CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells

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CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells Han Xie a , Melissa Chua b , Intekhab Islam a , Ricardo Bentini c , Tong Cao a , José Carlos Viana-Gomes c , Antônio Hélio Castro Neto c , Vinicius Rosa a,c,∗ a b c

Faculty of Dentistry, National University of Singapore, Singapore Nanyang Polytechnic, Singapore Centre for Advanced 2D Materials and Graphene Research Centre, National University of Singapore, Singapore

a r t i c l e

i n f o

a b s t r a c t

Article history:

Objective. The objective was to investigate the potential of graphene (Gp) to induce odonto-

Received 5 April 2016

genic and osteogenic differentiation in dental pulp stem cells (DPSC).

Received in revised form

Methods. Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass

9 September 2016

(Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium

Accepted 9 September 2016

from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation

Available online xxx

of DPSC on all substrates were evaluated after 14 and 28 days by alizarin red S staining, qPCR, immunofluorescence and FACS. Statistics were performed with two-way ANOVA and

Keywords:

multiple comparisons were performed using Tukey’s post hoc test at a pre-set significance

Tissue engineering

level of 5%.

Mesenchymal stem cells

Results. After 14 and 28 days, Gp induced higher levels of mineralization as compared to

Differentiation

Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes

Bone

and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl

Osteoblast

(p < 0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and

Carbon

increased bone-related gene and protein on Gl.

Scaffold

Significance. Graphene induced osteogenic and not odontoblastic differentiation of DPSC

Osteogenesis

without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research. © 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Graphene is a one atom thick two-dimensional honeycomb structure made of pure carbon. It may be the thinnest,

strongest and stiffest imaginable material ever created [1–3]. It can be obtained via chemical vapor deposition (CVD). This is a scalable method for production of large scale and high quality graphene that can be transferred to various substrates [4]. Due to its cytocompatibility and large

∗ Corresponding author at: Faculty of Dentistry, Oral Sciences, National University of Singapore, 11 Lower Kent Ridge Road, Singapore, 119083 Tel.: +65 6779 5555 ext 1650; Fax: +65 6778 5742. E-mail address: [email protected] (V. Rosa). http://dx.doi.org/10.1016/j.dental.2016.09.030 0109-5641/© 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

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surface area (∼2600 m2 g−1 ) that allows functionalization, CVD-grown graphene has emerged as an interesting platform that supports and promotes neurogenic, cardiomyogenic and adipogenic differentiation of mesenchymal stem cells (MSC) [5–8]. CVD-grown graphene (Gp) can enhance osteogenic differentiation of MSC either as two-dimensional films or three-dimensional substrates (e.g. self-supporting graphene hydrogel films and foams) [8–10]. MSC cultured on glass coated with Gp presented higher differentiation as compared to those cultured on polyethylene terephthalate (PET) and polydimethylsiloxane (PDMS) substrates regardless the presence of BMP-2 [11]. Similar phenomenon was observed for Gp and graphene oxide substrates compared to PDMS, but the effects were only observed with the use of osteogenic medium [12]. Conversely, another study showed that Gp and graphene oxide-based substrate could enhance osteogenic differentiation of caprine bone marrow-derived mononuclear cells. Nonetheless, the differentiation was compromised in the presence of osteogenic medium [13]. These controversial findings can be attributed to: i) the direct and multiple comparison of the outcomes obtained using Gp to other substrates that differ physically and chemically from it (e.g. PDMS, graphene oxide, PET, Si/SiO2 , SiO2 ) and ii) the frequent use of chemical inducers for osteogenic differentiation (e.g. dexamethasone, ␤-glycerophosphate, BMP-2) that may mask the effects promoted by Gp alone [8,11–14]. These create a plethora of scenarios that limits the understanding of the effects arising exclusively from Gp on cell differentiation hindering its bioapplications at large. Although Gp can stimulate the secretion of mineralized matrix in some types of MSC, it is not known if the material can favor odontoblastic differentiation. Dental pulp stem cells (DPSC) are analogous to bone marrow stem cells on the expression profiles for more than 4000 genes and present similar expression for a several markers such as fibroblast growth factor 2, alkaline phosphatase, collagen type I, osteocalcin and others [15,16]. As DPSC can be obtained from extracted tooth, under local anesthesia and without esthetic damage, they emerge as an interesting model for tissue engineering research [15,17–19]. The objective of this study was to evaluate the potential of Gp to induce odontoblastic or osteogenic differentiation of DPSC without the use of any chemical inductors. The hypothesis is that Gp induces odontogenic differentiation of DPSC.

Table 1 – Experimental set-up. Name

Source (S)

Group 1 Group 2 Group 3 Group 4

Gl Gl Gp Gp

Materials and methods

2.1.

Substrate preparation and characterization

Graphene (Gp) was produced by CVD using a custom-built furnace in a Class 1000 clean room facility at NUS Centre for Advanced 2D Materials and Graphene Research Centre as previously described [10,20]. Briefly, Gp was coated on copper foils at 1000 ◦ C in a mixture of hydrogen and methane gas. After, the copper foil was etched in 1.5% ammonium persulfate for 8 h and the film transferred to deionized water for 24 h. The transfer was completed by gently contacting the Gp film with a glass coverslip (Schott D263 M Glass Coverslips, Ted Pella

Gl Gp Gl Gp

Inc., USA) followed by incubation in isopropanol for 3 h. All the samples were characterized by atomic force microscopy (Dimension Icon AFM equipped with a ScanAsyst, Bruker, Germany) and Raman spectroscopy (Raman Microscope CRM 200, Witec, Germany) at room temperature with an excitation laser source of 532 nm.

2.2.

DPSC culture and culture system

The use of human DPSC (DPF003 single donor, All-cells, USA) in this study was approved by the Institutional Biosafety Committee (2014-00762) and NUS Institutional Review Board (NUS 2094). The DPSC (passage 3 to 5) were characterized for CD34, CD73, CD90, CD105 (Millipore, USA) and the results are analogous to those recently published by our group [21]. For all the assays described here, the cells were cultured under basal growth media [Dulbecco’s modified Eagle’s medium (Invitrogen, USA), supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen)] without the use of medium or chemical inducers for osteogenic differentiation. To evaluate the effects of graphene on stem cell differentiation, we have design an experiment comprising four groups composed by two substrates made of either glass (Gl) or graphene (Gp). Each group had a Source (S) substrate where the cells were used to condition the basal culture media that was subsequently used to treat cells seeded on the substrate Destiny (D). The culture procedure is illustrated in Fig. 1: 7 × 103 DPSC were seeded on both substrates of each group with 2 mL of culture medium (i), after 1 day, the media from D was discarded (ii) and 1 mL of the medium from S was transferred to D (iii). Finally, 1 mL of fresh culture medium was added to both S and D (iv). The steps ii to iv were repeated every other day for 28 days. For all the tests described hereafter, three independent samples were prepared for each S and D substrate analyzed.

2.3. DPSC

2.

Destiny (D) → → → →

Odontogenic and osteogenic differentiation of

For all the experiments described here, we analyzed the outcomes from both S and D substrates of each group described in Table 1. Mineralization was quantitatively assessed via alizarin red S staining after 14 and 28 days. Cells were washed by phosphate buffered saline (Invitrogen) and fixed with 4% paraformaldehyde (room temperature, 20 min). After washing with deionized water, cells were stained with 40 mmol/L of alizarin red (Sigma–Aldrich, USA) in distilled water (pH of 4.2 maintained with ammonium hydroxide) and kept at 37 ◦ C for 30 min. Samples were treated with 10% cetylpyridinium chloride solution (Sigma–Aldrich) at room temperature for

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Fig. 1 – (i) On day 0 DPSC were seeded in both substrates S (orange) and D (blue) with 2 ml of culture medium; (ii) Day 1: all the medium from D was discarded and (iii) 1 ml of medium from S was transferred to D; (iv) 1 ml of fresh culture medium was added to both substrates. Steps ii to iv were repeated every other day for 28 days.

15 min and the absorbance was measured by microplate reader (Infinite M200 PRO, Tecan, Germany) at a wavelength of 540 nm. Three individual readings were obtained from each substrate analyzed. The mean of absorbance was normalized to genomic DNA content obtained with DNAzol (Invitrogen) and measured using a spectrophotometer (NanoDrop ND-1000 Spectrophotometer, ThermoScientific, USA). Gene expressions for odonto and osteogenic-related genes were obtained based on the Cq method for calculating relative gene expression from quantification cycle values obtained by quantitative real-time PCR analysis. The oligonucleotide primer sequences are shown in Table 2. After 14 and 28 days in the culture system, DPSC were harvested, total RNA was isolated (Purelink RNA Mini Kit, Invitrogen) and cDNA synthesis was performed (iScript RT Supermix, Bio-Rad, USA). Two housekeeping genes were used as controls (␤-actin and GAPDH). As there were no significant changes in their expression for any of the conditions tested (substrates and time points) the data were normalized against GAPDH. Three individual real-time

PCR reactions were performed for each of the substrates analyzed. Protein expression of RUNX2, collagen type I (COL I) and osteocalcin (OCN) were evaluated after 14 and 28 days. Briefly, samples were fixed with 4% paraformaldehyde for 20 min and incubated overnight with primary antibodies RUNX2 (1:200), COL (1:200) and OCN (1:200) (Abcam, United Kingdom) at 4 ◦ C. The secondary antibody labelled by FITC was used and incubated at 37 ◦ C for 60 min. The specimens were counterstained with DAPI (Invitrogen). Fluorescent staining was imaged using a confocal microscope (FV1000, Olympus Optical, Japan). The spontaneous osteogenic shift was confirmed by fluorescence-activated cell sorting analysis (FACS, BD LSRFortessa, BD Biosciences, Germany). Briefly, 3 × 105 DPSC were cultured exclusively on Gl or Gp with basal growth media for 28 days. After, cells were detached (TrypLE, Invitrogen), incubtated with unconjugated human monoclonal antibodies osteopontin (OPN) and OCN (1:200 Abcam) and reacted with FITC-conjugated goat anti-mouse Ig-G and PE-conjugated goat anti-rabbit secondary antidoby (1:2000 for both). Data

Table 2 – Oligonucleotide primer sequences utilized in the RT-PCR. Gene

Primer

Sequence

Collagen type I (COL I)

Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse

5 -CTGACCTTCCTGCGCCTGATGTCC-3 5 -GTCTGGGGCACCAACGTCCAAGGG-3 5 -CACTGGCGCTGCAACAAGA-3 5 -CATTCCGGAGCTCAGCAGAATAA-3 5 -ATGAGAGCCCTCAGACTCCTC-3 5 -CGGGCCGTAGAAGCGCCGATA-3 5 -ACACAAGACGAACCGTAAGCC-3 5 -CACATGGGCCGTGTAGAGTC-3 5 -GGAGGAGTGTTCGTGAACGG-3 5 -CGGCTGATGTCACACGGTC-3 5 -CTCCGAGTTGGACGATGAGG-3 5 -TCATGCCTGCACTGTTCATTC-3 5 -ATGAGAAGTATGACAACAGCC-3 5 -AGTCCTTCCACGATACCAA-3 5 -CAGGCTGTGCTATCCCTGTA-3 5 -CATACCCCTCGTAGATGGGC-3

Runt-related transcription factor 2 (RUNX2) Osteocalcin (OCN) Homo sapiens msh homeobox 1 (MSX1) Paired box 9 (PAX 9) Dentin matrix acidic phosphoprotein 1 (DMP-1) GAPDH ␤-actin

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Fig. 2 – Raman fingerprints (A) and AFM characterization (B).

were analyzed with FlowJo software (v. 10.1, FlowJo LLC, USA). Negative controls were substrates devoid of cells. Statistical analyses were performed with two-way ANOVA and multiple comparisons were performed using Tukey’s post hoc test at a pre-set significance level of 5% (SigmaStat 2.0, USA).

3.

Results and discussion

3.1.

Sample characterization

Results for sample characterization (Raman fingerprints and AFM) are shown in Fig. 2. The peaks at 1587 cm−1 (G band) and 2500–2800 cm−1 (2D band) show that monolayer Gp was successfully produced. The first band arises from the stretching of the C C bond whereas the 2D band is present graphitic materials. AFM shows that Gp covered effectively the Gl substrate and presented some wrinkles and ripples.

3.2.

Graphene induces mineralization in DPSC

First we checked whether Gp could drive DPSC to deposit mineralized matrix without the use of osteogenic medium or chemical inducers. The higher absorbances observed on Group 4 as compared to Group 1 for both 14 and 28 days

confirm that Gp can induce DPSC to spontaneously secrete mineralized matrix (Fig. 3). Notably, when we used the culture medium from Gp to treat cells on Gl (Group 3), the latter presented higher absorbance as compared to all substrates from Group 1 and 2 after 28 days. On the other hand, the mineralization on Gp was negatively affected by the medium obtained from Gl (Group 2).

3.3. Graphene prevents odontogenic and induces osteogenic differentiation of DPSC Graphene can induce mineralization in DPSC (Fig. 3). Nonetheless, dentin and bone are considerably made of a mineral compound (hydroxyapatite) that is associated with the matrix produced by odontoblasts and osteoblasts [16,17,22,23]. As DPSC are capable to differentiate into these both cells types, we used them to confirm whether graphene induces osteoblastic or odontogenic differentiation of DPSC. Firstly we observed that odontoblastic-related genes (MSX1, PAX-6 and DMP-1) were significantly downregulated on GpS as compared to GlS in all groups analyzed (Fig. 4). An exception for this trend was observed for MSX-1 on GpD (Group 2) after 28 days. This may be related to the longer exposure to medium from Gl that primarily does not compromise its expression. MSX-1 is essential for the development of teeth,

Fig. 3 – (A) Alizarin red S staining. The media obtained from DPSC on Gp increased the mineralization of cells on Gl (Group 3) after 28 days (* denotes statistical difference between the groups, p < 0.05). (B) Alizarin red S staining evinced the presence of calcium-rich deposits in the DPSCs cultured on Gp (28 days). Please cite this article in press as: Xie H, et al. CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells. Dent Mater (2016), http://dx.doi.org/10.1016/j.dental.2016.09.030

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Fig. 4 – Expression of odontoblastic-related genes in DPSC. Except for MSX-1 after 28 days, cells on Gp experienced downregulation of all genes in both time points evaluated (“↓”: lower than GlS; * denotes statistical difference between the groups, p < 0.05).

Fig. 5 – Expression of osteoblastic-related markers on DPSC using the co-culture system (“↑”: higher than GlS; * denotes statistical difference between the groups, p < 0.05).

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Fig. 6 – Protein expression. GpS presented higher expression of all proteins (green) as compared to GlS in both time points. After 28 days, the expression of all proteins were increased on GlD (Group 3) as compared to GlS (Group 1). The medium obtained from glass decreased the expression on GpD (Group 3) as compared to GpS (nuclei were stained with DAPI (blue); scale bar = 50 ␮m). craniofacial structures and/or limb structures in embryos [24]. This highly conserved gene is a target activator of PAX-9 and the deletion of them causes tooth agenesis in mice [25,26]. PAX-6 is expressed in ectoderm-derived tissues of tooth germs and oral epithelia adjacent to the tooth germs during mouse tooth development [27]. The low expression of these markers expressed during tooth formation may indicate the lack of commitment of DPSC towards dental-related lineages when cultured on Gp. The decrease in DMP-1 expression reiterates this point. DMP-1 is highly expressed during odontogenesis and in mature odontoblasts capable to produce dentin

in vivo and its deficiency leads to dentinogenesis imperfecta [16,28–30]. Gp upregulates both gene and protein expressions of RUNX2 and OCN (Figs. 5 and 6). RUNX2 is a transcription factor that is essential for osteogenic differentiation and skeletal morphogenesis [31–33]. It is detected in preosteoblasts and can upregulate the expression of OCN [32,33]. In vitro studies have shown that the overexpression of RUNX2 downregulates the expression of dentin sialophosphoprotein preproprotein by odontoblasts [34]. The 6-fold increase in OCN gene expression on GpS after 28 days can be an indicator of osteoblastic

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Fig. 7 – Osteogenic differentiation of DPSC cultured with basal growth media on Gl and Gp. DPSC presented higher protein expression of both markers for mature osteoblasts on Gp compared to cells cultured on Gl.

differentiation. The graphene-induced osteogenic differentiation was further confirmed in the protein level. The FACS analysis showed that the DPSC on Gp presented significantly higher levels of both OPN and OCN as compared to Gl (Fig. 7). These are well-recognized signature proteins for the presence and function of mature osteoblasts [35,36]. Fig. 6 shows that, although significantly higher, the expressions observed for RUNX2 and OCN on GpD (Group 2) are not as strong as those from GpS (Groups 3 and 4). That is because GlS does not induce osteogenic differentiation of DPSC (Fig. 7). The use of the medium from Gl only dilutes the amount of the cytokines and growth factors originally present on GpD, therefore compromising the differentiation. In addition, the expression of the osteo-related genes and proteins analyzed were higher on Gl when this was treated with medium obtained from Gp (Group 3) comparing to GlS (Group 1 and 2). This confirms the ability of graphene to induce osteogenic differentiation in cells seeded on an inert substrate. This can be an attractive characteristic for the healing of bony defects as graphene may not only improve osteoblastic differentiation locally but also influence its surroundings. As we have not used any chemical inducers to stimulate the differentiation, it is very likely that the effects aforesaid arise from the physical characteristics of Gp. It is known that stem cells present lineage commitment with extreme sensitivity to tissue-level elasticity [37]. Moreover, the increase in the substrate stiffness can enhance osteoblastic differentiation of bone marrow MSC by upregulating the genetic expression of RUNX2 and OCN through ␣2 -integrin-ROCK-FAK-ERK1/2 axis [38]. Stem cells from human exfoliated deciduous teeth (SHED) present higher potential for osteogenic differentiation as the rigidity of the substrate increases [39]. The calcium content in DPSC cultured in a rigid hydrogel (G = 3600 Pa) was at

least 3.5 times higher comparing to DPSC seeded in hydrogels with G ≤ 1570 Pa [40]. Conversely, the odontogenic differentiation of SHED and DPSC without the stimuli from bioactive substances is often achieved using soft substrates (e.g. collagen, poly(lactide-co-glycolic acid), and other peptides and hydrogels [28,40,41]). Hence, it can be possible that the high Young’s modulus of monolayer Gp (1.0–2.4 TPa [3,42,43]) contributes to trigger preferentially the osteogenic differentiation of DPSC. Apart from the elastic properties, the presence of wrinkles and ripples on the surface of the material might act as anchor points leading to cytoskeleton tension that could trigger pathways sensitive to mechanical stimulation [11]. In fact, Gp increases the genetic expression of BMP-2 [44] that is associated with osteogenic differentiation upon cytoskeleton tension [37,45]. Future studies shall focus on the understanding of the mechanotransduction cascades affecting osteogenic differentiation on graphene.

4.

Conclusion

The differentiation of stem cells into osteoblastic or odontogenic lineages are not independent processes: the events governing to one cell fate inhibit the mechanisms leading the differentiation to another lineage. Although DPSC are originated in the dental pulp, graphene decreased the expression of the odontogenic-related genes studied. Notably, the down-regulation of DMP-1 indicates that DPSC on graphene are not prone to differentiate into odontoblastic-like cells. Therefore the hypothesis was rejected. Thus, CVDgrown graphene films may not consist as a platform for endodontic and pulp regeneration research when aiming to promote odontoblastic differentiation of DPSC. Nonetheless,

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Gp induced the expression of osteoblastic-related genes and proteins and can be an interesting material to be used for bone tissue engineering and regeneration.

Acknowledgements This research is supported by the National Research Foundation, Prime Minister’ Office, Singapore, under its Medium Sized Centre Programme. V.R. was supported by the grant from the National University of Singapore, Singapore (R221-000-091-112) and National University Health System, Singapore (R-221-000-074-515). A.H.C.N. acknowledges NRFCRP award “Novel 2D materials with tailored properties: beyond graphene” (R-144-000-295-281). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

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Please cite this article in press as: Xie H, et al. CVD-grown monolayer graphene induces osteogenic but not odontoblastic differentiation of dental pulp stem cells. Dent Mater (2016), http://dx.doi.org/10.1016/j.dental.2016.09.030