Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells

CARBON

x x x ( 2 0 1 3 ) x x x –x x x

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/carbon

Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells Omid Akhavan a b

a,b,* ,

Elham Ghaderi a, Mahla Shahsavar

a

Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box 14588-89694, Tehran, Iran

A R T I C L E I N F O

A B S T R A C T

Article history:

Graphene nanogrids (fabricated by graphene nanoribbons obtained through oxidative

Received 7 January 2013

unzipping of multi-walled carbon nanotubes) were used as two-dimensional selective tem-

Accepted 6 March 2013

plates for accelerated differentiation of human mesenchymal stem cells (hMSCs), isolated

Available online xxxx

from umbilical cord blood, into osteogenic lineage. The biocompatible and hydrophilic graphene nanogrids showed high actin cytoskeleton proliferations coinciding with patterns of the nanogrids. The amounts of proliferations were found slightly better than proliferation on hydrophilic graphene oxide (GO) sheets, and significantly higher than non-uniform proliferations on hydrophobic reduced graphene oxide (rGO) sheets and polydimethylsiloxane substrate. In the presence of chemical inducers, the reduced graphene oxide nanoribbon (rGONR) grid showed a highly accelerated osteogenic differentiation of the hMSCs (a patterned differentiation) in short time of 7 days in which the amount of the osteogenesis was 2.2 folds greater than the differentiation (a uniform differentiation) on the rGO sheets. We found that although in the absence of any chemical inducers the graphene nanogrids showed slight patterned osteogenic differentiations, the graphene sheets could not present any differentiation. Therefore, the highly accelerated differentiation on the rGONR grid was assigned to both its excellent capability in adsorption of the chemical inducers and physical stresses induced by the surface topographic features of the nanogrids.  2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Graphene with fascinating physicochemical characteristics (such as the highest surface area (2600 m2/g) [1] and Young’s modulus (0.5–1 TPa) among all known materials [2], extraordinary electrical [3] and thermal [4] conductivities, capability of bio-functionalization and mass production [5]) has received increasing attentions, especially in various areas of biophysics and biotechnology [6]. For example, graphene has been successfully applied in ultra sensitive biosensing [5,7], disease diagnosis [8], efficient antibacterial [9–14] and antiviral

materials [15], cancer targeting [16–18] and photothermal therapy [19–21], drug delivery [22–24], and tissue engineering [25–28]. In tissue engineering and regenerative medicine, human mesenchymal stem cell (hMSC) is one of the best versatile primary cells with highly active as well as repetitive proliferations and capabilities of differentiation into various cell lineages including osteoblasts, adipocytes and chondrocytes [29]. hMSCs can be found in different tissues such as fat, muscle and especially bone marrow [30]. Furthermore, it has been recently demonstrated that umbilical cord blood (UCB) can be

* Corresponding author at: Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran. Fax: +98 21 66022711. E-mail address: [email protected] (O. Akhavan). 0008-6223/$ - see front matter  2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.carbon.2013.03.010

Please cite this article in press as: Akhavan O et al. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon (2013), http://dx.doi.org/10.1016/j.carbon.2013.03.010

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applied as a reliable alternative source for isolating young hMSCs [31]. It has been reported that, managing suitable growth factor inducers [30], mechanical properties of materials [32], and/or substrate topography [33], can result in effective differentiation of hMSCs into desired cell lineages. Although growth factor inducers can well guide differentiation of stem cells, they often need weeks and even months of cell incubation for obtaining maturated distinct lineages [34]. This means that growth factor inducers alone cannot induce efficient and fast differentiation, and so, we need to develop more efficient techniques for increasing differentiation of stem cells. In this regard, it was found that nanomaterials can significantly affect proliferation, differentiation and lineage specification of hMSCs [35,36]. Among the various nanomaterials, graphene (G) sheets grown by chemical vapor deposition (CVD) exhibited excellent properties in promoting adhesion and osteogenic differentiation of hMSCs [37]. Concerning this, it was shown that CVDgrown G sheets can be used as biocompatible platforms in accelerated osteogenic differentiation of hMSCs with a rate comparable to differentiation under the influence of, e.g., BMP-2 growth factor, over a period of 15 days of incubation [38]. Then, Lee et al. [39] demonstrated that CVD-grown G sheets can highly stimulate osteogenic differentiation of hMSCs in the presence of chemical growth factors which can highly adsorb on G (such as, dexamethasone and b-glycerol phosphate anion) after a shorter time of incubation (12 days), as compared to graphene oxide (GO) sheets (prepared by Hummers’ method) which exhibited a significant lower osteogenic differentiation (due to their low capability in enough adsorption of the growth factors). They also showed that although osteogenic differentiation on G can be comparable to the differentiation induced by the chemical inducers on polydimethylsiloxane (PDMS) substrate (the former was nearly half of the latter), but the amount of differentiation on G in the presence of the chemical inducers was much higher ( one order in magnitude) than the differentiation in the absence of the inducers. As a disadvantage, they [39] found that G sheets cannot adsorb enough ascorbic acid as one of the chemical inducers required in formation of mature osteoblasts [40]. Beside G and GO, other nanomaterials such as carbon nanotubes (CNTs) [41] and gold nanoparticles [42] could stimulate osteogenic differentiation through a stress mechanism (operative by interaction of nanomaterials with cell membrane and binding to proteins of cytoplasm) which can activate the p38 mitogen-activated protein kinase signaling pathway for adjusting the expression of genes containing osteogenic transcription. In fact, it is demonstrated when stem cells are under the influence of physical stresses induced by topographic features of their substrate, differentiation into specific cell lineages can be promoted [43–45]. For example, Dalby et al. [33] reported that hMSCs proliferated on randomized poly-(methylmethacrylate) (PMMA) nanopatterns showed hMSC osteoblastic morphologies after 21 days of incubation in a standard cell culture media and in the absence of chemical inducers. In addition to such synthetic nanomaterials, it was reported that bionanomaterials e.g., tobacco mosaic virus and turnip yellow mosaic, can also enhance stem cell differentiation [46].

The novel nanostructures of graphene such as graphene nanoribbons (elongated strips of graphene with a high length-to-width ratio and straight edges) [47], graphene nanomeshes [48,49], and graphene nanoplatelets [50] were recently used in cancer cell imaging and photothermal therapy [18,19], but there has been no report about application of such nanostructures in tissue engineering. In this work, at first, we reported fabrication of a graphene nanogrid using graphene nanoribbons synthesized through unzipping of CNTs. Then, graphene oxide nanoribbon (GONR) and reduced graphene oxide nanoribbon (rGONR) grids were used as two-dimensional (2D) nanopattern templates for accelerated proliferation and osteogenic differentiation of hMSCs both in the absence and in the presence of some chemical inducers. To have a benchmark, GO and rGO sheets (originally synthesized by Hummers’ method and contained no nanopattern) were also investigated under the same conditions. The effects of physical stresses induced by the surface topography of the nanogrids on the differentiation of the cells were studied. In addition, capability of the nanogrids in high adsorption of the chemical growth factors was investigated and compared to that of the graphene sheets and PDMS substrate.

2.

Experimental

2.1.

Synthesis of GONRs

Multi-wall carbon nanotube (MWCNT) powder (with purity of 95%, outer mean diameter of 10–30 nm and length of <5–15 lm [51,52], produced by io.li.tec) was applied as received. The other chemicals were purchased from Merck and used as received. At first, 100 mg MWCNTs were suspended in 100 mL conc. sulfuric acid (H2SO4) for 12 h and then 500 mg potassium permanganate (KMnO4) was added to the mixture. Caution: It is danger to exceed 0.5 wt.% KMnO4 into H2SO4, because at high concentrations (e.g., 7 wt.%) the mixture can explode under heating [53]. H2SO4 was used for further exfoliation of the nanotubes as well as the subsequently produced graphene sheets. The mixture was stirred for 60 min at room temperature and then heated in an oil bath at 55 C for 30 min, at 65 C for 60 min and finally at 70 C for 20 min. Then, the solution was allowed to be cooled and stabilized at room temperature, while the oil bath was removed. The reaction progress of the solution was monitored through its color change so that after completion of the reactions, the green color of permanganate in acid disappeared and color of the solution changed from black to brown. Then, the obtained solution added slowly to cool DI water including 1 vol.% hydrogen peroxide (H2O2). The solution was filtered by PTFE membrane (5.0 lm pore size) and washed several times by diluted H2SO4, ethanol and DI water. To have a benchmark, GO aqueous suspension was also synthesized using a modified Hummers’ method as its details reported elsewhere [54].

2.2.

Reduction of GONRs

To achieve a chemically reduced GONR suspension, the obtained GONR powder (0.1 mg/mL) was dispersed in DI water

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CARBON

containing 1 vol.% conc. ammonium hydroxide (NH4OH) and 1 vol.% hydrazine monohydrate (N2H4ÆH2O) within a flask and heated at 85 C for 60 min. Caution: Hydrazine is a highly dangerous reductant and must be applied with particular attentions [55]. Bovine serum albumin (BSA) can be used for stabilization and further reduction of rGO [56]. Here, we used 50 lL of 2 mg/ mL BSA along with another 30 min sonication for stabilization of the rGONRs. Then the reduced suspension was centrifuged at 20 g for 60 min to eliminate any aggregates and/or multilayered rGONRs. The supernatant was gathered after centrifugation and washed several times with DI water to obtain rGONR powders. The same procedure was applied to reduce the GO sheets ito rGO ones.

2.3.

Fabrication of graphene nanogrids

The graphene nanogrids were fabricated by a paintbrushing method using the desired GONR or rGONR suspension (1 mg/mL) onto a PDMS (Dow Corning) substrate which already kept in a freezer at temperature of 6 C. To deposit the nanoribbons on the substrate with the same alignments, at first, the suspension was spread over the substrate by repeating unidirectional (e.g., away from the painter) strokes of a soft paintbrush. The deposited substrate was dried at 105 C in air for 12 min, and softly washed by DI water in a direction the same as the direction of the brushing. Then, to obtain the structure of the grid, the same deposition method repeated, but in a direction vertical to the last one. Once again the sample was dried and washed in a direction the same as the direction of the last brushing. It should be noted that the GO and rGO sheets were deposited on the PDMS using a dropcasting method, as previously described elsewhere [11].

2.4.

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Material characterization

Morphology of the graphene nanoribbons was examined by using scanning electron microscopy (SEM, Philips XL30). Topography and height profile of the nanoribbons were investigated by using atomic force microscopy (AFM; Digital Instruments NanoScope V) in tapping mode. X-ray photoelectron spectroscopy (XPS) was applied to study the chemical states of the nanoribbons. The data were obtained through a hemispherical analyzer supplied by an Al Ka X-ray source operating at energy of 1486.6 eV and a vacuum better than 10–7 Pa. The XPS peaks were deconvolution by using Gaussian components after a Shirley background subtraction. The O/C atomic ratios of the samples were determined using peak area ratio of the XPS core levels and the sensitivity factor (SF) of each element in XPS. Raman spectroscopy was managed at room temperature using a HR-800 Jobin–Yvon with 532 nm NdYAG excitation source for investigation on the carbon structure of the graphene nanoribbons. The samples for material characterization were prepared by drop-casting the desired suspension onto a cleaned Si3N4/Si(1 0 0) substrate. The hydrophilicity and/or hydrophobicity of the samples were investigated using contact angle measurements at room temperature.

2.5.

Isolation and proliferation of hMSCs

The hMSCs were isolated from UCB of an infant with informed consent. The UCB mononuclear cells were obtained by negative immunodepletion of CD3+, CD14+, CD19+, CD38+, CD66b+, and glycophorin A+ cells using a commercial kit. The cells were centrifuged at 20 g for 10 min to discard the supernatants. Culture of the isolated cells (105 cells/cm2) on the samples were performed in a culture medium (Dulbecco’s modified Eagle medium containing 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mM L-glutamine, and 10 ng/mL basic fibroblast growth factor) at 37 C in a 5% CO2 atmosphere. The culture medium was refreshed every 2 or 3 days up to 1 week. Morphology of the hMSCs cells proliferated on the samples was evaluated through fluorescence staining the cells. 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 0 ,6-diamidino-2-phenylindole (DAPI) for staining the nucleus of the cells (blue color). Then, the stained cells were monitored using a confocal fluorescence microscope (Zeiss LSM 510 confocal). The surface density of the cells was determined through counting the number of blue-stained nucleus of the cells per surface area. All experiments were repeated at least three times.

2.6.

Osteogenic differentiation

Osteogenesis of hMSCs (2 · 103 cells/cm2) was induced by adding 104 mM dexamethasone, 10 mM b-glycerophosphate, and 0.1 mM ascorbic acid into the culture medium. The prepared culture medium was refreshed every 2 or 3 days up to 1 week. After that, the induced cells were stained with Alizarin Red and monitored by the optical microscope. To quantitatively investigate the osteogenic differentiation, at first surface of the samples washed by DI water. Then, optical absorbance of the samples containing the stained cells was examined by using a NanoDrop (ND-1000) UV–vis spectrophotometer at wavelength of 450 nm (as kmax) before and after washing. To obtain a normalized optical absorbance, the optical absorbance of each sample after washing was normalized by the absorbance achieved before washing.

2.7. Adsorption of nanoribbons

chemical inducers by

graphene

At first, PDMS, GO, rGO, GONR, rGONR, GONR-PDMS, or rGONR-PDMS suspension (1 mg/mL in phosphate buffer saline which sonicated for 60 min for obtaining a homogeneous suspension) was added into 10 mM dexamethasone, 1 M b-glycerophosphate, 0.1 M ascorbic acid. The two latter suspensions (with 20 wt.% PDMS) were prepared to have a model corresponding to the GONR or rGONR grids in which there are PDMS regions for surface interactions. The obtained mixtures were stirred up to 24 h at room temperature. Then, each mixture was centrifuged at 6000 rpm for 30 min. The supernatant of the centrifuged suspension was transformed into a quartz cell for UV spectrophotometry in wavelength range of 200–

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CARBON

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250 nm. The amount of adsorption for each chemical inducer was evaluated based on the change observed in the optical absorption of each suspension relative to the optical absorption of the initially applied inducer before adding the PDMS, GO, rGO, GONR, or rGONR.

3.

Results and discussion

Fig. 1a presents SEM image of the aligned GONRs deposited on a Si3N4/Si(1 1 1) substrate by using a paint-brushing method. It shows formation of some one-dimensional features with length of 10 lm and width of 50–200 nm. Fig. 1b and c shows SEM and AFM images of GONR grids formed by two successive paint-brushing in which the last brushing is vertical to the first one. Fig. 1d shows AFM image of the GONR grid in a closer window. Height profile diagrams of the AFM images (Fig. 1e and f) indicate that the thickness of the nanoribbons (1.0 nm) is corresponding to the typical thickness of monolayer graphene oxide (0.8 nm) [57,58]. The AFM height profile histogram of the GONR grids (Fig. 1g) indicate that the number of layers of the nanoribbons is <6. In fact, based on

the area under the curve of histogram, one-third of the nanoribbons were monolayer and/or bilayer. SEM and AFM images of the GO sheets with thicknesses of 1 nm and lateral sizes of 2 lm can be found elsewhere [11,59]. The chemical states of the graphene nanoribbons were investigated by using XPS. Fig. 2A shows deconvoluted C(1s) peaks of the GONR and the rGONR grids as compared to that of the starting material, i.e., the MWCNTs. The deconvoluted peak centered at 285.0 eV was assigned to the C–C and C@C bonds. The other peaks centered at the binding energies of 286.4, 287.2, 288.2 and 289.5 eV were assigned to the C–OH, C–O–C, C@O, and O@C–OH oxygen-containing functional groups, respectively (see, e.g., [60–62]). All the peaks (except the peaks relating to the CC bonds) dramatically decreased in the rGONR grid, indicating completely remove of the oxygen-containing functional groups by the reduction process. Although the oxygen-containing peaks showed significant reductions, they were still partially remained in the main peak, particularly the C@O one which can be attributed to the attachment of BSA (incorporated in the reduction process) onto the rGONRs [56]. Meantime, another peak component

Fig. 1 – SEM images of GONRs aligned on a Si3N4/Si(1 0 0) substrate by a paint-brushing method in (a) one direction and (b) two vertical directions (the GONR grid), and AFM images of the GONR grid in a (c) wide and (d) close window. (e) and (f) Exhibit height profile diagrams of the lines marked in the AFM images shown in (c) and (d), respectively. (g) Shows height profile histogram of the GONR grids. Please cite this article in press as: Akhavan O et al. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon (2013), http://dx.doi.org/10.1016/j.carbon.2013.03.010

BE

(A)

O = C OH C=O C O C C OH C N C C&C=C

(B) Intensity (arb. unit)

Intensity (arb. unit)

c)

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b)

2

D

G

D/G intensity ratio

CARBON

1

0 MWCNT

GONR

rGONR

2D c) b) a)

a) 282

284

286

288

290

292

1000

1500

Binding Energy (eV)

140

2000

2500

3000

-1

Raman shift (cm )

(C)

Contact angle Roughness

2

1.5 100 80

1

60 40

Roughness (nm)

Contact angle (degree)

120

0.5

20 0

0 PDMS

GO

rGO

GONR

rGONR

Fig. 2 – (A) XPS and (B) Raman spectra of (a) MWCNT powder, (b) GONR and (c) rGONR. The inset of (A) shows D/G intensity ratio. (C) Contact angle and surface roughness of PDMS and GO, rGO, GONR and rGONR on the PDMS substrate.

appeared at 286.1 eV which can be attributed to formation of C–N bond on surface of the hydrazine-reduced GONRs (see, for example, [62,54]). Based on the SF-modified peak area ratios of the C(1s) and O(1s) core levels, the O/C ratio was found to decrease from 63% to 32% upon reduction. The latter ratio is still much higher than the oxygen content of the MWCNTs (5%), substantially due to the BSA attachment and/or the edge carboxylic acid moieties. The XPS results of the GO and rGO sheets can be found elsewhere [63]. The carbon structure of the nanoribbons was studied by using Raman spectroscopy, as shown in Fig. 2B. It is seen that, the G band (1580 cm–1) was broadened and the D band (1350 cm–1) strongly increased after oxidation of the MWCNTs. By reduction of the GONRs, one may expect that the structure of the nanoribbons is improved through repairing the aromaticity and so decreasing the D/G ratio. In this work, it was found that the D/G ratio increased after reduction which is consistent with some previous studies on reduction of GONRs and GO by hydrazine [63,64]. The increase of the D/G ratio from 1.46 to 1.67 (see the inset of Fig. 2B) can be attributed to the effect of reduction on improving the aromaticity through enhancing the number of smaller aromatic domains (as a responsible for the D peak), but not necessarily improving the overall size (as a responsible for the G peak)

[64]. The almost overall edge effects of the nanoribbons can further promote the mechanism of enhancing the number of smaller aromatic domains. In addition, the increase of the defects on surface of the hydrazine-reduced nanoribbons can be partially attributed to formation of the C–N bonds (see the XPS analysis). Raman spectra of carbon materials also present a 2D band (described by the adopted double resonant model [65,66]) sensitive to stacking of graphene layers [67]. The 2D band of single-layer graphene locates at 2679 cm1, while for multilayer graphene (containing 2–4 layers) the 2D band broadens and shifts to higher wavenumbers by 19 cm1 [68]. Fig. 2B shows that the 2D band of the MWCNTs locates around 2695 cm1 corresponding to their multilayer structure, while 2D band of the nanoribbons (both the GONRs and rGONRs) is around 2680 cm1 indicating formation of single-layer nanoribbons from the multilayer starting material. Raman spectra of the GO and rGO sheets can be found elsewhere [63]. It is well established that hydrophobic substrates (such as PDMS) show low aqueous sample loading [69] and high resistance to adsorption of proteins [70]. Since serum proteins mediate cellular adhesion, there are some correlations between the hydrophilicity of a substrate and cell proliferation on the substrate. Concerning this, water contact angle (CA)

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CARBON

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and surface roughness (SR) of the samples was measured and compared to those of the PDMS substrate. Fig. 2C indicates hydrophobic nature of the PDMS substrate (with CA 119.2 ± 2.3 and SR  1.51 nm) as well as rGO sheets deposited on the PDMS substrate (with CA  93.2 ± 4.6 and SR  0.98 nm), while for the GO sheets deposited on the PDMS substrate we found a hydrophilic property with CA  32.6 ± 1.6 and SR  1.21 nm, due to the presence of the oxygen-containing functional groups on surface of the sheets. In fact, no strong correlation was found between surface roughness and the measured values of CA. Fig. 2C also shows that although the PDMS substrate possesses a hydrophobic nature, the GONR and rGONR grids present hydrophilic properties with CA  18.9 ± 1.0 and 48.1 ± 4.6, respectively. The hydrophilic property of the rGONR grids/PDMS, in contrast to the hydrophobic property of the rGO/PDMS, and the better hydrophilic property of the GONR grids/PDMS than the GO/ PDMS can be assigned to the edge effects of the nanoribbons including defects and significant presence of the oxygen-containing functional groups on the edges providing electrostatic interactions. The morphology of the grids resulted in only 20– 30% increase in SR of the corresponding sheets (i.e., once again, no significant contribution of SR in the CA). To investigate about the effects of GONR and rGONR grids on proliferation and morphology of the cells, the hMSCs were deposited on the nanogrids and also the PDMS substrate as a

PDMS

control sample. Fig. 3 shows fluorescent images of the hMSCs proliferated on GONR and rGONR grids as well as PDMS substrate after 1, 3, 5 and 7 days. The actin cytoskeleton fibers of the cells stained by rhodamin–phalloidin show a red color, while the nucleus of the cells stained by DAPI show a blue color. The fluorescent images show that morphology of the hMSCs proliferated on the nanogrids and PDMS substrate are completely different. At day 1, no significant difference in the morphology of the cultured cells was observed, unless the number of cells proliferated on the nanogrids (especially the GONR grids) was significantly higher than that proliferated on the PDMS (as shown in Fig. 4 based on the number of blue-stained nucleus of the cells). However, after 3 days, the morphology of the hMSCs on PDMS appeared nearly spherical without a filopodia extension, while the cells proliferated on the GONR and rGONR grids exhibited a significant cellular protrusion (see Fig. 3). The cells cultured on the both GONR and rGONR grids showed spindle-like morphology and patterned compatible with the pattern of the grids, while the cells cultured on the PDMS irregularly spread out over the surface and showed some large spindle-like morphology after 7 days. The density of the cells proliferated on the both types of graphene nanogrids were found to be significantly higher than that proliferated on the PDMS. This can be assigned to hydrophobic property of PDMS resulting in poor aqueous material loading and so high resistance to adsorption of pro-

GONR

rGONR

1 day

3 days

5 days

7 days

Fig. 3 – Fluorescent images of actin cytoskeleton of hMSCs stained with RhP and DAPI after 1, 3, 5, and 7 days proliferation on PDMS substrate and GONR and rGONR grids. Scale bars are 10 lm. Please cite this article in press as: Akhavan O et al. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon (2013), http://dx.doi.org/10.1016/j.carbon.2013.03.010

CARBON

Surface density of hMSCs (number/cm2)

1.E+10

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1 day 3 days 5 days 7 days

1.E+09

1.E+08

1.E+07

1.E+06

PDMS

GONR

rGONR

GO

rGO

Fig. 4 – Surface density of hMSCs on PDMS substrate (as control), GONR and rGONR grids, and GO and rGO sheets (for comparison) after 1, 3, 5, and 7 days proliferation.

teins of the culture medium, in contrast to the hydrophobic property of the GONR and also rGONR grids. Although the density of the cells on the GONR grid was slightly higher than that on the rGONR grid, the difference became less pronounced by increasing the proliferation time to 7 days. To have a better comparison, Fig. 4 also presents the amount of surface density of the hMSCs cultured on GO and rGO sheets. Although the hydrophilic GO sheets showed a high density proliferation of the cells with cytoskeleton fibers as large as the fibers observed on the PDMS, the both types of hydrophilic graphene nanogrids still presented a significantly higher density, as an advantage of the nanogrids in the cell proliferation. The better proliferation of the cells on the hydrophobic rGO sheets, as compared to the PDMS with the same hydrophobicity, can be assigned to partial interaction of the p-electron cloud of the rGO sheets with the inner core of the proteins of the culture medium [39]. To study the osteogenic differentiation, the hMSCs deposited on the samples were induced to differentiate into osteoblasts in a medium supplemented by dexamethasone, bglycerolphosphate and ascorbic acid. In fact, dexamethasone is a synthetic glucocorticoid usually applied in changing the expression levels of many proteins and enzymes contributed in bone differentiation [71,72]. In addition, dexamethasone along with b-glycerolphosphate can show synergistical effects through interaction with intracellular alkaline phosphatase enzymes for synthesizing new mineralized bone tissues. It was also reported that ascorbic acid can help to post-differentiation resulting in formation of mature osteoblasts [40]. Fig. 5A shows that although no significant osteogenic differentiation was found on surface of the PDMS control substrate after 1 week, the extent of the induced differentiation of the hMSCs cultured on the rGONR grids was highly significant and even greater than that cultured on the GONR grids after 7 days. In addition, the bone tissues were differentiated in a pattern coinciding with the nanogrids. Fig. 5B shows that in the absence of the induced agents, the osteogenic differentiation significantly decreased, but there was still considerable differentiation on the GONR and rGONR grids in which, once

7

again, the latter exhibited higher differentiation than the former. The spectrophotometric quantification shown in Fig. 5C confirmed the higher osteogenic differentiation of the hMSCs on the rGONR grid than the GONR one. In fact, the extent of the induced differentiation on the GONR and rGONR grids was 6.4 and 16.3 folds differentiation on the PDMS, although for the noninduced condition, these values were 3.4 and 2.7, respectively. The better cell differentiation on the GONR grids than the reduced one in the noninduced condition can be assigned to the better hydrophilicity of the former. To have a better comparison, we also cultured the hMSCs on GO and rGO sheets deposited on the PDMS substrate. It was found that, the extents of the induced osteogenic differentiation on the GO and rGO sheets were 3.5 and 7.4 folds differentiation on the PDMS, while for the noninduced condition they were 1.8 and 1.1 folds, respectively (as shown in Fig. 5C). The standard coverslips were also used as another control sample to reconfirm the cell differentiation on the graphene-based materials. These results confirmed the induction effect of the topographic features of the nanogrids on the differentiation of the hMSCs both in the presence and in the absence of chemical inducers. Concerning the effect of the topographic features on the differentiation, it was suggested that many ripples and wrinkles on the large scale (>100 nm) of CVD-grown G are responsible in protein adsorption, cell adhesion, proliferation, and differentiation [38]. In fact, observation of no cell differentiation on highly oriented pyrolytic graphite (HOPG) samples was assigned to absence of such localized out-of-plane deformations in HOPG [38]. In this work, the edge defects of graphene nanoribbons could provide suitable out-of-plane deformations (on 100 nm scales (see Fig. 1d)) for topographical induction of the osteogenic differentiation, coinciding with the patterns of the nanoribbons. Table 1 also shows a comparison between the results of this work and the comparable results of other reported data from literatures. Comparing the graphene-based materials listed in Table 1, the rGONR grid can also show a high amount of differentiation ratio (16.3), and so, it can be used as one of the most effective substrates in osteogenic differentiation. In addition, the rGONR could provide one of the fastest osteogenic differentiations of hMSCs reported up to now (i.e., 7 days). Meanwhile, the rGONR grid can provide a selective osteogenic differentiation resulting in a patterned differentiation coinciding with the pattern of the nanoribbons. Concerning this, very recently, patterning hMSCs differentiated into neuronal lineage by printing PDMS barriers on fluorinated graphene films was reported [73]. Since it was demonstrated that stiffness of substrates can influence the differentiation of stem cells [74], various stiffness of the GONR, rGONR, GO, and rGO may affect the cell differentiation (in addition to the topographic effects). However, it was recently reported that due to the thinness of graphene sheets, the substrate supporting graphene (here, PDMS substrate) controls the overall stiffness of the deposited graphene sheets. For example, elasticity of PDMS (1:10), G and GO on the PDMS was reported in the same order of magnitude (3– 7 MPa), and the elasticity of G on SiO2 was reported two orders of magnitude higher than its elasticity on PDMS [39]. Since the substrate used in this work (i.e., PDMS) was identical through-

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CARBON

PDMS

x x x ( 2 0 1 3 ) x x x –x x x

GONR

rGONR

(A)

(a)

(b)

(c)

(B)

(a)

(b)

(c)

(D) 0.3

0.6

Induced Noninduced

Normalized optical absorbance of normal cells stained by RhP @ 450 nm

Normalized optical absorbance of cells stained by Alizarin Red @ 450 nm

(C)

0.2

0.1

0

Noninduced Induced

0.5 0.4 0.3 0.2 0.1 0

PDMS

GONR

rGONR

GO

rGO

Coverslip

PDMS

GONR

rGONR

GO

rGO

Coverslip

Fig. 5 – Osteogenic differentiation of hMSCs monitored by Alizarin Red staining after 1 week incubation (A) with induction and (B) without induction on (a) PDMS substrate and (b) GONR and (c) rGONR grids. Scale bars are 10 lm. Normalized optical absorbance of C) the differentiated cells stained by Alizarin Red and (D) the normal cells stained by RhP, at wavelength of 450 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

out the experiments, probable differences in stiffness of the graphene sheets and nanoribbons would not be significant factors in the cell differentiation. To further study the effects of the chemical inducers on the osteogenic differentiation as compared to the normal proliferation, the actin cytoskeleton fibers of the normal cells of the samples incubated with and without the chemical inducers were stained by RhP after 1 week incubation. Fig. 5D shows the normalized optical absorbance of each sample for the induced and noninduced conditions. It was found that the chemical inducers applied for the cell differentiation not only could induce the osteogenic differentiation (Fig. 5C), but also could suppress proliferation of the normal cells (Fig. 5D). The data given in Fig. 5D are also consistent with the results shown in Figs. 3 and 4. Using ultraviolet (wavelength of 200–250 nm) spectrophotometry, the capacities of the various samples (PDMS, GO, rGO, GONR, rGONR. GONR-PDMS and rGONR-PDMS) for adsorption of the various osteogenic chemical inducers (dexa-

methasone, b-glycerophosphate and ascorbic acid) were evaluated, as shown in Fig. 6. It was found that the rGONR (as the best material for the osteogenic differentiation of the hMSCs in this work) could adsorb the highest amount of dexamethasone per weight of the rGONR independent of the exposure time (see Fig. 6a). For example, the rGONR adsorbed 905 mg dexamethasone per gram of the rGONR after 24 h exposure time, while the PDMS substrate could adsorb only 107 mg dexamethasone per gram of PDMS at the same conditions. Using Fig. 6, it can be concluded that the amount of adsorption of each sample after 24 h approached to a saturated amount for that sample. For adsorption of b-glycerolphosphate (Fig. 6b), although adsorption on the rGONR (7.6 g per gram of rGONR after 24 h) was found slightly lower than the adsorption on the rGO (8.7 g per gram of rGO) and comparable with the adsorption on the PDMS (6.4 g per gram of PDMS), the adsorption on the rGONR-PDMS (9.8 g per gram of rGONRPDMS) showed slightly higher than the adsorption on the other substrates. Fig. 6c shows that although adsorption of

Please cite this article in press as: Akhavan O et al. Graphene nanogrids for selective and fast osteogenic differentiation of human mesenchymal stem cells. Carbon (2013), http://dx.doi.org/10.1016/j.carbon.2013.03.010

CARBON

9

xxx (2013) xxx–xxx

Table 1 – Comparing the characteristcs of graphene-based nanomaterials in osteogenic differentiation of hMSCs. Sample

Refs. Period of Differentiation Patterned differentiation differentiation ratio1 (day)

Inducer

CVD-grown G/Glass

– BMP-2 CVD-grown-G/SiO2/Si – BMP-2 CVD-grown G/PET – BMP-2 CVD-grown G/PDMS – BMP-2

15

15.8 1.00 15.2 1.04 20.2 1.13 16.7 1.20

No

[38]

CVD-grown G/PDMS

– 12 Dexamethasone, b-glycerolphosphate, ascorbic acid – Dexamethasone, b-glycerolphosphate, ascorbic acid

1.08 7.86

No

[39]

No

[This work]

GO/PDMS

GO/PDMS

rGO/PDMS

GONR/PDMS

rGONR/PDMS

– Dexamethasone, ascorbic acid – Dexamethasone, ascorbic acid – Dexamethasone, ascorbic acid – Dexamethasone, ascorbic acid

7

0.46 2.55

b-glycerolphosphate,

1.81 3.56

b-glycerolphosphate,

1.10 7.47

b-glycerolphosphate,

3.46 6.45

b-glycerolphosphate,

2.67 16.3

Yes

1

Ratio of the amount of cell differentiation (obtained through the optical absorbance of the stained cells) on graphene layer to the amount of differentiation on the substrate.

ascorbic acid on the rGONR (173 mg per gram of rGONR after 24 h) was significantly higher than rGO (67 mg per gram of rGO), it was not comparable to the adsorption on the PDMS (554 mg per gram of PDMS) as the material showed the highest adsorption of ascorbic acid. Nevertheless, the GONR-PDMS and the rGONR-PDMS could present adsorptions comparable to the PDMS (519 and 417 mg per gram of corresponding substrate after 24 h). Based on the results shown in Fig. 5, the rGONR grid (especially under the effects of the chemical inducers) exhibited the best osteogenic differentiation of the hMSCs as compared to other materials studied in this work. Furthermore, based on the results presented in Fig. 6a, it was found that the rGONR possesses the ability of high adsorption of dexamethasone (as compared to the other materials) which can be assigned to the p–p interaction between the aromatic rings of dexamethasone and the rGONR basal plane. The slightly better capability of the rGONR than rGO in adsorption of dexamethasone can be attributed to possibility of formation a chemical bonding between dexamethasone and the hydroxyl bonds remained on the edge defects of the rGONR through formation of C–O–C bond (the mechanism by which dexamethasone can adsorb on the GO and the GONR in which the p–p interaction are not so provided). Indeed, we found nearly 2-fold increase of the C–O–C bond of the rGONR after 24 h exposure to dexamethasone by using XPS. These results demonstrate the high importance of the adsorption of dexamethasone by substrate in promotion of osteogenic differen-

tiation of hMSCs, as previously reported [75]. Since the difference in adsorption of dexamethasone on the rGONR and the rGO was not so much corresponding to the high difference observed in the cell differentiation on rGONR and rGO (see Fig. 5), the physical stress induced by the surface topographic features of the nanogrids should be considered as another important parameter in the high osteogenic differentiation on the rGONR grids, as previously reported for other nanopatterns (see, e.g. [33]). Fig. 6b presents that adsorption of b-glycerolphosphate on the rGO and the PDMS was comparable to the adsorption on the rGONR and even the rGONR-PDMS. This means that a high adsorption of b-glycerolphosphate alone cannot significantly promote the osteogenic differentiation of hMSCs and to do this, enough adsorbed dexamethasone along with bglycerolphosphate is required to interact with intracellular alkaline phosphatase enzyme. The high adsorption of b-glycerolphosphate on rGONR, rGO and even PDMS can be assigned to the electrostatic attraction between these materials and the phosphate ions of b-glycerolphosphate. On the other hand, the low adsorption of b-glycerolphosphate on the GO which possesses a high surface density of oxygencontaining functional groups can be attributed to the electrostatic repulsion between the oxygen and phosphate ions. The lowest adsorption of ascorbic acid on the rGO and then the rGONR (shown in Fig. 6c) can be assigned to existence of nearly no oxygen-containing functional groups on surface of these nanomaterials for participation in hydrogen

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10

CARBON

Adsorbed mass ratio

0.8

(B) 12

10 mM dexamethasone

1h 6h 12 h 24 h

1 M beta glycerophosphate 1h 6h 12 h 24 h

10 Adsorbed mass ratio

(A) 1

x x x ( 2 0 1 3 ) x x x –x x x

0.6

0.4

0.2

8

6

4

2

0

0 PDMS

GONR rGONR

GO

(C)

rGO

1

Adsorbed mass ratio

0.8

GONR- rGONRPDMS PDMS

PDMS

GONR rGONR

GO

rGO

GONR- rGONRPDMS PDMS

0.1 M ascorbic acid

1h 6h 12 h 24 h

0.6

0.4

0.2

0 PDMS

GONR rGONR

GO

rGO

GONR- rGONRPDMS PDMS

Fig. 6 – Adsorption of (A) dexamethasone, (B) b-glycerolphosphate anion and (c) ascorbic acid as chemical agents applied in osteogenic induction of hMSCs on PDMS, GO, rGO, GONR, rGONR, GONR-PDMS (20 wt.%) and rGONR-PDMS (20 wt.%).

bonding to the –OH moieties of the acid. On the other hand, since PDMS, GO, GONR, GONR-PDMS and rGONR-PDMS possess such functional groups, they could highly adsorb ascorbic acid. Now, as one of the reasons, the much better osteogenic differentiation of hMSCs on the rGONR grid than the rGO can be also assigned to higher adsorption of ascorbic acid on the former through hydrogen bonding to the oxygen groups originated from the available region of the PDMS substrate and/or the oxygen groups located on the edge defect of the nanoribbons.

4.

Conclusions

Graphene nanogrids (graphene nanoribbons deposited on PDMS in vertically crossed directions) were synthesized and applied as selective 2D templates in accelerated proliferation and differentiation of hMSCs. The biocompatibility of the hydrophilic rGONR grids was tested through monitoring the high actin cytoskeleton proliferation of the hMSCs. Although the size of the cytoskeleton fibers proliferated on the nanogrids was significantly smaller than those proliferated on the graphene sheets and PDMS, the amount of the patterned proliferation on the nanogrids was comparable to the un-patterned proliferation on the hydrophilic GO sheets, and significantly higher than the nonuniform proliferations on

the hydrophobic rGO sheets and PDMS substrate. In the presence of the chemical inducers, the rGONR grids showed the fastest osteogenic differentiation of the hMSCs (a patterned differentiation) reported up to now (corresponding to the short incubation time of 7 days). This time is nearly half of the time required for osteogenesis on rGO sheets. In addition, the amount of differentiation after 7 days was found 2.2 folds greater than the differentiation on the rGO sheets (a uniform differentiation). The excellent characteristic of the rGONR grid in the fast osteogenic differentiation was attributed to both capability of the rGONR grids in high adsorption of the chemical inducers (especially ascorbic acid which could not be adsorbed by rGO sheets) and the physical stress induced by the surface topographic features of the nanogrids. These results can promote further investigation on selective and/or patterned differentiation of stem cells on diverse morphologies of the graphene structures as biocompatible and implantable scaffolds even with 3D structures.

Acknowledgements O. Akhavan would like to thank the Research Council of Sharif University of Technology and also Iran Nanotechnology Initiative Council for financial support of the work.

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CARBON

xxx (2013) xxx–xxx

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