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Flexible bactericidal graphene oxide–chitosan layers for stem cell proliferation
Applied Surface Science 301 (2014) 456–462
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Flexible bactericidal graphene oxide–chitosan layers for stem cell proliferation M. Mazaheri a , O. Akhavan b,c,∗ , A. Simchi a,c a b c
Department of Materials Science and Engineering, Sharif University of Technology, PO Box 11365-9466, Tehran, Iran Department of Physics, Sharif University of Technology, PO Box 11155-9161, Tehran, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, PO Box 14588-89694, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 16 February 2014 Accepted 16 February 2014 Available online 24 February 2014 Keywords: Graphene Chitosan Antibacterial nanocomposites Stem cells
a b s t r a c t Graphene oxide (GO)–chitosan composite layers with stacked layer structures were synthesized using chemically exfoliated GO sheets (with lateral dimensions of ∼1 m and thickness of ∼1 nm), and applied as antibacterial and flexible nanostructured templates for stem cell proliferation. By increasing the GO content from zero to 6 wt%, the strength and elastic modulus of the layers increased ∼80% and 45%, respectively. Similar to the chitosan layer, the GO–chitosan composite layers showed significant antibacterial activity (>77% inactivation after only 3 h) against Staphylococcus aureus bacteria. Surface density of the actin cytoskeleton fibers of human mesenchymal stem cells (hMSCs) cultured on the chitosan and GO(1.5 wt%)–chitosan composite layers was found nearly the same, while it significantly decreased by increasing the GO content to 3 and 6 wt%. Our results indicated that although a high concentration of GO in the chitosan layer (here, 6 wt%) could decelerate the proliferation of the hMSCs on the flexible layer, a low concentration of GO (i.e., 1.5 wt%) not only resulted in biocompatibility but also kept the mechanical flexibility of the self-sterilized layers for high proliferation of hMSCs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Human mesenchymal stem cells (hMSCs) are thought to be multipotent cells, which can replicate as undifferentiated cells having the potential to differentiate to lineages of mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma [1]. hMSCs are critical for numerous groundbreaking therapies in the field of regenerative medicine. Combination of stem cells with biomaterial scaffolds provides a promising strategy for engineering tissues and cellular delivery [2–4]. Some natural biomaterials such as polysaccharides play important role in maintaining the structure of the extracellular matrix and have been investigated for use as a potential scaffold material for stem cell transplantation [3]. Chitosan (poly (1,4-b-d-glucopyranosamine)) is a N-deacetylated cationic polysaccharide that has attracted much attention for various biomedical applications, mainly because of its excellent antimicrobial [5], non-toxicity, high biocompatibility and bioadsorbility properties [6,7]. Positive surface charges of
∗ Corresponding author at: Sharif University of Technology, Department of Physics, Azadi, Tehran 11155-9161, Iran. Tel.: +98 21 66164566; fax: +98 21 66022711.. E-mail address: [email protected] (O. Akhavan). http://dx.doi.org/10.1016/j.apsusc.2014.02.099 0169-4332/© 2014 Elsevier B.V. All rights reserved.
chitosan and its biocompatibility possess an ability to support the cell growth effectively [8]. Nevertheless, chitosan has some drawbacks and its mechanical properties are not good enough for some biomedical applications. To improve the mechanical properties of chitosan and other biopolymers, various inorganic fillers have been utilized [9–11]. Meanwhile, carbon nanostructures such as carbon nanotubes [11,12], graphene [13], graphene oxide (GO) [14,15], nanodiamonds [16], and fullerene [15] have been the focus of recent studies due to their promising properties. Particularly, GO is very attractive due to its extraordinary mechanical properties (high Young’s modulus and hardness, and excellent flexibility) together with lower cost compared to other carbon nanostructures [17]. GO consists of graphene sheets which are chemically functionalized with hydroxyl and epoxy groups [17,18]. Carbonyl groups are also present as carboxylic acids along the sheet edges. The existence of oxygen-containing groups makes GO hydrophilic and dispersive into some polar solvents forming intercalated composites with polar molecules through the strong interaction. Epoxide, carboxyl, and hydroxyl groups present on the basal plane and edges of GO enable greater interactions with proteins through covalent, electrostatic, and hydrogen bonding [19,20]. On the other hand, GO have been proved to exhibit better biocompatibility than reduced graphene oxide [20,21]. Thin GO sheets can potentially serve as a
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biocompatible, transferable, and implantable platform that have the potential to mediate stem cell lineage specification for tissue regeneration [19]. Recent studies have also shown that GO can be effective preconcentration platforms for accelerated stem cell growth and differentiation through molecular interactions [19,22,23]. Thus, GO is a promising nanofiller for improving the properties of chitosan without hampering its biocompatibility. Fabrication of graphene–chitosan composite layers has been the focus of recent studies. Hu et al. [24] used microwave irradiation to prepare chitosan modified graphene sheets. The results indicated that chitosan is covalently grafted onto the surface of graphene sheets by amido bonds. Ganesh et al. [25] and Yang et al. [26] showed that the graphene or graphene oxide sheets prefer to disperse well within the chitosan matrix, improving its mechanical strength. He et al. [27] fabricated porous graphene oxide–chitosan materials with a high adsorbing ability to metal ions having enhanced compressive strength. On the other hand, Bush et al. [28] demonstrated that ultra-low graphene loading in chitosan-based composite causes a dramatic increase in the wettability of chitosan. Fan et al. [29] showed that the graphene–chitosan composites are biocompatible to L929 cells. Albeit the potential applications and advantages of GO–reinforced chitosan layers for biomedicine, little work has been performed on their biocompatibility and interactions with stem cells. So, the present work aimed at preparing chitosan layers contained GO to get more insight into the effect of GO content in chitosan matrix on the stem cell proliferation, antibacterial and mechanical properties.
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temperature for an additional 2 h to obtain a homogeneous solution. The GO–chitosan layers with various GO contents (0, 1.5, 3 and 6 wt%) were fabricated by a solution-casting method. The composite layers were dried at 50 ◦ C overnight to completely remove acetic acid. 2.4. Material characterization Topography of the GO sheets and the GO–chitosan composites was characterized by atomic force microscopy (AutoProbe CPResearch, Veeco, Plainview, NY, USA). For atomic force microscopy (AFM), the samples were prepared by drop-casting a diluted GO suspension on a freshly cleaved mica substrate. Raman Spectroscopy (Senterra, Bruker, Leipzig, Germany) of the GO sheets was carried out at room temperature using a 528 nm laser excitation source. The mechanical properties of the GO–chitosan layers were investigated using a universal tensile test instrument (STM20, Santam, Tehran, Iran). Specimens for the tensile test were cut into strips of 10.0 mm wide and 60 mm long from the layers with thickness of 0.3 mm. The tensile tests were performed in the air using a 50 N load cell at a crosshead speed of 2 mm/min. Five tests were performed for each composition and the average of the results were reported as the mechanical properties with standard deviation. Scanning electron microscopy (SEM, Mira, TESCAN, Cranberry TWP, USA) operating at 2.0 kV was used to study the surface morphology and fractured cross-section of the layers. 2.5. Antibacterial test
2. Experimental 2.1. Reagents Chitosan with a medium molecular weight of 90–150 kDa and a degree of deacetylation above 80% was prepared from SigmaAldrich (Munich, Germany). The graphite used in this study was obtained from Qingdao Haida Graphite Co. (Qingdao, China) and other reagents and chemicals were purchased from Merck Co. (Darmstadt, Germany). 2.2. Synthesis of graphene oxide GO was synthesized using the modified Hummers’ method [30]. Briefly, 0.5 g graphite was vigorously stirred for 10 min in 50 mL concentrated H2 SO4 in an ice-water bath. Then, 0.5 g NaNO3 and 3 g KMnO4 were slowly added into the solution and stirred for 2 h in the ice-water bath. After removing the bath, 100 mL deionized (DI) water was added into the flask during ∼1 h while the solution was stirred and its temperature was kept at 98 ◦ C. The resultant mixture was further stirred for 2 h at 98 ◦ C. The temperature was reduced to 60 ◦ C and then 3 ml H2 O2 (30 wt% aqueous solution) was added. The mixture was cooled to room temperature, diluted with DI water and left overnight. The obtained GO was filtered (grade No.40 filter paper, Whatman, Kent, UK) and washed with HCl (10 vol%) solution and then DI water to remove the residual acid. GO sheets were obtained by ultrasonication of the filtered product in DI water at power of 600 W for 1 h. The obtained dispersion was centrifuged 2 cycles at 5000 rpm for 20 min to remove unexfoliated GO. 2.3. Preparation of GO–chitosan composites A chitosan aqueous solution was prepared by dissolving 1 g chitosan powder in 1.0 v/v% acetic acid solution. Then, GO suspension (0.1 mg/mL) was slowly added into the chitosan aqueous solution during a vigorous stirring. The obtained solution was agitated in an ultrasonic bath for approximately 10 min and stirred at room
Antibacterial performance of the chitosan and GO–chitosan layers was investigated against Staphylococcus aureus (S. aureus, BBRC 100501 ) as a Gram-positive bacterium. Before antibacterial assay, all samples were sterilized by 70% ethanol spray and dried under a sterile hood. The bacteria were cultured on a nutrient broth at 37 ◦ C for 24 h. Then, a portion of the bacterial suspension was diluted to 107 CFU/mL. For antibacterial drop-testing, each sample was placed into a sterilized Petri dish. Then, 100 L of the bacterial broth culture was seeded onto the samples at 37 ◦ C. After 3 h, the bacteria were washed out in 9.9 mL saline solution. After sonication for 30 s, each bacterial suspension was spread on a nutrient agar plate and incubated at 37 ◦ C for 24 h to count the surviving bacterial colonies by using an optical microscope. The total number of the colonies was determined by area based estimation. The reported data were the average value of three separate tests ± standard deviations. 2.6. Cell viability assay The hMSCs were isolated from umbilical cord blood (UCB) of an infant with informed consent. The UCB single-nuclear cells were obtained by negative immunodepletion using a commercial kit (RosetteSep, StemCell Technologies, USA). The cells were centrifuged at 8000 rpm for 10 min to discard the supernatants. Cells were cultured in an environment under 5% CO2 at 37 ◦ C and fed with Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, 10 ng/mL basic fibroblast growth factor and 2 mM l-glutamine. The viability of the cells exposed to the layers (∼1000 cells/cm2 ) was evaluated by using a fluorescence staining cell method after 7 days of incubation. In this method, the cells were fixed in 5% paraformaldehyde, stained with rhodamin–phalloidin (RhP) for staining the actin cytoskeleton fibers of the cells (red color) and 4 ,6-diamidino-2-phenylindole
1 Biochemical and Bioenvironmental Research Center Culture Collection (a local culture collection).
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Fig. 1. (a) AFM image and (b) height profile of exfoliated GO sheets on a mica substrate, (c) Raman spectrum of the GO sheets, and (d) a digital image of the flexible GO–chitosan layer.
(DAPI) for staining the nucleus of the cells (blue color). Then, morphology of the cytoskeleton fibers were monitored using a confocal fluorescence microscope (LSM 510 confocal, Zeiss, Jena, Germany) through a blue light excitation filter. The surface density of the cells was evaluated through counting the number of blue-stained nucleus of the cells. All experiments were repeated at least three times.
3. Results and discussion
from strong hydrogen bonding between oxygen functional groups on surface of the GO sheets, e.g., epoxy and hydroxyl groups, and chitosan amino groups [26,34]. Moreover, the presence of GO can increase chain entanglement and lead to higher tensile strength. The elongation at break decreased from 18% in chitosan layer to 13% in the GO(6 wt%)–chitosan composite layer. In fact, GO as a reinforcement material can limit the elongation of the polymer chains and constrain the deformation of the polymeric matrix. Top-view and cross-sectional SEM images of the chitosan and GO(6 wt%)–chitosan layers are shown in Fig. 3. It was found that,
The topography of the synthesized GO sheets was studied by AFM, as shown in Fig. 1a and b. The thickness of the sheets is smaller than 1 nm consistent with the typical thickness of single-layer GO (∼0.8 nm) [31,32]. The lateral dimensions of the GO sheets vary from several hundred nanometers to several micrometers indicating the high aspect ratio of the GO sheets. The Raman spectrum of the GO sheets (Fig. 1c) shows both D and G bands, as expected. The relative intensities of the two lines depend on the type of graphitic material. The 1584 cm−1 band (the G band) is assigned to the E2g phonons of the in-plane sp2 structure of carbon materials and the band located at 1319 cm−1 (the D band) is attributed to the breathing mode of –point phonons of A1g symmetry originated from structural defects induced by hydroxyl and/or epoxide bonds on the GO sheets [33]. The results of uniaxial tensile testing of the GO–chitosan layers are shown in Fig. 2. The elastic modulus (E), fracture strength () and elongation at break (εb ) of the layers are summarized in Table 1. As seen, the strength (E and ) of the chitosan layer is distinctly lower than that of the GO–containing chitosan layers. Addition of small amounts of the GO sheets (e.g., 6 wt%) resulted in significant change in the mechanical properties (∼80% and 45% increase in the and E values). Such changes can be originated
Fig. 2. Engineering tensile stress–strain curves for chitosan and GO–chitosan composite layers with different GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (a and b) Top-view and (c and d) cross-sectional SEM images of (a and c) chitosan and (b and d) GO(6 wt%)–chitosan layers.
Table 1 Mechanical properties of chitosan and GO–chitosan layers. Specimen
(MPa)
E (GPa)
εb (%)
Chitosan GO(1.5 wt%)–chitosan GO(3 wt%)–chitosan GO(6 wt%)–chitosan
51 ± 8 74 ± 5 80 ± 8 92 ± 6
2.4 ± 0.2 2.8 ± 0.2 3.2 ± 0.1 3.5 ± 0.2
18 ± 4 14 ± 2.3 13.5 ± 1.5 13 ± 2
the surface of the chitosan layers was very smooth while the surface of the composite layers wrinkled (see Fig. 3a and b). Differences in the morphology of the fracture surfaces are also noticeable (Fig. 3c and d). The composite layer clearly exhibits a layered structure (with thicknesses ∼200–500 nm) which can be originated from the morphology of the stacked GO sheets, while in the chitosan layer distinguishing the layered structure is not so clear. To quantitatively investigate the surface topography of the GO–chitosan, AFM was used. Fig. 4 shows typical surface morphology of the layers. The surface roughness parameters including average roughness (Ra ) and root mean square of roughness (Rq ) derived from AFM studies are reported in Table 2. Results show Table 2 Roughness parameters of the layers. Specimen
Ra (nm)
Rq (nm)
Chitosan GO(1.5 wt%)–chitosan GO(3 wt%)–chitosan GO(6 wt%)–chitosan
1.6 ± 0.1 2.4 ± 0.2 12.9 ± 1.1 17.5 ± 2.9
2.1 ± 0.1 3.0 ± 0.3 16.3 ± 1.5 21.3 ± 2.8
the effect of GO addition on the surface topography of the chitosan layer. The Ra of chitosan layer is considerably lower than those of the composite ones. For example, Ra significantly increased from 1.6 ± 0.1 nm to 17.5 ± 2.9 nm as the GO content increased from zero to 6 wt%. This can be attributed to the variation in the structure of the chitosan layer by adding the GO sheets, as also observed in Fig. 3. Surface roughness plays an important role in biomedical applications as the rougher surface of the GO–chitosan composite should influence cellular adhesion, differentiation, and proliferation. Increasing the surface roughness, increase the surface area of implants compared to larger smooth surfaces. The increased surface area increases cell attachment and augments the biomechanical interlocking between bone tissue and the implant [35,36]. Fig. 5 exhibits the viability of S. aureus bacteria after 3 h contact with the layers. All specimens showed reducion of the number of bacteria over 77%. Chitosan significantly inhibit the growth of the bacteria (as also shown in Fig. 5), although their inhibitory effects difference with molecular weight and the kind of bacteria [21]. A possible mechanism can be considered based on disruption of cell membrane that leads to leakage of cytoplasmic contents as a result of interaction between positively charged chitosan and negatively charged proteins as well as phospholipids in microbial cell membrane [37]. Another possible reason can be assigned to permeation of chitosan oligosaccharides into the cell nucleus, interfering with the RNA transcription mechanism and thus with the synthesis of proteins [38]. Consistently, the GO–chitosan layers showed significant antibacterial activities. The antibacterial activity of the layers increased by increasing the graphene content, consistent with the antibacterial property of the graphene materials reported previously [39,40]. The bacterial inhibition on surface of the layers can
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Fig. 4. AFM images of GO–chitosan layers with (a) 0, (b) 1.5, (c) 3, and (d) 6 wt% GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
be attributed to both membrane and oxidative stress [41] and/or the direct contact of the sharp edges of the GO sheets (vertically aligned ones) with bacterial cell membrane. In fact, S. aureus as a Gram-positive bacterium is more sensitive to the direct contact interaction of the graphene sheets than Gram-negative bacteria, as previously reported [42]. Hence, increasing the surface roughness can enhance the antibacterial activity [43]. To investigate in vitro biocompatibility of the layers, hMSCs were selected because they can be differentiated into other cells like
Fig. 5. Antibacterial activity of the chitosan and GO–chitosan layers against of S. aureus bacteria.
osteoblast, chondrocytes, adipocytes, etc. Thus, the stem cells play increasingly prominent roles in tissue repair and regeneration, and the interactions between stem cells and biomaterials are becoming one of the most important issues [44]. Fig. 6 shows fluorescent images of the actin cytoskeleton fibers of hMSCs stained with RhP (red color). It is seen that the presence of graphene oxide did not influence the shape of the cells. Although the surface density of the cytoskeleton fibers on the chitosan and GO(1.5 wt%)–chitosan layers are nearly the same, the surface density significantly decreased by increasing the GO content to 3 and 6 wt%. On the other hand, Fig. 7 shows that the numbers of hMSC nuclei (obtained through counting the cell nuclei stained by DAPI) are nearly unchanged by increasing the GO content to 3 wt%. Only a slight decrease in the number of cell nuclei was observed on the surface of GO (6 wt%)–chitosan layer. These results suggested that a high concentration of GO in the chitosan layer (here, 6 wt%) could slightly inhibit the proliferation of hMSCs and cause low cytotoxicity to hMSCs. There are also consistent with the findings of Kim et al. [34] that showed proliferation rate of hMSCs decreased with incorporation of higher amounts of graphene into the chitosan substrates [45]. Chen et al. [35] also reported a moderate toxicity of GO to organisms (∼20% cell growth inhibition induced by the sheets) [46]. Several factors such as concentration, size and shape can influence the cytotoxicity of graphene [47]. The generation of reactive oxygen species (ROS) is also known as a common toxicity mechanism of carbon materials [48]. Meantime, there is a correlation between cell death and ROS generation in or near cells and the concentration of carbon materials [49]. On the other hand, the extent of GO aggregation
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Fig. 6. Fluorescent images of actin cytoskeleton fibers of hMSCs cultured on GO–chitosan layers with (a) 0, (b) 1.5, (c) 3 and (d) 6 wt% GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. The percent of hMSC nuclei (obtained through counting the cell nuclei stained by DAPI) on GO–chitosan layers with various GO contents.
could have a great impact on the biological/toxicological responses [47,50]. Therefore, it is expected that ROS generation and probability of restacking of GO sheets would increase at high concentrations. Consequently, enhanced cytotoxicity of GO(6 wt%)–chitosan layers compared to chitosan was observed. Meanwhile, a low concentration of GO(i.e., 1.5 wt%) not only resulted in a biocompatible surface showing a high proliferation of hMSCs (similar to the chitosan), but also yielded a better mechanical properties (i.e., E and ) than the chitosan layer (see Fig. 2). 4. Conclusions In this study, layers of chitosan reinforced with GO sheets were prepared by solution casting methods to use as substrates for stem cell proliferation. The main findings are summarized below. • The addition of GO sheets into chitosan affected the surface topography of the prepared layers. The surface roughness parameter was increased by a factor of >15 when 6 wt% GO sheets were added. SEM studies also revealed that the surface of the composite layers is wrinkled due to staking configuration of GO sheets in the polymer matrix.
• Mechanical characterization by tensile testing indicated that GO sheets significantly improve the mechanical strength (Young’s modulus and tensile strength) of chitosan, possibly due to the strong hydrogen bonds between the GO and the polymer amine groups. • Antibacterial assay of the layers showed remarkable inhibition of the bacterial growth against S. aureus bacteria. The GO sheets improved the bactericidal capacity of chitosan. • Fluorescent images of actin cytoskeleton fibers of hMSCs cultured on layers revealed a high cell density on the surface of chitosan and GO (1.5 wt%)–chitosan layers. However, the cells cultured on the composite layers with higher GO contents showed lower proliferation. • Since GO sheets improved the mechanical strength and stem cell proliferation of chitosan layers, the composite materials could be considered as a promising platforms for hMSC culture. The most suitable concentration of GO sheets to promote hMSCs proliferation for tissue engineering applications was found 1.5 wt%. Acknowledgments The authors would like to thank the Research Council of Sharif University of Technology for supporting the work. References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, et al., Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] T.R. Nayak, H. Andersen, V.S. Makam, C. Khaw, S. Bae, X. Xu, et al., Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells, ACS Nano 5 (2011) 4670–4678. [3] S.M. Willerth, E.S.E. Sakiyama, Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery, in: StemBook, 2008 ed., Harvard Stem Cell Institut., Cambridge, MA, 2008. [4] O. Akhavan, E. Ghaderi, M. Shahsavar, Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells, Carbon 59 (2013) 200–211. [5] Y. Wu, S. Yu, F. Mi, C. Wu, S. Shyu, Preparation and characterization on mechanical and antibacterial properties of chitsoan/cellulose blends, Carbohydrate Polymers 57 (2004) 435–440. [6] E.D.S. Costa-ju, M.M. Pereira, H.S. Mansur, Properties and biocompatibility of chitosan films modified by blending with PVA and chemically crosslinked, Journal of Materials Science Materials in Medicine 20 (2009) 553–561. [7] G.A.F. Roberts, Chitin Chemistry, Palgrave Macmillan, London, UK, 1992.
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Contents lists available at ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Flexible bactericidal graphene oxide–chitosan layers for stem cell proliferation M. Mazaheri a , O. Akhavan b,c,∗ , A. Simchi a,c a b c
Department of Materials Science and Engineering, Sharif University of Technology, PO Box 11365-9466, Tehran, Iran Department of Physics, Sharif University of Technology, PO Box 11155-9161, Tehran, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, PO Box 14588-89694, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 20 January 2014 Received in revised form 16 February 2014 Accepted 16 February 2014 Available online 24 February 2014 Keywords: Graphene Chitosan Antibacterial nanocomposites Stem cells
a b s t r a c t Graphene oxide (GO)–chitosan composite layers with stacked layer structures were synthesized using chemically exfoliated GO sheets (with lateral dimensions of ∼1 m and thickness of ∼1 nm), and applied as antibacterial and flexible nanostructured templates for stem cell proliferation. By increasing the GO content from zero to 6 wt%, the strength and elastic modulus of the layers increased ∼80% and 45%, respectively. Similar to the chitosan layer, the GO–chitosan composite layers showed significant antibacterial activity (>77% inactivation after only 3 h) against Staphylococcus aureus bacteria. Surface density of the actin cytoskeleton fibers of human mesenchymal stem cells (hMSCs) cultured on the chitosan and GO(1.5 wt%)–chitosan composite layers was found nearly the same, while it significantly decreased by increasing the GO content to 3 and 6 wt%. Our results indicated that although a high concentration of GO in the chitosan layer (here, 6 wt%) could decelerate the proliferation of the hMSCs on the flexible layer, a low concentration of GO (i.e., 1.5 wt%) not only resulted in biocompatibility but also kept the mechanical flexibility of the self-sterilized layers for high proliferation of hMSCs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Human mesenchymal stem cells (hMSCs) are thought to be multipotent cells, which can replicate as undifferentiated cells having the potential to differentiate to lineages of mesenchymal tissues including bone, cartilage, fat, tendon, muscle, and marrow stroma [1]. hMSCs are critical for numerous groundbreaking therapies in the field of regenerative medicine. Combination of stem cells with biomaterial scaffolds provides a promising strategy for engineering tissues and cellular delivery [2–4]. Some natural biomaterials such as polysaccharides play important role in maintaining the structure of the extracellular matrix and have been investigated for use as a potential scaffold material for stem cell transplantation [3]. Chitosan (poly (1,4-b-d-glucopyranosamine)) is a N-deacetylated cationic polysaccharide that has attracted much attention for various biomedical applications, mainly because of its excellent antimicrobial [5], non-toxicity, high biocompatibility and bioadsorbility properties [6,7]. Positive surface charges of
∗ Corresponding author at: Sharif University of Technology, Department of Physics, Azadi, Tehran 11155-9161, Iran. Tel.: +98 21 66164566; fax: +98 21 66022711.. E-mail address: [email protected] (O. Akhavan). http://dx.doi.org/10.1016/j.apsusc.2014.02.099 0169-4332/© 2014 Elsevier B.V. All rights reserved.
chitosan and its biocompatibility possess an ability to support the cell growth effectively [8]. Nevertheless, chitosan has some drawbacks and its mechanical properties are not good enough for some biomedical applications. To improve the mechanical properties of chitosan and other biopolymers, various inorganic fillers have been utilized [9–11]. Meanwhile, carbon nanostructures such as carbon nanotubes [11,12], graphene [13], graphene oxide (GO) [14,15], nanodiamonds [16], and fullerene [15] have been the focus of recent studies due to their promising properties. Particularly, GO is very attractive due to its extraordinary mechanical properties (high Young’s modulus and hardness, and excellent flexibility) together with lower cost compared to other carbon nanostructures [17]. GO consists of graphene sheets which are chemically functionalized with hydroxyl and epoxy groups [17,18]. Carbonyl groups are also present as carboxylic acids along the sheet edges. The existence of oxygen-containing groups makes GO hydrophilic and dispersive into some polar solvents forming intercalated composites with polar molecules through the strong interaction. Epoxide, carboxyl, and hydroxyl groups present on the basal plane and edges of GO enable greater interactions with proteins through covalent, electrostatic, and hydrogen bonding [19,20]. On the other hand, GO have been proved to exhibit better biocompatibility than reduced graphene oxide [20,21]. Thin GO sheets can potentially serve as a
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biocompatible, transferable, and implantable platform that have the potential to mediate stem cell lineage specification for tissue regeneration [19]. Recent studies have also shown that GO can be effective preconcentration platforms for accelerated stem cell growth and differentiation through molecular interactions [19,22,23]. Thus, GO is a promising nanofiller for improving the properties of chitosan without hampering its biocompatibility. Fabrication of graphene–chitosan composite layers has been the focus of recent studies. Hu et al. [24] used microwave irradiation to prepare chitosan modified graphene sheets. The results indicated that chitosan is covalently grafted onto the surface of graphene sheets by amido bonds. Ganesh et al. [25] and Yang et al. [26] showed that the graphene or graphene oxide sheets prefer to disperse well within the chitosan matrix, improving its mechanical strength. He et al. [27] fabricated porous graphene oxide–chitosan materials with a high adsorbing ability to metal ions having enhanced compressive strength. On the other hand, Bush et al. [28] demonstrated that ultra-low graphene loading in chitosan-based composite causes a dramatic increase in the wettability of chitosan. Fan et al. [29] showed that the graphene–chitosan composites are biocompatible to L929 cells. Albeit the potential applications and advantages of GO–reinforced chitosan layers for biomedicine, little work has been performed on their biocompatibility and interactions with stem cells. So, the present work aimed at preparing chitosan layers contained GO to get more insight into the effect of GO content in chitosan matrix on the stem cell proliferation, antibacterial and mechanical properties.
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temperature for an additional 2 h to obtain a homogeneous solution. The GO–chitosan layers with various GO contents (0, 1.5, 3 and 6 wt%) were fabricated by a solution-casting method. The composite layers were dried at 50 ◦ C overnight to completely remove acetic acid. 2.4. Material characterization Topography of the GO sheets and the GO–chitosan composites was characterized by atomic force microscopy (AutoProbe CPResearch, Veeco, Plainview, NY, USA). For atomic force microscopy (AFM), the samples were prepared by drop-casting a diluted GO suspension on a freshly cleaved mica substrate. Raman Spectroscopy (Senterra, Bruker, Leipzig, Germany) of the GO sheets was carried out at room temperature using a 528 nm laser excitation source. The mechanical properties of the GO–chitosan layers were investigated using a universal tensile test instrument (STM20, Santam, Tehran, Iran). Specimens for the tensile test were cut into strips of 10.0 mm wide and 60 mm long from the layers with thickness of 0.3 mm. The tensile tests were performed in the air using a 50 N load cell at a crosshead speed of 2 mm/min. Five tests were performed for each composition and the average of the results were reported as the mechanical properties with standard deviation. Scanning electron microscopy (SEM, Mira, TESCAN, Cranberry TWP, USA) operating at 2.0 kV was used to study the surface morphology and fractured cross-section of the layers. 2.5. Antibacterial test
2. Experimental 2.1. Reagents Chitosan with a medium molecular weight of 90–150 kDa and a degree of deacetylation above 80% was prepared from SigmaAldrich (Munich, Germany). The graphite used in this study was obtained from Qingdao Haida Graphite Co. (Qingdao, China) and other reagents and chemicals were purchased from Merck Co. (Darmstadt, Germany). 2.2. Synthesis of graphene oxide GO was synthesized using the modified Hummers’ method [30]. Briefly, 0.5 g graphite was vigorously stirred for 10 min in 50 mL concentrated H2 SO4 in an ice-water bath. Then, 0.5 g NaNO3 and 3 g KMnO4 were slowly added into the solution and stirred for 2 h in the ice-water bath. After removing the bath, 100 mL deionized (DI) water was added into the flask during ∼1 h while the solution was stirred and its temperature was kept at 98 ◦ C. The resultant mixture was further stirred for 2 h at 98 ◦ C. The temperature was reduced to 60 ◦ C and then 3 ml H2 O2 (30 wt% aqueous solution) was added. The mixture was cooled to room temperature, diluted with DI water and left overnight. The obtained GO was filtered (grade No.40 filter paper, Whatman, Kent, UK) and washed with HCl (10 vol%) solution and then DI water to remove the residual acid. GO sheets were obtained by ultrasonication of the filtered product in DI water at power of 600 W for 1 h. The obtained dispersion was centrifuged 2 cycles at 5000 rpm for 20 min to remove unexfoliated GO. 2.3. Preparation of GO–chitosan composites A chitosan aqueous solution was prepared by dissolving 1 g chitosan powder in 1.0 v/v% acetic acid solution. Then, GO suspension (0.1 mg/mL) was slowly added into the chitosan aqueous solution during a vigorous stirring. The obtained solution was agitated in an ultrasonic bath for approximately 10 min and stirred at room
Antibacterial performance of the chitosan and GO–chitosan layers was investigated against Staphylococcus aureus (S. aureus, BBRC 100501 ) as a Gram-positive bacterium. Before antibacterial assay, all samples were sterilized by 70% ethanol spray and dried under a sterile hood. The bacteria were cultured on a nutrient broth at 37 ◦ C for 24 h. Then, a portion of the bacterial suspension was diluted to 107 CFU/mL. For antibacterial drop-testing, each sample was placed into a sterilized Petri dish. Then, 100 L of the bacterial broth culture was seeded onto the samples at 37 ◦ C. After 3 h, the bacteria were washed out in 9.9 mL saline solution. After sonication for 30 s, each bacterial suspension was spread on a nutrient agar plate and incubated at 37 ◦ C for 24 h to count the surviving bacterial colonies by using an optical microscope. The total number of the colonies was determined by area based estimation. The reported data were the average value of three separate tests ± standard deviations. 2.6. Cell viability assay The hMSCs were isolated from umbilical cord blood (UCB) of an infant with informed consent. The UCB single-nuclear cells were obtained by negative immunodepletion using a commercial kit (RosetteSep, StemCell Technologies, USA). The cells were centrifuged at 8000 rpm for 10 min to discard the supernatants. Cells were cultured in an environment under 5% CO2 at 37 ◦ C and fed with Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, 10 ng/mL basic fibroblast growth factor and 2 mM l-glutamine. The viability of the cells exposed to the layers (∼1000 cells/cm2 ) was evaluated by using a fluorescence staining cell method after 7 days of incubation. In this method, the cells were fixed in 5% paraformaldehyde, stained with rhodamin–phalloidin (RhP) for staining the actin cytoskeleton fibers of the cells (red color) and 4 ,6-diamidino-2-phenylindole
1 Biochemical and Bioenvironmental Research Center Culture Collection (a local culture collection).
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Fig. 1. (a) AFM image and (b) height profile of exfoliated GO sheets on a mica substrate, (c) Raman spectrum of the GO sheets, and (d) a digital image of the flexible GO–chitosan layer.
(DAPI) for staining the nucleus of the cells (blue color). Then, morphology of the cytoskeleton fibers were monitored using a confocal fluorescence microscope (LSM 510 confocal, Zeiss, Jena, Germany) through a blue light excitation filter. The surface density of the cells was evaluated through counting the number of blue-stained nucleus of the cells. All experiments were repeated at least three times.
3. Results and discussion
from strong hydrogen bonding between oxygen functional groups on surface of the GO sheets, e.g., epoxy and hydroxyl groups, and chitosan amino groups [26,34]. Moreover, the presence of GO can increase chain entanglement and lead to higher tensile strength. The elongation at break decreased from 18% in chitosan layer to 13% in the GO(6 wt%)–chitosan composite layer. In fact, GO as a reinforcement material can limit the elongation of the polymer chains and constrain the deformation of the polymeric matrix. Top-view and cross-sectional SEM images of the chitosan and GO(6 wt%)–chitosan layers are shown in Fig. 3. It was found that,
The topography of the synthesized GO sheets was studied by AFM, as shown in Fig. 1a and b. The thickness of the sheets is smaller than 1 nm consistent with the typical thickness of single-layer GO (∼0.8 nm) [31,32]. The lateral dimensions of the GO sheets vary from several hundred nanometers to several micrometers indicating the high aspect ratio of the GO sheets. The Raman spectrum of the GO sheets (Fig. 1c) shows both D and G bands, as expected. The relative intensities of the two lines depend on the type of graphitic material. The 1584 cm−1 band (the G band) is assigned to the E2g phonons of the in-plane sp2 structure of carbon materials and the band located at 1319 cm−1 (the D band) is attributed to the breathing mode of –point phonons of A1g symmetry originated from structural defects induced by hydroxyl and/or epoxide bonds on the GO sheets [33]. The results of uniaxial tensile testing of the GO–chitosan layers are shown in Fig. 2. The elastic modulus (E), fracture strength () and elongation at break (εb ) of the layers are summarized in Table 1. As seen, the strength (E and ) of the chitosan layer is distinctly lower than that of the GO–containing chitosan layers. Addition of small amounts of the GO sheets (e.g., 6 wt%) resulted in significant change in the mechanical properties (∼80% and 45% increase in the and E values). Such changes can be originated
Fig. 2. Engineering tensile stress–strain curves for chitosan and GO–chitosan composite layers with different GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (a and b) Top-view and (c and d) cross-sectional SEM images of (a and c) chitosan and (b and d) GO(6 wt%)–chitosan layers.
Table 1 Mechanical properties of chitosan and GO–chitosan layers. Specimen
(MPa)
E (GPa)
εb (%)
Chitosan GO(1.5 wt%)–chitosan GO(3 wt%)–chitosan GO(6 wt%)–chitosan
51 ± 8 74 ± 5 80 ± 8 92 ± 6
2.4 ± 0.2 2.8 ± 0.2 3.2 ± 0.1 3.5 ± 0.2
18 ± 4 14 ± 2.3 13.5 ± 1.5 13 ± 2
the surface of the chitosan layers was very smooth while the surface of the composite layers wrinkled (see Fig. 3a and b). Differences in the morphology of the fracture surfaces are also noticeable (Fig. 3c and d). The composite layer clearly exhibits a layered structure (with thicknesses ∼200–500 nm) which can be originated from the morphology of the stacked GO sheets, while in the chitosan layer distinguishing the layered structure is not so clear. To quantitatively investigate the surface topography of the GO–chitosan, AFM was used. Fig. 4 shows typical surface morphology of the layers. The surface roughness parameters including average roughness (Ra ) and root mean square of roughness (Rq ) derived from AFM studies are reported in Table 2. Results show Table 2 Roughness parameters of the layers. Specimen
Ra (nm)
Rq (nm)
Chitosan GO(1.5 wt%)–chitosan GO(3 wt%)–chitosan GO(6 wt%)–chitosan
1.6 ± 0.1 2.4 ± 0.2 12.9 ± 1.1 17.5 ± 2.9
2.1 ± 0.1 3.0 ± 0.3 16.3 ± 1.5 21.3 ± 2.8
the effect of GO addition on the surface topography of the chitosan layer. The Ra of chitosan layer is considerably lower than those of the composite ones. For example, Ra significantly increased from 1.6 ± 0.1 nm to 17.5 ± 2.9 nm as the GO content increased from zero to 6 wt%. This can be attributed to the variation in the structure of the chitosan layer by adding the GO sheets, as also observed in Fig. 3. Surface roughness plays an important role in biomedical applications as the rougher surface of the GO–chitosan composite should influence cellular adhesion, differentiation, and proliferation. Increasing the surface roughness, increase the surface area of implants compared to larger smooth surfaces. The increased surface area increases cell attachment and augments the biomechanical interlocking between bone tissue and the implant [35,36]. Fig. 5 exhibits the viability of S. aureus bacteria after 3 h contact with the layers. All specimens showed reducion of the number of bacteria over 77%. Chitosan significantly inhibit the growth of the bacteria (as also shown in Fig. 5), although their inhibitory effects difference with molecular weight and the kind of bacteria [21]. A possible mechanism can be considered based on disruption of cell membrane that leads to leakage of cytoplasmic contents as a result of interaction between positively charged chitosan and negatively charged proteins as well as phospholipids in microbial cell membrane [37]. Another possible reason can be assigned to permeation of chitosan oligosaccharides into the cell nucleus, interfering with the RNA transcription mechanism and thus with the synthesis of proteins [38]. Consistently, the GO–chitosan layers showed significant antibacterial activities. The antibacterial activity of the layers increased by increasing the graphene content, consistent with the antibacterial property of the graphene materials reported previously [39,40]. The bacterial inhibition on surface of the layers can
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Fig. 4. AFM images of GO–chitosan layers with (a) 0, (b) 1.5, (c) 3, and (d) 6 wt% GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
be attributed to both membrane and oxidative stress [41] and/or the direct contact of the sharp edges of the GO sheets (vertically aligned ones) with bacterial cell membrane. In fact, S. aureus as a Gram-positive bacterium is more sensitive to the direct contact interaction of the graphene sheets than Gram-negative bacteria, as previously reported [42]. Hence, increasing the surface roughness can enhance the antibacterial activity [43]. To investigate in vitro biocompatibility of the layers, hMSCs were selected because they can be differentiated into other cells like
Fig. 5. Antibacterial activity of the chitosan and GO–chitosan layers against of S. aureus bacteria.
osteoblast, chondrocytes, adipocytes, etc. Thus, the stem cells play increasingly prominent roles in tissue repair and regeneration, and the interactions between stem cells and biomaterials are becoming one of the most important issues [44]. Fig. 6 shows fluorescent images of the actin cytoskeleton fibers of hMSCs stained with RhP (red color). It is seen that the presence of graphene oxide did not influence the shape of the cells. Although the surface density of the cytoskeleton fibers on the chitosan and GO(1.5 wt%)–chitosan layers are nearly the same, the surface density significantly decreased by increasing the GO content to 3 and 6 wt%. On the other hand, Fig. 7 shows that the numbers of hMSC nuclei (obtained through counting the cell nuclei stained by DAPI) are nearly unchanged by increasing the GO content to 3 wt%. Only a slight decrease in the number of cell nuclei was observed on the surface of GO (6 wt%)–chitosan layer. These results suggested that a high concentration of GO in the chitosan layer (here, 6 wt%) could slightly inhibit the proliferation of hMSCs and cause low cytotoxicity to hMSCs. There are also consistent with the findings of Kim et al. [34] that showed proliferation rate of hMSCs decreased with incorporation of higher amounts of graphene into the chitosan substrates [45]. Chen et al. [35] also reported a moderate toxicity of GO to organisms (∼20% cell growth inhibition induced by the sheets) [46]. Several factors such as concentration, size and shape can influence the cytotoxicity of graphene [47]. The generation of reactive oxygen species (ROS) is also known as a common toxicity mechanism of carbon materials [48]. Meantime, there is a correlation between cell death and ROS generation in or near cells and the concentration of carbon materials [49]. On the other hand, the extent of GO aggregation
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Fig. 6. Fluorescent images of actin cytoskeleton fibers of hMSCs cultured on GO–chitosan layers with (a) 0, (b) 1.5, (c) 3 and (d) 6 wt% GO contents. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 7. The percent of hMSC nuclei (obtained through counting the cell nuclei stained by DAPI) on GO–chitosan layers with various GO contents.
could have a great impact on the biological/toxicological responses [47,50]. Therefore, it is expected that ROS generation and probability of restacking of GO sheets would increase at high concentrations. Consequently, enhanced cytotoxicity of GO(6 wt%)–chitosan layers compared to chitosan was observed. Meanwhile, a low concentration of GO(i.e., 1.5 wt%) not only resulted in a biocompatible surface showing a high proliferation of hMSCs (similar to the chitosan), but also yielded a better mechanical properties (i.e., E and ) than the chitosan layer (see Fig. 2). 4. Conclusions In this study, layers of chitosan reinforced with GO sheets were prepared by solution casting methods to use as substrates for stem cell proliferation. The main findings are summarized below. • The addition of GO sheets into chitosan affected the surface topography of the prepared layers. The surface roughness parameter was increased by a factor of >15 when 6 wt% GO sheets were added. SEM studies also revealed that the surface of the composite layers is wrinkled due to staking configuration of GO sheets in the polymer matrix.
• Mechanical characterization by tensile testing indicated that GO sheets significantly improve the mechanical strength (Young’s modulus and tensile strength) of chitosan, possibly due to the strong hydrogen bonds between the GO and the polymer amine groups. • Antibacterial assay of the layers showed remarkable inhibition of the bacterial growth against S. aureus bacteria. The GO sheets improved the bactericidal capacity of chitosan. • Fluorescent images of actin cytoskeleton fibers of hMSCs cultured on layers revealed a high cell density on the surface of chitosan and GO (1.5 wt%)–chitosan layers. However, the cells cultured on the composite layers with higher GO contents showed lower proliferation. • Since GO sheets improved the mechanical strength and stem cell proliferation of chitosan layers, the composite materials could be considered as a promising platforms for hMSC culture. The most suitable concentration of GO sheets to promote hMSCs proliferation for tissue engineering applications was found 1.5 wt%. Acknowledgments The authors would like to thank the Research Council of Sharif University of Technology for supporting the work. References [1] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, et al., Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [2] T.R. Nayak, H. Andersen, V.S. Makam, C. Khaw, S. Bae, X. Xu, et al., Graphene for controlled and accelerated osteogenic differentiation of human mesenchymal stem cells, ACS Nano 5 (2011) 4670–4678. [3] S.M. Willerth, E.S.E. Sakiyama, Combining stem cells and biomaterial scaffolds for constructing tissues and cell delivery, in: StemBook, 2008 ed., Harvard Stem Cell Institut., Cambridge, MA, 2008. [4] O. Akhavan, E. Ghaderi, M. Shahsavar, Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells, Carbon 59 (2013) 200–211. [5] Y. Wu, S. Yu, F. Mi, C. Wu, S. Shyu, Preparation and characterization on mechanical and antibacterial properties of chitsoan/cellulose blends, Carbohydrate Polymers 57 (2004) 435–440. [6] E.D.S. Costa-ju, M.M. Pereira, H.S. Mansur, Properties and biocompatibility of chitosan films modified by blending with PVA and chemically crosslinked, Journal of Materials Science Materials in Medicine 20 (2009) 553–561. [7] G.A.F. Roberts, Chitin Chemistry, Palgrave Macmillan, London, UK, 1992.
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