaces of the GO/UHMWPE composites with different amounts of GO: (a) 0 wt.%, (b) 0.1 wt.%, (c) 0.3 wt.%, (d) 0.5 wt.% and (e) 1 wt.% and (f) a magnified image of the GO/UHMWPE with 0.5 wt.% GO.

UHMWPE composites would have improved friction performance with coordination metal material compared with unfilled UHMWPE material. 3.3. Tensile properties As we know, pure UHMWPE has good ductility. Under tension condition at room temperature, it will yield first and then draw followed by strain hardening effect and finally rupture at a relative long elongation [36]. Fig. 6 showed the typical stress–strain curves of the GO/ UHMWPE composites. As the curves showed, different additions of GO sheets had different effects on the tension performance of the composites. Detailed information about the tensile properties was shown in Table 1. As shown in the inset (Fig. 6), although the amounts of GO

in all composites were low, the enhancement of yield strength by the GO addition was realized. However, it should be mentioned that, the ultimate tensile strength and tensile elongation both decreased once GO sheets were added into the UHMWPE even with a very low amount of 0.1 wt.%, and then the values of the ultimate tensile strength and tensile elongation increased with the increase of GO content up to 0.5 wt.%. The GO/UHMWPE composite with the GO content of 0.5 wt.% had the maximum values, which are slightly higher than those of the pure UHMWPE. As the content of GO further increased to 1.0 wt.%, these values decreased instead of increased forward. These results were similar with other graphene/GO-reinforced polymer composites. Fan et al. [37] observed that graphene-reinforced chitosan composites showed improvement in the elastic modulus and hardness, but their values were

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6.2

0.35

0% 0.1% 0.3% 0.5% 1%

0.30 0.25

5.8

Absorption

5.6 5.4

0.20 0.15 0.10

5.2

0.05 0.00

5.0 0.0

0.2

0.4

0.6

0.8

1.0

24h

GO wt.% Fig. 5. The microhardness of the GO/UHMWPE composites with different amounts of GO.

Stress (MPa)

45

24.0 23.5

0% 0.1% 0.3% 0.5% 1%

23.0

40

22.5

35

22.0

30

21.5

0.10

0.15

0.20

0.25

96h

Fig. 7. Viability of the MC3T3-E1 cells on the GO/UHMWPE composites with different amounts of GO at different incubation times.

parts of the polymer chains were adsorbed onto the GO surface due to the ultrahigh surface area of GO sheets. For those composites with little GO content, GO sheets were randomly distributed and only helped to improve the yield strength. For the composite with 0.5 wt.% GO, its layered structure was favorable to load transfer from polymer matrix to GO. Moreover, the relative loosened layered structure could absorb some energy from the external forces. However, the UHMWPE chains might be destroyed as the content of GO increased and the plastic deformation of the composites would reduce.

55 50

48h

Time

25 20 15 10

3.4. Biocompatibility

5 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Strain (mm/mm) Fig. 6. Typical stress–strain curves of the GO/UHMWPE composites. Inset is the magnified curves of the marked area.

irregular with the addition of graphene contents. Wang et al. [38] gave stress–strain curves of GO/polybenzimidazole composites, which indicated the tensile strength had an optimal value when GO content was 0.3 wt.% and too high or too low GO content would reduce the performance of materials. To explain the mechanical properties of the GO/ UHMWPE composites, a mechanism of particle reinforced polymer material could be used due to the strong interaction between GO and the UHMWPE matrix. As a result,

MTT assay is a cytotoxicity test method by evaluating the number of living cells and the strength to living cells metabolism [39]. Fig. 7 showed the relationship between the absorption of cell plate and the cell culture time. The observed results revealed that all composite samples were suitable for the proliferation of the MC3T3-E1 cells in a fast rate. There was no obvious change in the adsorption on the GO/UHMWPE composites compared with that on the pure UHMWPE, indicating that the addition of GO sheets into UHMWPE had no negative effect on the cell growth. It can be definitely attributed to the excellent intrinsic biocompatibility and the hydrophilic nature of GO material. Fig. 8 showed the cell attachment on the GO/UHMWPE composite with 0.5 wt.% GO at the incubation periods of 24, 48 and 96 h, respectively. It was clear that MC3T3-E1 cells well adhered and proliferated on the GO/UHMWPE scaffold with the increase of the culture time. After 96 h

Table 1 Mechanical properties of the GO/UHMWPE composites with different amounts of GO. Sample designation

Yield strength (MPa)

Ultimate tensile strength (MPa)

Elongation at break (%)

UHMWPE GO/UHMWPE GO/UHMWPE GO/UHMWPE GO/UHMWPE

22.88 ± 0.77 23.03 ± 0.46 23.42 ± 1.08 23.70 ± 1.16 23.38 ± 1.06

30.45 ± 1.27 22.82 ± 0.76 24.15 ± 1.56 30.61 ± 1.72 26.55 ± 0.80

2.71 ± 0.47 0.88 ± 0.32 1.20 ± 0.24 2.76 ± 0.53 1.53 ± 0.48

(0.1%) (0.3%) (0.5%) (1%)


Micro-hardness (Hv)

6.0

1032



Fig. 8. Fluorescent microscopy images of the MC3T3-E1 cells on the GO/UHMWPE composite with 0.5 wt.% GO for 24 h (a), 48 h (b) and 96 h (c).

Fig. 9. SEM images of the MC3T3-E1 cells on the GO/UHMWPE composite with 0.5 wt.% GO for 24 h (a), 48 h (b) and 96 h (c).

incubation, the sample surface was almost covered with cells. As shown in Fig. 9, after the cells were seeded directly on the GO/UHMWPE scaffold and incubated for 24 h, the scaffold surface appeared to be adhered with some cells. The cells maintained the normal morphology with some pseudopods around them, which were probably produced from the cells. With the increase of culture time, the cells secreted extracellular matrices (ECM), where cells were able to adhere, grow and spread on the scaffold surface. As we know, various carbon materials have been proven to be promising for biomedical applications such as tissue engineering and implants [40,41], in part because of their inherent biocompatibility. Also, as we described before, GO and graphene have been proved to exhibit excellent biocompatibility [28,42]. Fan et al. [37] found that graphene/chitosan composites had good biocompatibility and cells could adhere and grow on the composite films as well as on pure chitosan film; Liu et al. [43] found that, because of the existence of the hydrophilic groups (carboxylic acid, hydroxyl and epoxide groups), GO sheets could promote the attachment and proliferation of human cells, especially retinal pigment epithelium (RPE) cells. Therefore, we believed that the preservation of biocompatibility for the GO/UHMWPE composites was attributed to the excellent intrinsic biocompatibility and the hydrophilic nature of GO. 4. Conclusions A simple liquid-phase mixing followed by hot-pressing was used to prepare GO/UHMWPE composites, where GO sheets were well dispersed into polymer matrix. The addition of GO with small amounts could obviously increase the microhardness and the GO/UHMWPE composite with 0.5 wt.% GO had the best tensile strength. Moreover, the GO/UHMWPE composites exhibited good biocompatibility. Therefore, the combined advantages of the GO/UHMWPE composites, including improved mechanical properties

and good biocompatibility, make them promising materials for artificial joints applications. Acknowledgements The authors acknowledge financial support from the Natural Science Foundation of China (51005225) and the Top Hundred Talents Program of Chinese Academy of Sciences.

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