Abstract
Objective
The objective was to investigate the potential of graphene (Gp) to induce odontogenic and osteogenic differentiation in dental pulp stem cells (DPSC).
Methods
Gp was produced by chemical vapor deposition. DPSC were seeded on Gp or glass (Gl). Cells were maintained in culture medium for 28 days. Every two days, culture medium from Gp was used to treat cells on Gl and vice versa. Mineralization and differentiation 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 multiple comparisons were performed using Tukey’s post hoc test at a pre-set significance level of 5%.
Results
After 14 and 28 days, Gp induced higher levels of mineralization as compared to Gl. Odontoblastic genes (MSX-1, PAX and DMP) were down-regulated and osteogenic genes and proteins (RUNX2, COL and OCN) were significantly upregulated on Gp comparing to Gl ( p < 0.05 for all cases). Medium from Gp induced downregulation of odontoblastic genes and increased bone-related gene and protein on Gl.
Significance
Graphene induced osteogenic and not odontoblastic differentiation of DPSC without the use of chemical inducers for osteogenesis. Graphene has the potential to be used as a substrate for craniofacial bone tissue engineering research.
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 . 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 . Due to its cytocompatibility and large surface area (∼2600 m 2 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) .
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) . 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 . 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 . 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 . 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/SiO 2 , SiO 2 ) and ii) the frequent use of chemical inducers for osteogenic differentiation ( e.g. dexamethasone, β-glycerophosphate, BMP-2) that may mask the effects promoted by Gp alone . 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 . 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 .
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.
2
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 . 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 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 . 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 × 10 3 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
Odontogenic and osteogenic differentiation of DPSC
For all the experiments described here, we analyzed the outcomes from both S and D substrates of each group described in Table 1 .
| Name | Source (S) | Destiny (D) | |
|---|---|---|---|
| Group 1 | Gl | → | Gl |
| Group 2 | Gl | → | Gp |
| Group 3 | Gp | → | Gl |
| Group 4 | Gp | → | Gp |
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 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 ΔΔCq 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 (β-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.
| Gene | Primer | Sequence |
|---|---|---|
| Collagen type I (COL I) | Forward Reverse |
5′-CTGACCTTCCTGCGCCTGATGTCC-3′ 5′-GTCTGGGGCACCAACGTCCAAGGG-3′ |
| Runt-related transcription factor 2 (RUNX2) | Forward Reverse |
5′-CACTGGCGCTGCAACAAGA-3′ 5′-CATTCCGGAGCTCAGCAGAATAA-3′ |
| Osteocalcin (OCN) | Forward Reverse |
5′-ATGAGAGCCCTCAGACTCCTC-3′ 5′-CGGGCCGTAGAAGCGCCGATA-3′ |
| Homo sapiens msh homeobox 1 (MSX1) | Forward Reverse |
5′-ACACAAGACGAACCGTAAGCC-3′ 5′-CACATGGGCCGTGTAGAGTC-3′ |
| Paired box 9 (PAX 9) | Forward Reverse |
5′-GGAGGAGTGTTCGTGAACGG-3′ 5′-CGGCTGATGTCACACGGTC-3′ |
| Dentin matrix acidic phosphoprotein 1 (DMP-1) | Forward Reverse |
5′-CTCCGAGTTGGACGATGAGG-3′ 5′-TCATGCCTGCACTGTTCATTC-3′ |
| GAPDH | Forward Reverse |
5′-ATGAGAAGTATGACAACAGCC-3′ 5′-AGTCCTTCCACGATACCAA-3′ |
| β-actin | Forward Reverse |
5′-CAGGCTGTGCTATCCCTGTA-3′ 5′-CATACCCCTCGTAGATGGGC-3′ |
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 × 10 5 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 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).
2
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 . 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 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 . 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 × 10 3 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.
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