Graphene accelerates osteoblast attachment and biomineralization

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Graphene accelerates osteoblast attachment and biomineralization

Jia Ren, Xiaogang Zhang and Yao Chen^♠

School of Mechanical and Electric Engineering & Collaborative Innovation Center of Suzhou Nano Science and Technology, So- ochow University, Suzhou 215123, China

Received 5 October 2016 Accepted 25 October 2016

♠Corresponding Author E-mail: chenyao@suda.edu.cn Tel: +86-512-6758-1872

Open Access

pISSN: 1976-4251 eISSN: 2233-4998

Article Info

Abstract

In this paper, the in vitro biocompatibility of graphene film (GF) with osteoblasts was evaluated through cell adhesion, viability, alkaline phosphatase activity, F-actin and vinculin expressions, versus graphite paper as a reference material. The results showed that MG-63 cells exhibited stronger cell adhesion, better proliferation and viability on GF, and osteoblasts cultured on GF exhibited vinculin expression throughout the cell body. The rougher and wrinkled surface morphology, higher elastic modulus and easy out-of-plane deformation associated with GF were considered to promote cell adhesion. Also, the biomineralization of GF was assessed by soaking in simulated body fluid, and the GF exhibited enhanced mineralization ability in terms of mineral deposition, which almost pervaded the entire GF surface.

Our results suggest that graphene promotes cell adhesion, activity and the formation of bone- like apatite. This research is expected to facilitate a better understanding of graphene-cell interactions and potential applications of graphene as a promising toughening nanofiller in bioceramics used in load-bearing implants.

Key words: graphene film, osteoblast, adhesion, viability, mineralization

1. Introduction

In recent years, carbonaceous nanomaterials including graphene and carbon nanotubes (CNTs) have attracted much attention as an important class of novel materials. They have been widely designed for structural engineering and functional device applications due to their extraordinary mechanical strength, and excellent electrical and thermal conductivities [1-4]. In particular, the choice of carbonaceous nanomaterials as toughening nanofillers in brittle ceramics seems to be an attractive option because graphene or CNT bridging around crack faces can lower the driving force for crack propagation [5-10].

Among various ceramics, the inferior fracture toughness of hydroxyapatite (HA), which otherwise has excellent bioactivity and osteointegration, limits its wide application in the load-bearing implants used in the orthopedic field. To improve their toughness, HA-based composites have been developed with some bio-inert reinforcements such as Al2O3, TiO2, and Ti. However, it is well known that an ideal reinforcement in HA should significantly improve the mechanical properties without compromising its original biocompatibility [11].

Compared with the conventional toughening agents above, it is well recognized that the main advantage of CNTs or graphene in HA-based composites is the very low filler loading required to achieve a significant toughening effect. This feature opens a promising avenue in methods for toughening HA-based composites.

Nevertheless, the biocompatibility of CNTs is still under debate due to their cytotoxic responses in organic environments, although some researchers have ascribed the cytotoxicity of CNTs to the presence of metallic catalyst particles rather than the CNT itself [11]. In this respect, advances in the synthesis of pure and large-scale graphene via chemical vapor deposition or micromechanical exfoliation of graphite are expected to fulfill the vital DOI: http://dx.doi.org/

DOI:10.5714/CL.2017.22.042 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/

by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Three-dimensional porous graphene materials for environmental applications Muruganantham Rethinasabapathy, Sung-Min Kang, Sung-Chan Jang and Yun Suk Huh

KCS Korean Carbon Society carbonlett.org

pISSN: 1976-4251 eISSN: 2233-4998

REVIEWS VOL. 22 April 30 2017

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Matsuoka et al. [18], in which the adhesive strength was assessed by the ratio of the adherent cell numbers after using 0.1% trypsin-EDTA treatment, to the untreated cell numbers.

After being cultured for 12 h, the cells were washed with phosphate buffer saline (PBS) twice and treated with 0.1%

trypsin-EDTA solution (Invitrogen) for 5 min, 10 min, 20 min, and 30 min at 37±1°C in a humidified 5% CO2 atmosphere. Then, each sample was washed twice with PBS to remove the detached cells. The cells adhered on the substrate were fixed with 4% paraformaldehyde and stained with rhodamine-phalloidin (Invitrogen). The cell numbers in ten pho- tographs of each substrate were counted, and measurements were performed on each sample.

The F-actin and vinculin of the adherent MG-63 cells on the GF and GP were labeled with a fluorescent dye to observe the actin cytoskeleton and focal adhesion, respectively. MG-63 cells were seeded on each sample for 12 h, and then were washed with PBS and fixed with 4% paraformaldehyde for 30 min.

Thereafter, the cells were permeabilized with 0.1% Triton X-100 for 30 min and then incubated with 2 U/mL of rhodamine-phalloidin (Invitrogen) for 60 min to label F-actin. Subsequently, the cells were incubated with monoclonal anti-vinculin-FITC anti- body (Sigma, USA) for 12 h to label vinculin. The fluorescently stained cells were visualized using a fluorescence microscope (Olympus IX71, Japan).

Cell proliferation and viability was evaluated by WST-1 test (Cell Proliferation and Cytotoxicity Assay Kit; Beyo- time, China). After the MG-63 cells were incubated on each sample for 1 d, 3 d and 7 d, the cells were rinsed twice with PBS. Thereafter, 10 μL of WST-1 solution and 90 μL of medium (final dilution, 1:10) were added to each sample followed by another 2 h incubation. Supernatants were quantified using a spectrophotometer (SpectraMax Paradigm Multi-Mode De- tection Platform; Molecular Devices, USA) at 450 nm with a reference wavelength of 625 nm. Results of the WST-1 were reported as optical density.

Osteoblast differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. The cells were seeded on the samples and cultured for 1 d, 3 d, and 7 d, rinsed twice with PBS, and then permeabilized with 0.1% Triton X-100 for 30 min. ALP activity was measured by using a Lab Assay ALP kit (Beyotime) according to the manufacturer’s protocol. After 4 h incubation, supernatants were quantified spectrophotometrically at 405 nm.

The osteoconductivity of the carbonaceous materials was assessed by apatite mineralization in a SBF solution, which was prepared according to the well-known Kokubo composition [19]. Both samples were soaked in SBF and kept at 37±1°C in a humidified atmosphere of 5% CO2 for 1 d, 3 d, and 7 d, respectively. After the soaking period, samples were carefully washed with deionized water three times and dried at ambient temperature. Mineralization products were char- acterized by X-ray diffraction (X’Pert-Pro MPD; PANalyti- cal, the Netherlands) using Cu Kα radiation with a scanning rate of 2°/min at a range from 20° to 50°, and their morphology were examined using scanning electron microscopy (Hi- tachi S-4700, Japan).

Numerical data were analyzed using Student’s t test. Statisti- cal significance was considered to be P<0.05.

requirements for biomedical applications. In any case, graphene reinforced HA composites have been recognized as a promising material in this field [12-15].

In our previous work [12,13], we reported on the fracture toughness of graphene reinforced HA composite synthesized using spark plasma sintering, which exhibited an ~80%

improvement compared to pure HA, even when the added graphene was only 1.0% weight.

For biomaterials used in orthopedic applications, cell attachment is an initial and critical requirement, since it is essential for other cell functions, such as proliferation and mineral formation. Very recently, various investigations on the biocompatibility of graphene have demonstrated its positive effects on cell adhesion, proliferation and differentiation, while certain forms and derivatives of graphene have induced significant cell toxicity, which is considered beneficial for anticancer activity [16-19]. Kalbacova et al. [16] found human osteoblasts and mesenchymal stromal cells had improved adherence on large single layer graphene as compared with those cells on a SiO2 substrate, with the initial presence/absence of fetal bovine serum. Aryaei et al. [17] also showed that graphene is beneficial for osteoblast attachment and proliferation.

Despite the abovementioned advances in the biocompatibility of graphene, there are still concerns regarding cell functions on the surface of graphene, and especially the effect of graphene substrates on cell behavior.

Given the present scenario, the aim of this investigation was to explore the biocompatibility of a graphene film (GF) through in vitro experiments, including osteoblast adhesion, osteoblast proliferation and bone-like apatite deposited in a simulated body fluid (SBF) solution. For this study, graphite paper (GP) with the same composition as graphene was selected as the reference material.

2. Experimental

Chemical vapor deposition (CVD) grown GF (thickness, 20–

50 μm; diameter, 3 mm) from Nanjing Emperor Nano Material (China) were employed, and GP (thickness, ~1 mm; diameter, 3 mm) was selected as a reference material. Atomic force microscopy (AFM; MultiView 1000, Nanonics Imaging Ltd.) was used to measure the surface morphology and roughness of the GF and GP.

Human osteoblast-like cell lines (MG-63) were collect- ed from the Orthopaedic Institute of Soochow University, China. All aqueous solutions were prepared with deionized water. MG-63 human osteoblast cells were cultured in Dul- becco’s Modified Eagle’s Medium (Thermo Scientific, USA) supplemented with 10% fetal bovine serum (Invitrogen, USA) at 37±1°C in a humidified 5% CO2 atmosphere. When the cultured cells reached confluence, they were detached using 0.25% trypsin-ethylenediaminetetraacetic acid (trypsin- EDTA) solution (Invitrogen) and were used for the designed experiments described below, where the carbonaceous materials were placed into a 96-well plate and seeded with a density of 2×10⁴ cells/mL.

The adhesive strength of the MG-63 cells to a carbonaceous material was tested based on the method proposed by

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roughness and curled nanostructure of the MWCNTs, given the same chemical composition of MWCNTs and SWCNTs [18].

Cell adhesion on the surface of a solid material is mainly me- diated by the focal adhesion protein and anchor points of the cytoskeleton. Therefore, F-actin and vinculin expressions on both carbon materials were visualized. It is clear from Fig. 3a and c that the number of osteoblasts adhered to the GF is much higher than that on the GP. Moreover, cultured osteoblasts with spindle- like morphology are spread in a flat fashion on the GF (Fig. 3b), whereas the osteoblast cultured on the surface of the GP seems to exhibit a smaller cell body and limited spreading (Fig. 3d).

This indicates that the cells attached to the graphene exhibit spreading. Furthermore, it is clear that the F-actin (orange-red) is much denser and more widely spread throughout the cell bodies on the GF as compared to the GP cultured osteoblasts. Most importantly, vinculin (green) is distributed throughout the cell bodies cultured on the GF, while it is only located in a limited area within a cell on the GP, as clearly depicted in Fig. 3b and d.

From the results described above, the stronger adhesion of osteoblasts to the GF might be ascribed to the substrate structure, considering that GF and GP share the same composition. The importance of the presence of nano and submicron roughness comes from the widely accepted hypothesis that nanostructures can enhance the absorption of adhesion proteins [20], which are thought to mediate the adhesion of anchorage-dependent cells such as osteoblasts. As such, the nano and submicron textured surface of the GF offers a greater number of opportunities for these proteins to interlock with nano/submicron asperities. This is evident given the formation of cytoskeleton with F-actin, and the focal adhesion with vinculin throughout the cell body on the GF (Fig. 3).

Also, it has been demonstrated that substrate stiffness significantly affects the cell attachment and proliferation, and that stiffer substrates are more cell-friendly [21,22]. It is well known that the elastic modulus of graphene (~1 TPa) is much higher than that of graphite, and cells can sense the substrate down to several micrometers [17]. Therefore it is expected that a GF with a thickness of ~1 mm would be easily sensed by the cultured osteoblasts.

3. Results and Discussion

As shown in Fig. 1 in the AFM images of the GF and GP (capture area 50 μm×50 μm), the roughness factor of the GF was determined to be about root mean square=731±133 nm, while for the GP root mean square=336±23 nm. It is interest- ing to note that a number of wrinkles are visible on the surface of the GF (Fig. 1a), which contributed to the greater level of nano-roughness of the GF, as compared with the comparatively smooth surface of the GP (Fig. 1b).

Fig. 2 shows the ratio of adherent cells after 5–30 min treatment with 0.1% trypsin-EDTA. It is clear that the ratio of the adherent cells on both samples decreases with increasing treatment time. It should be noted that the ratio of adherent cells on the GF is higher than that on GP at each time point; in particular,

~15% of the cells cultured on the GF remain after 5 min of treatment. The results imply that the GF exhibits relatively stronger adhesive strength with osteoblasts.

Similarly, Matsuoka et al. [18] reported that the cell adhesive strength of multi-walled carbon nanotubes (MWCNTs) was higher than that of single-walled carbon nanotubes (SWCNTs), and it has been speculated that the increase is the result of the

Fig. 1. Atomic force microscopy images of as received graphene film (a) and graphite paper (b).

Fig. 2. Ratio of the adherent cell numbers after 5–30 min treatment with 0.1% trypsin-ethylenediaminetetraacetic acid treatment. Value are presented as mean±standard error (n=3). ^*P<0.05 between graphene film and graphite paper.

Fig. 3. Low- and high-magnification fluorescent images of F-actin (orange-red) and vinculin (green) of adherent osteoblasts on graphene film (a, b) and graphite paper (c, d).

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mention that osteoblasts are anchorage-dependent cells, i.e., they will die if they do not become well-adhered to the surface of a solid material.

As for the ALP activity, it is evident from Fig. 4b that the ALP activity of the cells on the GF is relatively higher than those cultured on the GP substrate at day 3 and day 7, although it was not remarkable on day 1 of the culture. Hence, the WST and ALP results show strong evidence that the surface of the GF substrate can provide favorable conditions for osteoblast adhesion, growth and proliferation.

Fig. 5 shows the X-ray diffraction spectra of the two substrates mineralized in SBF at 37°C. It can be seen that no apatite forms on the surfaces of the GF and GP after 1 d and 3 d immersion (Fig. 5a and b), and the peak of HA is present when the two substrates were immersed in SBF for 7 d. Nevertheless, it should be noted that the peak intensity of HA on the surface of the GF is much higher than that on the surface of the GP (Fig.

However, it should be noted that, so far, no evidence has con- firmed that cells can sense these substrates with an elastic modulus higher than few hundred kilopascals, and this needs further investigation. Moreover, Aryaei et al. [17] reported that graphene can sustain stress and allow for easy out-of-plane deformation, and subsequently contribute to the formation of strong anchor points of the cytoskeleton [23]. Therefore, the enhanced cell adhesion to graphene might be ascribed to the combination of several features: the rougher and wrinkled surface morphology, higher elastic modulus and easy out-of-plane deformation associated with the GF.

The viability of osteoblasts cultured on both substrates is depicted in Fig. 4a. Despite negligible changes in the viability of the cells cultured on both GF and GP, respectively, at day 1 and day 3, it should be pointed out that the viability of osteoblasts cultured on the GF increased by up to ~13.2% as compared with those cultured on the GP substrate at day 7. It is important to

Fig. 4. (a) Viability of MG-63 cells cultured on graphene film (GF) and graphite paper (GP) for 1 d, 3 d, and 7 d and (b) alkaline phosphatase activity of MG- 63 cells cultured on GF and GP for 1 d, 3 d, and 7 d. Value are presented as mean±standard error (n=3). ^*P<0.05 between GF and GP.

Fig. 5. X-ray diffraction results of (a) as-received graphene film (GF) and graphite paper (GP) samples and GF and GP soaked in simulated body fluid for (b) for 1 d and (c) for 7 d (c).

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the entire surface of the graphene, imparting good osteoconductivity to the graphene. These findings are expected to advance the potential application of graphene as a toughening nanofiller in bioceramics used in load-bearing implants.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Acknowledgements

Y.C. would like to acknowledge financial supports from the National Natural Science Foundation of China (51471113, 51275326) and Science and Technology Support Program of Ji- angsu Province, China (BE2013062).

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In this study, GF and GP were used as substrates to evaluate biocompatibility and biomineralization through a cell adhesion strength test, WST-1 assay, ALP activation and SBF immersion.

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