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The promising application of graphene oxide as coating materials in orthopedic implants: preparation, characterization and cell behavior
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015019 (http://iopscience.iop.org/1748-605X/10/1/015019) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 202.28.191.34 This content was downloaded on 13/02/2015 at 14:41
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 015019
doi:10.1088/1748-6041/10/1/015019
Paper
received
3 September 2014 re vised
6 January 2015
The promising application of graphene oxide as coating materials in orthopedic implants: preparation, characterization and cell behavior
accep ted for publication
8 January 2015 published
10 February 2015
Changhong Zhao1, Xiuzhen Lu1, Carl Zanden2 and Johan Liu1,2,3 1
SMIT Center, School of Mechatronic Engineering and Automation and Key Laboratory of Advanced Display and System Applications, Shanghai University, No 20, Chengzhong Road, Shanghai 201800, People’s Republic of China 2 Bionano Systems Laboratory, Department of Microtechnology and Nanoscience (MC2), Chalmers University of Technology, SE-412 96 Gothenburg, Sweden E-mail: [email protected] Keywords: graphene oxide, coatings, cellular activity, biocompatibility, differentiation
Abstract To investigate the potential application of graphene oxide (GO) in bone repair, this study is focused on the preparation, characterization and cell behavior of graphene oxide coatings on quartz substrata. GO coatings were prepared on the substrata using a modified dip-coating procedure. Atomic force microscopy (AFM), scanning electron microscopy (SEM) and Raman spectroscopy results demonstrated that the as-prepared coatings in this study were homogeneous and had an average thickness of ~67 nm. The rapid formation of a hydroxyapatite (HA) layer in the simulated body fluid (SBF) on GO coated substrata at day 14, as proved by SEM and x-ray diffraction (XRD), strongly indicated the bioactivity of coated substrata. In addition, MC3T3-E1 cells were cultured on the coated substrata to evaluate cellular activities. Compared with the non-coated substrata and tissue culture plates, no significant difference was observed on the coated substrata in terms of cytotoxicity, viability, proliferation and apoptosis. However, interestingly, higher levels of alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion were observed on the coated substrata, indicating that GO coatings enhanced cell differentiation compared with non-coated substrata and tissue culture plates. This study suggests that GO coatings had excellent biocompatibility and more importantly promoted MC3T3-E1 cell differentiation and might be a good candidate as a coating material for orthopedic implants.
1. Introduction Orthopedic implants often fail due to different reasons: poor osseointegration at the tissue-implant interface, loosening of artificial implants caused by the generation of wear debris and infections [1]. Among them, the lack of bone tissue integration and implantrelated infection are two major complications that may be encountered after implant insertion [2]. The surface properties of the implant, such as composition and morphology, play a crucial role in the success or failure of the implant [2, 3]. Ideally, implant surfaces should enhance osteoblast functions and concomitantly inhibit bacterial colonization. Numerous surface modification strategies, particularly surface coating, have been developed to promote bone modeling 3
Author to whom all correspondence should be addressed
© 2015 IOP Publishing Ltd
and prevent bacterial inhibition simultaneously. Unfortunately, up to now there is not a general solution to satisfy the requirements for an ideal implant surface. For instance, even though polymer-coated or modified implant surfaces combined with therapeutic agents provided encouraging properties and functions to improve device-tissue integration and reduce foreign body reactions and infections [4–9], they have poor wear resistance and worse, some degradation products, such as lactic acid, may generate acidic conditions and downregulate osteoblasts [10]. Bioactive ceramics such as hydroxyapatite have an excellent osteogenic effect. However, both the mechanical properties and the adhesion of the coatings to the implant substrata need to be improved [11]. Further advances in this field will require concurrent development in surface modification techniques and better candidates as surface coating materials.
Biomed. Mater. 10 (2015) 015019
C Zhao et al
A new carbon-based material, grapheme, since it was first deposited by mechanical exfoliation in 2004, has become one of the most exciting research topics owing to its unique properties such as high conductivity, transparency, mechanical strength and good tribological characteristics [12–16]. As one of the most important graphene derivatives, GO chemically exfoliated from oxidized graphite is considered a promising material for biomedical applications and has already shown potential for use as biosensors [17], bioimaging [18], drug delivery [19–21] and substrates for stem cell differentiation [22] due to its excellent aqueous processability, amphiphilicity, surface functionalizability, fluorescence quenching ability and low costs. Overall, GO meets most of the requirements for excellent implant coating materials, such as good biocompatibility, excellent mechanical strength and tribological characteristics. Moreover, some studies have shown that GO had inherent antibacterial properties [23]. This unique property can be used to prevent implant-induced infections. On the other hand, the toxicity of GO at a cellular level is still under debate. Some studies focused on GO dispersions indicated that GO was not cytotoxic unless at high concentrations [24, 25]. Nevertheless, only limited information is available regarding the cellular events on GO coatings, particularly on osteoblast adhesion, viability and differentiation on GO coatings. We conduct this study to investigate the potential of GO as an implant coating material. A fast dip-coating method was used to develop GO coatings on quartz substrata. Formation of hydroxyapatite on coated and non-coated substrata was evaluated by soaking substrata in SBF solution. Cell-material interaction was studied by culturing preosteoblast MC3T3-E1 cells on substrata.
2. Experimental section 2.1. Materials GO was purchased from XFNANO Materials Tech Co, Ltd (Nanjing, China) and suspended in ultrapure water to obtain GO aqueous dispersions w ith definite concentration by sonication. 3-aminopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulfuric acid, hydrogen peroxide solution (30% w/w in water), ethanol and acetone were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). Quartz substrata with a special size (22.0 mm in diameter, 1 mm in thickness) were purchased from Lianyungang Langston Quartz Glass Co, Ltd (Lianyungang, China). All chemicals were used without further purification. 2.2. Preparation of GO coatings on quartz substrata A modified dip-coating method combining electrostatic adsorption and solvent evaporation was used to prepare the coatings on substrata. To improve the water adsorption and hydrophilicity, quartz substrata were treated with piranha solution (hydrogen peroxide / 2
concentrated sulfuric acid 1:3;v/v) for 30 min at 100 °C, then washed with acetone, ethanol and ultrapure water successively and dried under nitrogen gas. The cleaned substrata were immersed in 5% anhydrous ethanol solution of APTES for 30 min, washed with ethanol and water and dried with nitrogen gas, followed by drying completely at 120 °C with nitrogen gas. GO sheets were immobilized on the APTES-treated substrate via electrostatic interaction by immersing the substrate in the GO aqueous suspension (1 mg ml−1) for 1 h, washing with water and ethanol and drying with nitrogen gas. Then, another 50 μl GO suspension was dropped on each substrate. The GO coating was finally formed through the natural evaporation of water at room temperature and then dried at 120 °C with nitrogen gas to remove the residual water and stabilize the GO coating layer. In the following sections, the GO-coated substrata and the quartz substrata without GO coating were abbreviated as coated substrata and non-coated substrata, respectively. 2.3. Characterizations of coated substrata The surface topography of coated substrata was observed using atomic force microscopy (AFM, SPM-9600, Shimadzu, Japan). Raman analysis of coated substrata was conducted using a Renishaw Invia Plus laser Raman spectrometer (Renishaw, UK). The surface morphology of coated substrata was observed using JSM 6700F field emission scanning electron microscopy (JEOL, Japan). 2.4. Formation of hydroxyapatite on substrata Coated and non-coated substrata were immersed in a 12-well plate filled with an SBF (pH = 7.42) solution. The 12-well plate was incubated in a cell culture incubator for 14 d, with the solution refreshed every second day. The SBF solution was prepared according to the previous protocol [26, 27]. After thoroughly washing with water three times, the substrata were dried under vacuum. SEM and an x-ray diffractometer (XRD) (D/max-2550 pc, Rigaku, Japan) were employed to investigate the mineral phase on substrata. 2.5. Cell culture The osteoblast-like cell line MC3T3-E1 was purchased from the Chinese Academy of Sciences Cell Bank (Beijing, China). Cells were cultured according to previous reports [27–30]. Briefly, cells were cultured in tissue culture plates containing alpha-minimal essential medium (alpha-MEM, Gibco BRL, Invitrogen, Life Technologies, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic solution containing 100 U ml −1 penicillin and 100 mg ml −1 streptomycin (Gibco BRL) in a cell incubator. In the following cell studies, all coated and non-coated substrata were sterilized overnight through exposure to UV light prior to in vitro study. 2.6. CCK8 assay A CCK8 assay (Beyotime Institute of Biotechnology, China) was performed to determine cell cytotoxicity
Biomed. Mater. 10 (2015) 015019
C Zhao et al
and proliferation on coated substrata, non-coated ones and tissue culture plates. For cytotoxicity testing, culture extracts were obtained by incubating substrata and tissue culture plates with alpha-MEM for 48 h in the cell incubator. After that, cells at a density of ~5000 cells/well were cultured in 96-well plates with culture extracts for 24 h and then washed with PBS and 10 μl of CCK-8 solution was added to each well and they were subsequently incubated for another 3 h in the cell incubator. The optical density (OD) of the CCK-8 solution in each well was recorded on a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm. For cell proliferation testing, cells at a density of ~20 000 cells/well were cultured on substrata with alpha-MEM in a 12-well culture plate and incubated for 1 and 2 d. At each time point, the medium was removed and cells were rinsed twice with PBS. CCK8 stock solution (10% of total volume) was added to 12-well plates and incubated for 3 h in the cell incubator. 100 μl of the reacted CCK8 stock solution in each well was transferred to 96-well plates and the absorbance was measured using the microplate reader at 450 nm. 2.7. Cell morphology The substrata in 12-well tissue culture plates were rinsed with alpha-MEM (1 ml per well) for 1 h in the cell incubator. Cells at a density of ~20 000 cells/well were seeded on the substrata and continuously incubated for 24 h. Non-coated substrata and tissue culture plates were selected as the controls. The cell morphology was recorded using optical microscopy. 2.8. Immunofluorescence microscopy Cells at a density of ~20 000 cells/well were seeded on each experimental substrate and cultured with alpha-MEM in the cell incubator for 24 h. Afterwards, 100 μl of the combined live/dead cell staining solution (100 μg ml −1 fluorescein diacetate and 50 μg ml −1 propidium iodide in PBS) was added to each well and incubated for 30 min at room temperature, then washed 3 times with PBS. Images were obtained using a BX51M optical microscope (Olympus Co, Japan) equipped with a fluorescence light source and filters. 2.9. Apoptosis assay An apoptosis kit (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, USA) was employed to detect apoptotic and necrotic cells. After incubation on coated substrata, non-coated ones and tissue culture plates in 12-well tissue culture plates for 48 h (cell seeding at a density of ~20 000 cells/well), cells were washed twice with PBS, then resuspended in annexin V-binding buffer and incubated with annexin V-FITC/PI in the dark for 15 min according to the manufacturer’s instructions. The stained cells were analyzed using the fluorescence-activated cell sorting method (MoFlo XDP, Beckman Coulter, USA). Subsequently, the 3
results were analyzed by flow cytometry using Summit software. 2.10. ALP activity and osteocalcin secretion Cell differentiation was characterized by determining both alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion. ALP activity is an early marker of osteoblast differentiation, whereas OC is a late marker. Cells (~20 000 cells/well) were cultured in triplicate (n = 3) on each sample group in differentiation media containing alpha-MEM medium supplemented with 50 mg ml−1 ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich). The ALP activity was determined using a commercial alkaline phosphatase substrate kit (Beyotime Institute of Biotechnology, China), which is a colorimetric assay based on measuring the enzymatic conversion of p-nitrophenyl phosphate to the yellowish product p-nitrophenol in the presence of ALP. At each time point, culture medium was removed; the cells were washed twice with PBS and lysed with cell lysis buffer (Sigma Aldrich). The cell lysate was mixed with a working assay solution at a ratio of 1:5, shaken for 1 min with a plate mixer and then incubated at 37 °C for 15 min. The reaction was stopped using 100 µl of stop solution and the absorbances were measured at 405 nm with a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo, USA). ALP activity was normalized to the total cellular protein, which was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). The result was a nanomole of p-nitrophenol liberated per microgram of total cellular protein per hour. For determination of OC secretion, cell culture medium was collected in contact with the cells and the amount of OC secreted was measured using the mouse osteocalcin EIA Kit (Biomedical Technologies, Stoughton, MA, USA) according to the manufacturer’s instructions. Data were expressed as nanograms of OC per milligram of cellular protein. 2.11. Statistical analysis For statistical analysis, six parallel tests were conducted for each group, except as otherwise noted. All data are expressed as the mean with the standard deviation (mean ± SD). Significance has been calculated using a Student’s t-test. * denotes a statistical significance (p 99%, single layer ratio: >99%, thickness: 0.8 ~ 1.2 nm, diameter: 1 ~ 5 μm) were purchased from Nanjing XFNANO Materials Tech Co, Ltd (Nanjing, China). The immobilized GO coatings on the substrate were analyzed using AFM and SEM. SEM and AFM observations showed that GO coatings consisting of multilayer GO sheets were
Biomed. Mater. 10 (2015) 015019
C Zhao et al
Figure 1. SEM image (a), Raman spectra (b), AFM image (c) and depth profile (d) of the coated substrate.
formed on the quartz substrate with high uniformity and coverage (figures 1(a) and (c)). Many of the GO layers overlapped adjacent GO layers along the edges and exhibited a continuous and wrinkled structure. The AFM profiles (figure 1(d)) showed that these coatings were ~67 nm in height. The Raman spectra demonstrated the presence of two broad peaks assigned to the G and D peaks (~1590 and ~1350 cm−1, respectively) as shown in figure 1(b). The G and D peaks reflect the main features in the Raman spectra of graphitic carbon-based materials. Basically, G and D peaks of graphene/GO signify sp2 hybridization (graphitic signature of carbon) and disorder due to the defects induced on the sp2 hybridized hexagonal sheet of carbon, respectively [31]. For GO, the strong D peak due to the defects from strong treatment with chemicals and amorphous carbon content is more significant (1300–1450 cm−1) with the sp2 cluster partly changed to sp3 [32]. 3.2. HA deposition Calcium phosphate materials, such as HA, that have an inorganic composition similar to bone, are bioactive and osteoconductive and promote direct cell attachment to bone [33–35]. Integration with bone tissue can be improved and accelerated by the presence of a calcium phosphate coating on the metal implant surface. The calcium phosphate coating can efficiently absorb special albumen available in serum, further enhance adherence and the differentiation ability of the osteoblast, promote the production of fibrinogen and accelerate osteoblast proliferation. A bioactive surface is considered to induce calcium phosphate deposition in SBF. To test the bioactivity of coated substrata, 4
biomimetic formation of hydroxyapatite was studied in an SBF solution. The surface morphology of substrata after 14 d was observed using SEM as shown in figure 2. The coated substrate was completely covered with compact spherical inorganic aggregates (figure 2(a)). SEM images at higher magnification clearly showed that the mineral sphere exhibits a porous nest-like structure (figures 2(c) and (d)). Without the GO coating treatment, the noncoated ones showed poor HA deposition and only a few isolated spherical particles were observed on the surface (figure 2(b)). XRD studies were carried out to further confirm the phase structure of the deposition on the coated and non-coated substrata (figure 3).The XRD patterns of the coated substrate after 14 d showed characteristics of the HA hexagonal phase (JCPDS card, no. 09-0432). The peaks at 25.8°, 28.1°,32.1° and 33.8° corresponded to the (0 0 2), (2 1 0), (2 1 1) and (2 0 2) reflections of HA. However, the non-coated substrata only showed the amorphous structure of the quartz substrate. The significant difference between the coated substrate and the non-coated one indicated that GO coating induced and accelerated the HA deposition, which demonstrated that GO coating could offer a bioactive surface for biological mineralization and bone formation. Functional surface chemical groups play important roles in nucleating calcium phosphate deposition on surgical titanium implants. For instance, surfaces modified with −PO4H2 and −COOH functional groups are reported to exhibit a stronger nucleating ability and induce apatite deposition [36]. The fast HA formation on coated substrata in this study might be due to hydrophilic functional groups, especially −COOH on
Biomed. Mater. 10 (2015) 015019
C Zhao et al
Figure 2. SEM images of coated substrata ((a): 10 000 × , (c): 2500 × , (d): 20 000×) and non-coated substrata ((b): 10 000×) after immersion in SBF for 14 d.
the other hand, the cell density on different substrata in figure 4 also showed that there was no prominent difference among coated substrata, non-coated substrata and tissue culture plates, indicating the excellent biocompatibility of GO coatings. The cell morphology was also evaluated using live/ dead staining with fluorescein diacetate (to stain live cells green) and propidium iodide (to stain dead cells red). Fluorescence microscopy revealed that most of the cells on coated substrata stayed alive, and both the cell density and morphology were similar to those on noncoated ones (figure 4(b)), which is consistent with the optical microscopy results. Figure 3. XRD patterns of coated substrata before (a) and after (c) immersion in SBF for 14 d and non-coated substrata (b) after immersion in SBF for 14 d.
GO coatings and the negative charged surface could absorb more Ca2+ to accelerate the Ca and P nucleating and crystal growth. Another factor contributing to HA deposition on coated substrata is the enhanced surface roughness [37]; in our study the roughness of coated substrata was determined from AFM height images to be 32.39 ± 5.11 nm, much higher than 4.37 ± 1.16 nm on non-coated substrata. The wrinkle-like GO sheets increased the surface roughness and provided more contact points for nucleating Ca and P. 3.3. Cell morphology The cell morphologies on coated substrata, noncoated ones and tissue culture plates were recorded to investigate the effect of GO coatings on cell adhesion and spreading (figure 4). After 24 h, cell morphological differences among those samples were not found. On 5
3.4. Cell cytotoxicity The cell cytotoxicity was determined using a CCK8 assay (figure 5), in which the optical density (OD) corresponded to the cell density in the culture CCK-8 solution. As shown in figure 5, a prominent difference among coated substrata, non-coated substrata and tissue culture plates was not observed after 24 h, indicating that GO coatings had no obvious toxic effects on MC3T3-E1 cells. 3.5. Cell proliferation and apoptosis Cell proliferation on coated substrata, non-coated ones and tissue culture plates was determined using a CCK-8 assay. A statistically significant increase in cell proliferation was found on all samples with increasing culture time (figure 6). Comparing the cell proliferation on coated substrata, non-coated ones and tissue culture plates against each other, there was no significant difference observed on both day 1 and day 2. Previously, A549 cells were reported
Biomed. Mater. 10 (2015) 015019
C Zhao et al
Figure 4. (a) Optical microscopic images (scale bar of 100 μm is applicable to all) and (b) fluorescent images (scale bar of 200 μm is applicable to all) of cells grown on the coated substrata, non-coated substrata and tissue culture plates.
The cell apoptosis assay was also performed to further evaluate the effect of GO coatings on cell growth. As demonstrated in figure 7, GO coatings did not induce any prominent apoptosis or necrosis compared with non-coated substrata and tissue culture plates. The percentages of apoptotic cells on coated substrata, noncoated ones and tissue culture plates were measured to be 8.77 ± 1.28%, 8.96 ± 1.09% and 9.43 ± 2.02%, respectively.
1.0 0.9
Absorbance (OD)
0.8 0.7 0.6 0.5 0.4 0.3 0.2
no nco ate d
co ate d
0.0
tis su ec ult ur ep lat e
0.1
Absorbance (OD)
Figure 5. Cell cytotoxicity of coated substrata, noncoated substrata and tissue culture plates, measured by CCK-8 assay.
1.2 1.0
coated non-coated tissue culture plate
*
*
*
0.8 0.6 0.4 0.2 0.0
day 1
day 2
Figure 6. Cell proliferation on coated, non-coated substrata and tissue culture plates at day 1 and day 2. *: p
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My IOPscience
The promising application of graphene oxide as coating materials in orthopedic implants: preparation, characterization and cell behavior
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015019 (http://iopscience.iop.org/1748-605X/10/1/015019) View the table of contents for this issue, or go to the journal homepage for more
Download details: IP Address: 202.28.191.34 This content was downloaded on 13/02/2015 at 14:41
Please note that terms and conditions apply.
Biomed. Mater. 10 (2015) 015019
doi:10.1088/1748-6041/10/1/015019
Paper
received
3 September 2014 re vised
6 January 2015
The promising application of graphene oxide as coating materials in orthopedic implants: preparation, characterization and cell behavior
accep ted for publication
8 January 2015 published
10 February 2015
Changhong Zhao1, Xiuzhen Lu1, Carl Zanden2 and Johan Liu1,2,3 1
SMIT Center, School of Mechatronic Engineering and Automation and Key Laboratory of Advanced Display and System Applications, Shanghai University, No 20, Chengzhong Road, Shanghai 201800, People’s Republic of China 2 Bionano Systems Laboratory, Department of Microtechnology and Nanoscience (MC2), Chalmers University of Technology, SE-412 96 Gothenburg, Sweden E-mail: [email protected] Keywords: graphene oxide, coatings, cellular activity, biocompatibility, differentiation
Abstract To investigate the potential application of graphene oxide (GO) in bone repair, this study is focused on the preparation, characterization and cell behavior of graphene oxide coatings on quartz substrata. GO coatings were prepared on the substrata using a modified dip-coating procedure. Atomic force microscopy (AFM), scanning electron microscopy (SEM) and Raman spectroscopy results demonstrated that the as-prepared coatings in this study were homogeneous and had an average thickness of ~67 nm. The rapid formation of a hydroxyapatite (HA) layer in the simulated body fluid (SBF) on GO coated substrata at day 14, as proved by SEM and x-ray diffraction (XRD), strongly indicated the bioactivity of coated substrata. In addition, MC3T3-E1 cells were cultured on the coated substrata to evaluate cellular activities. Compared with the non-coated substrata and tissue culture plates, no significant difference was observed on the coated substrata in terms of cytotoxicity, viability, proliferation and apoptosis. However, interestingly, higher levels of alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion were observed on the coated substrata, indicating that GO coatings enhanced cell differentiation compared with non-coated substrata and tissue culture plates. This study suggests that GO coatings had excellent biocompatibility and more importantly promoted MC3T3-E1 cell differentiation and might be a good candidate as a coating material for orthopedic implants.
1. Introduction Orthopedic implants often fail due to different reasons: poor osseointegration at the tissue-implant interface, loosening of artificial implants caused by the generation of wear debris and infections [1]. Among them, the lack of bone tissue integration and implantrelated infection are two major complications that may be encountered after implant insertion [2]. The surface properties of the implant, such as composition and morphology, play a crucial role in the success or failure of the implant [2, 3]. Ideally, implant surfaces should enhance osteoblast functions and concomitantly inhibit bacterial colonization. Numerous surface modification strategies, particularly surface coating, have been developed to promote bone modeling 3
Author to whom all correspondence should be addressed
© 2015 IOP Publishing Ltd
and prevent bacterial inhibition simultaneously. Unfortunately, up to now there is not a general solution to satisfy the requirements for an ideal implant surface. For instance, even though polymer-coated or modified implant surfaces combined with therapeutic agents provided encouraging properties and functions to improve device-tissue integration and reduce foreign body reactions and infections [4–9], they have poor wear resistance and worse, some degradation products, such as lactic acid, may generate acidic conditions and downregulate osteoblasts [10]. Bioactive ceramics such as hydroxyapatite have an excellent osteogenic effect. However, both the mechanical properties and the adhesion of the coatings to the implant substrata need to be improved [11]. Further advances in this field will require concurrent development in surface modification techniques and better candidates as surface coating materials.
Biomed. Mater. 10 (2015) 015019
C Zhao et al
A new carbon-based material, grapheme, since it was first deposited by mechanical exfoliation in 2004, has become one of the most exciting research topics owing to its unique properties such as high conductivity, transparency, mechanical strength and good tribological characteristics [12–16]. As one of the most important graphene derivatives, GO chemically exfoliated from oxidized graphite is considered a promising material for biomedical applications and has already shown potential for use as biosensors [17], bioimaging [18], drug delivery [19–21] and substrates for stem cell differentiation [22] due to its excellent aqueous processability, amphiphilicity, surface functionalizability, fluorescence quenching ability and low costs. Overall, GO meets most of the requirements for excellent implant coating materials, such as good biocompatibility, excellent mechanical strength and tribological characteristics. Moreover, some studies have shown that GO had inherent antibacterial properties [23]. This unique property can be used to prevent implant-induced infections. On the other hand, the toxicity of GO at a cellular level is still under debate. Some studies focused on GO dispersions indicated that GO was not cytotoxic unless at high concentrations [24, 25]. Nevertheless, only limited information is available regarding the cellular events on GO coatings, particularly on osteoblast adhesion, viability and differentiation on GO coatings. We conduct this study to investigate the potential of GO as an implant coating material. A fast dip-coating method was used to develop GO coatings on quartz substrata. Formation of hydroxyapatite on coated and non-coated substrata was evaluated by soaking substrata in SBF solution. Cell-material interaction was studied by culturing preosteoblast MC3T3-E1 cells on substrata.
2. Experimental section 2.1. Materials GO was purchased from XFNANO Materials Tech Co, Ltd (Nanjing, China) and suspended in ultrapure water to obtain GO aqueous dispersions w ith definite concentration by sonication. 3-aminopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sulfuric acid, hydrogen peroxide solution (30% w/w in water), ethanol and acetone were purchased from Sinopharm Chemical Reagent Co, Ltd (Shanghai, China). Quartz substrata with a special size (22.0 mm in diameter, 1 mm in thickness) were purchased from Lianyungang Langston Quartz Glass Co, Ltd (Lianyungang, China). All chemicals were used without further purification. 2.2. Preparation of GO coatings on quartz substrata A modified dip-coating method combining electrostatic adsorption and solvent evaporation was used to prepare the coatings on substrata. To improve the water adsorption and hydrophilicity, quartz substrata were treated with piranha solution (hydrogen peroxide / 2
concentrated sulfuric acid 1:3;v/v) for 30 min at 100 °C, then washed with acetone, ethanol and ultrapure water successively and dried under nitrogen gas. The cleaned substrata were immersed in 5% anhydrous ethanol solution of APTES for 30 min, washed with ethanol and water and dried with nitrogen gas, followed by drying completely at 120 °C with nitrogen gas. GO sheets were immobilized on the APTES-treated substrate via electrostatic interaction by immersing the substrate in the GO aqueous suspension (1 mg ml−1) for 1 h, washing with water and ethanol and drying with nitrogen gas. Then, another 50 μl GO suspension was dropped on each substrate. The GO coating was finally formed through the natural evaporation of water at room temperature and then dried at 120 °C with nitrogen gas to remove the residual water and stabilize the GO coating layer. In the following sections, the GO-coated substrata and the quartz substrata without GO coating were abbreviated as coated substrata and non-coated substrata, respectively. 2.3. Characterizations of coated substrata The surface topography of coated substrata was observed using atomic force microscopy (AFM, SPM-9600, Shimadzu, Japan). Raman analysis of coated substrata was conducted using a Renishaw Invia Plus laser Raman spectrometer (Renishaw, UK). The surface morphology of coated substrata was observed using JSM 6700F field emission scanning electron microscopy (JEOL, Japan). 2.4. Formation of hydroxyapatite on substrata Coated and non-coated substrata were immersed in a 12-well plate filled with an SBF (pH = 7.42) solution. The 12-well plate was incubated in a cell culture incubator for 14 d, with the solution refreshed every second day. The SBF solution was prepared according to the previous protocol [26, 27]. After thoroughly washing with water three times, the substrata were dried under vacuum. SEM and an x-ray diffractometer (XRD) (D/max-2550 pc, Rigaku, Japan) were employed to investigate the mineral phase on substrata. 2.5. Cell culture The osteoblast-like cell line MC3T3-E1 was purchased from the Chinese Academy of Sciences Cell Bank (Beijing, China). Cells were cultured according to previous reports [27–30]. Briefly, cells were cultured in tissue culture plates containing alpha-minimal essential medium (alpha-MEM, Gibco BRL, Invitrogen, Life Technologies, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic solution containing 100 U ml −1 penicillin and 100 mg ml −1 streptomycin (Gibco BRL) in a cell incubator. In the following cell studies, all coated and non-coated substrata were sterilized overnight through exposure to UV light prior to in vitro study. 2.6. CCK8 assay A CCK8 assay (Beyotime Institute of Biotechnology, China) was performed to determine cell cytotoxicity
Biomed. Mater. 10 (2015) 015019
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and proliferation on coated substrata, non-coated ones and tissue culture plates. For cytotoxicity testing, culture extracts were obtained by incubating substrata and tissue culture plates with alpha-MEM for 48 h in the cell incubator. After that, cells at a density of ~5000 cells/well were cultured in 96-well plates with culture extracts for 24 h and then washed with PBS and 10 μl of CCK-8 solution was added to each well and they were subsequently incubated for another 3 h in the cell incubator. The optical density (OD) of the CCK-8 solution in each well was recorded on a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 450 nm. For cell proliferation testing, cells at a density of ~20 000 cells/well were cultured on substrata with alpha-MEM in a 12-well culture plate and incubated for 1 and 2 d. At each time point, the medium was removed and cells were rinsed twice with PBS. CCK8 stock solution (10% of total volume) was added to 12-well plates and incubated for 3 h in the cell incubator. 100 μl of the reacted CCK8 stock solution in each well was transferred to 96-well plates and the absorbance was measured using the microplate reader at 450 nm. 2.7. Cell morphology The substrata in 12-well tissue culture plates were rinsed with alpha-MEM (1 ml per well) for 1 h in the cell incubator. Cells at a density of ~20 000 cells/well were seeded on the substrata and continuously incubated for 24 h. Non-coated substrata and tissue culture plates were selected as the controls. The cell morphology was recorded using optical microscopy. 2.8. Immunofluorescence microscopy Cells at a density of ~20 000 cells/well were seeded on each experimental substrate and cultured with alpha-MEM in the cell incubator for 24 h. Afterwards, 100 μl of the combined live/dead cell staining solution (100 μg ml −1 fluorescein diacetate and 50 μg ml −1 propidium iodide in PBS) was added to each well and incubated for 30 min at room temperature, then washed 3 times with PBS. Images were obtained using a BX51M optical microscope (Olympus Co, Japan) equipped with a fluorescence light source and filters. 2.9. Apoptosis assay An apoptosis kit (FITC Annexin V Apoptosis Detection Kit I, BD Biosciences, USA) was employed to detect apoptotic and necrotic cells. After incubation on coated substrata, non-coated ones and tissue culture plates in 12-well tissue culture plates for 48 h (cell seeding at a density of ~20 000 cells/well), cells were washed twice with PBS, then resuspended in annexin V-binding buffer and incubated with annexin V-FITC/PI in the dark for 15 min according to the manufacturer’s instructions. The stained cells were analyzed using the fluorescence-activated cell sorting method (MoFlo XDP, Beckman Coulter, USA). Subsequently, the 3
results were analyzed by flow cytometry using Summit software. 2.10. ALP activity and osteocalcin secretion Cell differentiation was characterized by determining both alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion. ALP activity is an early marker of osteoblast differentiation, whereas OC is a late marker. Cells (~20 000 cells/well) were cultured in triplicate (n = 3) on each sample group in differentiation media containing alpha-MEM medium supplemented with 50 mg ml−1 ascorbic acid (Sigma-Aldrich) and 10 mM β-glycerophosphate (Sigma-Aldrich). The ALP activity was determined using a commercial alkaline phosphatase substrate kit (Beyotime Institute of Biotechnology, China), which is a colorimetric assay based on measuring the enzymatic conversion of p-nitrophenyl phosphate to the yellowish product p-nitrophenol in the presence of ALP. At each time point, culture medium was removed; the cells were washed twice with PBS and lysed with cell lysis buffer (Sigma Aldrich). The cell lysate was mixed with a working assay solution at a ratio of 1:5, shaken for 1 min with a plate mixer and then incubated at 37 °C for 15 min. The reaction was stopped using 100 µl of stop solution and the absorbances were measured at 405 nm with a microplate reader (SPECTRA MAX PLUS 384 MK3, Thermo, USA). ALP activity was normalized to the total cellular protein, which was determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). The result was a nanomole of p-nitrophenol liberated per microgram of total cellular protein per hour. For determination of OC secretion, cell culture medium was collected in contact with the cells and the amount of OC secreted was measured using the mouse osteocalcin EIA Kit (Biomedical Technologies, Stoughton, MA, USA) according to the manufacturer’s instructions. Data were expressed as nanograms of OC per milligram of cellular protein. 2.11. Statistical analysis For statistical analysis, six parallel tests were conducted for each group, except as otherwise noted. All data are expressed as the mean with the standard deviation (mean ± SD). Significance has been calculated using a Student’s t-test. * denotes a statistical significance (p 99%, single layer ratio: >99%, thickness: 0.8 ~ 1.2 nm, diameter: 1 ~ 5 μm) were purchased from Nanjing XFNANO Materials Tech Co, Ltd (Nanjing, China). The immobilized GO coatings on the substrate were analyzed using AFM and SEM. SEM and AFM observations showed that GO coatings consisting of multilayer GO sheets were
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Figure 1. SEM image (a), Raman spectra (b), AFM image (c) and depth profile (d) of the coated substrate.
formed on the quartz substrate with high uniformity and coverage (figures 1(a) and (c)). Many of the GO layers overlapped adjacent GO layers along the edges and exhibited a continuous and wrinkled structure. The AFM profiles (figure 1(d)) showed that these coatings were ~67 nm in height. The Raman spectra demonstrated the presence of two broad peaks assigned to the G and D peaks (~1590 and ~1350 cm−1, respectively) as shown in figure 1(b). The G and D peaks reflect the main features in the Raman spectra of graphitic carbon-based materials. Basically, G and D peaks of graphene/GO signify sp2 hybridization (graphitic signature of carbon) and disorder due to the defects induced on the sp2 hybridized hexagonal sheet of carbon, respectively [31]. For GO, the strong D peak due to the defects from strong treatment with chemicals and amorphous carbon content is more significant (1300–1450 cm−1) with the sp2 cluster partly changed to sp3 [32]. 3.2. HA deposition Calcium phosphate materials, such as HA, that have an inorganic composition similar to bone, are bioactive and osteoconductive and promote direct cell attachment to bone [33–35]. Integration with bone tissue can be improved and accelerated by the presence of a calcium phosphate coating on the metal implant surface. The calcium phosphate coating can efficiently absorb special albumen available in serum, further enhance adherence and the differentiation ability of the osteoblast, promote the production of fibrinogen and accelerate osteoblast proliferation. A bioactive surface is considered to induce calcium phosphate deposition in SBF. To test the bioactivity of coated substrata, 4
biomimetic formation of hydroxyapatite was studied in an SBF solution. The surface morphology of substrata after 14 d was observed using SEM as shown in figure 2. The coated substrate was completely covered with compact spherical inorganic aggregates (figure 2(a)). SEM images at higher magnification clearly showed that the mineral sphere exhibits a porous nest-like structure (figures 2(c) and (d)). Without the GO coating treatment, the noncoated ones showed poor HA deposition and only a few isolated spherical particles were observed on the surface (figure 2(b)). XRD studies were carried out to further confirm the phase structure of the deposition on the coated and non-coated substrata (figure 3).The XRD patterns of the coated substrate after 14 d showed characteristics of the HA hexagonal phase (JCPDS card, no. 09-0432). The peaks at 25.8°, 28.1°,32.1° and 33.8° corresponded to the (0 0 2), (2 1 0), (2 1 1) and (2 0 2) reflections of HA. However, the non-coated substrata only showed the amorphous structure of the quartz substrate. The significant difference between the coated substrate and the non-coated one indicated that GO coating induced and accelerated the HA deposition, which demonstrated that GO coating could offer a bioactive surface for biological mineralization and bone formation. Functional surface chemical groups play important roles in nucleating calcium phosphate deposition on surgical titanium implants. For instance, surfaces modified with −PO4H2 and −COOH functional groups are reported to exhibit a stronger nucleating ability and induce apatite deposition [36]. The fast HA formation on coated substrata in this study might be due to hydrophilic functional groups, especially −COOH on
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Figure 2. SEM images of coated substrata ((a): 10 000 × , (c): 2500 × , (d): 20 000×) and non-coated substrata ((b): 10 000×) after immersion in SBF for 14 d.
the other hand, the cell density on different substrata in figure 4 also showed that there was no prominent difference among coated substrata, non-coated substrata and tissue culture plates, indicating the excellent biocompatibility of GO coatings. The cell morphology was also evaluated using live/ dead staining with fluorescein diacetate (to stain live cells green) and propidium iodide (to stain dead cells red). Fluorescence microscopy revealed that most of the cells on coated substrata stayed alive, and both the cell density and morphology were similar to those on noncoated ones (figure 4(b)), which is consistent with the optical microscopy results. Figure 3. XRD patterns of coated substrata before (a) and after (c) immersion in SBF for 14 d and non-coated substrata (b) after immersion in SBF for 14 d.
GO coatings and the negative charged surface could absorb more Ca2+ to accelerate the Ca and P nucleating and crystal growth. Another factor contributing to HA deposition on coated substrata is the enhanced surface roughness [37]; in our study the roughness of coated substrata was determined from AFM height images to be 32.39 ± 5.11 nm, much higher than 4.37 ± 1.16 nm on non-coated substrata. The wrinkle-like GO sheets increased the surface roughness and provided more contact points for nucleating Ca and P. 3.3. Cell morphology The cell morphologies on coated substrata, noncoated ones and tissue culture plates were recorded to investigate the effect of GO coatings on cell adhesion and spreading (figure 4). After 24 h, cell morphological differences among those samples were not found. On 5
3.4. Cell cytotoxicity The cell cytotoxicity was determined using a CCK8 assay (figure 5), in which the optical density (OD) corresponded to the cell density in the culture CCK-8 solution. As shown in figure 5, a prominent difference among coated substrata, non-coated substrata and tissue culture plates was not observed after 24 h, indicating that GO coatings had no obvious toxic effects on MC3T3-E1 cells. 3.5. Cell proliferation and apoptosis Cell proliferation on coated substrata, non-coated ones and tissue culture plates was determined using a CCK-8 assay. A statistically significant increase in cell proliferation was found on all samples with increasing culture time (figure 6). Comparing the cell proliferation on coated substrata, non-coated ones and tissue culture plates against each other, there was no significant difference observed on both day 1 and day 2. Previously, A549 cells were reported
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Figure 4. (a) Optical microscopic images (scale bar of 100 μm is applicable to all) and (b) fluorescent images (scale bar of 200 μm is applicable to all) of cells grown on the coated substrata, non-coated substrata and tissue culture plates.
The cell apoptosis assay was also performed to further evaluate the effect of GO coatings on cell growth. As demonstrated in figure 7, GO coatings did not induce any prominent apoptosis or necrosis compared with non-coated substrata and tissue culture plates. The percentages of apoptotic cells on coated substrata, noncoated ones and tissue culture plates were measured to be 8.77 ± 1.28%, 8.96 ± 1.09% and 9.43 ± 2.02%, respectively.
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Figure 5. Cell cytotoxicity of coated substrata, noncoated substrata and tissue culture plates, measured by CCK-8 assay.
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Figure 6. Cell proliferation on coated, non-coated substrata and tissue culture plates at day 1 and day 2. *: p
