Enhanced Stem Cell Osteogenic Differentiation by Bioactive Glass Functionalized Graphene Oxide Substrates

1Department of Geriatric Dentistry, Peking University School and Hospital of Stomatology, Beijing 100081, China 2Department of Prosthodontics, Peking University School and Hospital of Stomatology, Beijing 100081, China 3Department of Dental Materials, Peking University School and Hospital of Stomatology, Beijing 100081, China 4State Key Laboratory of Organic-Inorganic Composites, Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, Beijing University of Chemical Technology, Beijing 100029, China 5State Key Laboratory of New Ceramics and Fine Processing and Department of Materials Science and Engineering, Tsinghua University, Beijing 100084, China 6Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China 7National Engineering Laboratory for Digital and Material Technology of Stomatology, Beijing 100081, China 8Beijing Laboratory of Biomedical Materials, Peking University School and Hospital of Stomatology, Beijing 100081, China


Introduction
Graphene oxide (GO), a two-dimensional monolayer of carbon atoms intimately packed into a honeycomb lattice, has shown great promise for biomedical applications [1,2], such as cell imaging [3], drug delivery [4,5], and biomedical device [5,6].Furthermore, due to its unique nanostructures, remarkable mechanical properties, and large surface area [7][8][9], GO has gained tremendous attentions as a new nanoplatform for stem cell culture.Lee et al. indicated that GO substrates could promote adipogenic differentiation of MSCs because of their high affinity to insulin [10].Kim et al. demonstrated that GO substrates provide a suitable environment for ASCs adhesion, proliferation, and differentiation with presence of osteogenic chemical inducers [11,12].Recent work also demonstrated that GO can act as both an effective reinforcement and a bioactivator of biopolymers such as gelatin [13] and chitosan [14], offering the biopolymer-GO composites the possibility to manipulate bone cells behavior.However, in terms of stem cells osteogenesis, it remains a great challenge to functionalize GO as ideal substrate for the effective specification of stem cell fate to enhance bone repair.
Bioactive glasses (BG), with biomimetic composition and significant degradation [15], have been contemporarily confirmed to possess high efficiency in bonding with bone.In vivo experiments results showed that implanted BG produced no systemic or local toxicity [16,17], no inflammatory affection, and no repulsive response.Some in vitro studies suggested that the bioactivity of BG was associated with the formation of a crystalline hydroxyapatite surface layer and controlled release of ionic dissolution products [18,19].Furthermore, these ion dissolution and release of BG could exert a genetic control over the osteoblast cell cycle and the rapid expression of osteogenic genes [20,21].These discoveries have stimulated more extensive investigations of BG utilization for bone defect repair.BG decoration on carbon fibers has been demonstrated to promote hydroxyapatite nucleation, biomineralization, and cellular ALP activity [22].BG incorporation in PLGA was also reported to induce BGdose-dependent cell proliferation and ALP expression [23].As such, we hypothesized that the BG might present as a prospective candidate to functionalize GO so as to provide effective environments to modulate structure and function of GO and facilitate stem cells' osteogenic differentiation.
In this study, we designed and fabricated DGO/BG hybrid scaffold using sol-gel method as osteoinductive substrates to manipulate stem cell fate.SEM, Raman, and EDS characterization confirmed the incorporation of BG on GO without compromising their characteristic bands.The cellular activities analysis demonstrated that DGO/BG hybrid scaffolds could significantly promote the adhesion, spreading, and osteogenic differentiation of mesenchymal stem cells, due to their unique nanoscale topography, biomimetic composition, and mechanical cues.

Preparation and Characterization of Graphene.
Graphene oxide (GO) and its reduzate have been successfully prepared using an improved Hummers' method [24] and a reduction process using hydrazine hydrate and a chemical method for the simultaneous reduction and surface functionalization of GO using dopamine hydrochloride.TEM (HRTEM, H-7650B, Hitachi, Japan) was chosen to observe the structure and morphology of the samples.The composition and phase purity of the products were examined by X-ray diffraction spectroscopy (XRD, Rigaku D/max 2500 VB2+/PC, Japan), Raman spectra (LabRAM HR Evolution, HORIBA Jobin Yvon, France), and Fourier transform infrared spectroscopy (FTIR, Avatar 360, Nicolet Co., USA).

Synthesis and Characterization of Dopamine Functionalized Graphene
Oxide/Bioactive Glass (DGO/BG).DGO/BG composite with 1 wt.% and 5 wt.%GO was synthesized by sol-gel-melting method through the following steps just as Figure 1.Tetraethoxysilane (TEOS, Aldrich, USA), calcium nitrate tetrahydrate (CN, Aldrich, USA), and triethyl phosphate (TEP, Aldrich, USA) were used to prepare 58S type BG by sol-gel solution.Briefly, TEP was added into a mixed solvent of distilled water, absolute ethyl alcohol, and ammonia water and stirred at 80 ∘ C for 24 h to obtain a hydrolyzed TEP solution.CN and TEOS were then dissolved into the hydrolyzed TEP solution and stirred continuously at room temperature for 120 h to generate a sol-gel solution.The DGO was dissolved in dimethylformamide (DMF, analytic pure, 99.5 wt.%, Tianjin Fine Chemical Co., China), followed by ultrasonication for 2 h.The preprepared sol-gel solution was added into the solution of DGO to a total concentration of 1 wt.% and 5 wt.%DGO with different ratios.After 2 h of magnetic stirring, suspensions were dried on a hotplate at 80 ∘ C for 12 h.The composite was carbonized at 300 ∘ C for 1 h, 750 ∘ C for 2 h, and then at 1000 ∘ C for 3 h in N 2 atmosphere.Surface topography and elementary composition were observed by SEM and EMAX EX-300 system (Horiba, Japan) to confirm the successful reduction of resulting material as well as its immobilization with dopamine.

Preparation of DGO/BG-Based Substrates.
Coating of glass coverslips with DGO/BG was performed as described below.In order to clean substrate, the glass coverslip with 14 mm diameter was first immersed into the piranha solution (hydrogen peroxide/sulfuric acid with the volumetric ratio of 1 : 3) for 10 minutes at 120 ∘ C, rinsed with ethanol and water for 5 minutes, and then blown dried by nitrogen gas at room temperature.Then the coverslip was immersed in 3% toluene solution of 3-aminopropyltriethoxysilane (3-APTES) for 30 min for functionalization.At last, it was baked with nitrogen at 125 ∘ C after being rinsed with toluene, ethanol, and water for 5 minutes again.30 L DGO/BG was uniformly coated on a coverslip surface by Spin Processor (Smart Coater 100, Analysis China Co., LTD) and then dried with

Immunofluorescence Staining for
Cell Adhesion, Spreading, and Osteogenic Differentiation.Initial adhesion can be measured in early time ranging from 2 h to 1 d and the osteogenic differentiation usually in 7 d [25][26][27].To assess the initial adhesion and osteogenic differentiation of rBMSCs, the cells were incubated with DGO/BG disks for 3 h, 1 d, and 7 d.Immunocytochemistry samples were harvested at each time point and then cells were fixed with 4.0% (w/v) paraformaldehyde for 30 min, permeabilized with 0.1 wt.% (w/v) TritonX-100 for 10 min, and blocked with 3 wt.%bovine serum albumin (BSA) for 1 h at room temperature.Samples were incubated with primary antibodies in 1 wt.%BSA for 2 h against BMP2 (abcam, ab14933), OPN (abcam, ab8448), RUNX2 (abcam, ab76956), and/or vinculin (abcam, ab18058).After removing the primary antibody, samples were incubated with secondary antibodies for 1 h at room temperature: goat anti-rabbit IgG H&L (TRITC) preadsorbed (abcam, ab50598) and goat anti-mouse IgG H&L (FITC) (abcam, ab6785).Then cells were treated with Phalloidin-Atto 565 (Sigma) for 1 h at room temperature and then stained with DAPI (Roche) for 10 min.The figures were captured by laser scanning confocal microscopy (Zeiss, LSM 780, Germany).DAPI was used to quantify cell number.The initial adhesion was evaluated by counting the number of live cells per unit area on each substrate and each group was repeated for three times.

Statistical Analysis. Fluorescence microscopy images
were analyzed utilizing Matlab (MathWorks, Inc., R2011b) software.All quantitative data were expressed as mean ± standard deviation (SD).The one-way ANOVA was performed for statistical analysis.Differences between groups of *  < 0.05 were considered statistically significant and * *  < 0.01 was considered highly significant.

Characteristics of Graphene.
TEM images and digital photographs of graphene before and after the modification are shown in Figure 2(a).The GO sheets and DGO were smooth with folded shapes at the edges and aqueous dispersions of DGO showed a homodisperse state (Figure 2(a)(A), (B)).While following the chemical reduction, G sheets displayed a crumpled and wrinkled structure and the aqueous dispersions appeared to precipitate (Figure 2(a)(C)).
The composition and phase purity of the products were examined by XRD (Figure 2(b)).After reduction and modification, the typical GO peak at 11.28 ∘ disappears, and a new broad diffraction peak appears at 25 ∘ , corresponding to a decrease of interlayer spacing from 0.78 nm to 0.39 nm.Further characterization of the products was carried out by Raman.GO showed a D band at 1336 cm −1 and G band at 1586 cm −1 , respectively.After modification and reduction of GO, the intensity ratio of the D band and G band (ID/IG) increased significantly (Figure 2(c) and Supplementary Figure S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2016/5613980).FTIR was also used to characterize the products in Figure 2(d).The FTIR spectra of GO and DGO showed broad bands ranging from 3400 to 3500 cm −1 .The oxygen-containing functional groups of GO were revealed by the bands at 1054, 1226, 1617, and 1719 cm −1 .

Characterization of DGO/BG
. SEM images showed topography and distribution of pure BG and BG in DGO/BG composites with different loading ratio and reaction temperature (Figures 3 and 4(a), Supplementary Figure S2).In all cases the BG particles showed a round shape.As indicated, BG particles only emerged above reaction temperature of 600 ∘ C with sol-gel method (Figure 3(a)(A)).At 1000 ∘ C, even distribution of BG particles with diameter ranging from 50 to  100 nm was achieved on 1 wt.%DGO/BG; relatively speaking, 5 wt.%DGO/BG composite resulted in more heterogeneous microscale topography with nucleation of BG particles on GO (Figures 3(b)(C) and 3(c)(C)).It should be notified that BG particles appeared to be melted and more aggregated and at 1200 ∘ C, besides fusion and densification, there is also an increase in the particles' size (Figure 3(a)(D)).
Raman spectrum (Figure 4(c)) showed that the characteristic peak of BG is at ∼850 cm −1 .DGO has two broad peaks known as G and D bands (∼1590 cm −1 and ∼1335 cm −1 , resp.).Both 1 wt.%DGO/BG and 5 wt.%DGO/BG have three broad peaks including the characteristic peaks of DGO and BG without apparent shift, suggesting that these characteristic bands were not destroyed during the functionalization process.EDS spectra (Figure 4(b)) showed that C and O elements were detected in pure DGO, the elements of C, O, Si, and Ca were detected in pure BG, and all of this elements can be detected in 1 wt.%DGO/BG and 5 wt.%DGO/BG with different ratio.These results indicated that functionalization of GO with BG has yielded DGO/BG composite scaffold.

Adhesion and Spreading Patterns of rBMSCs on
Bioactive DGO/BG.rBMSCs cell morphology on different substrates after 3 h and 1 day of culture was observed and counted by CLSM after immunofluorescent staining (Figure 5).At 3 h, the rBMSCs presented a round shape on all substrates (Figure 5(a)).The cell area was slightly wider on the 1 wt.%DGO/BG scaffold than other groups.After 1 day, cells cultivated on 1 wt.% and 5 wt.%DGO/BG scaffold showed more spreading, larger area, and more confluence.Cells on DGO and BG substrates showed spindle-shaped morphology with constricted cell area (Figures 5(a) and 5(c)).

rBMSCs
Viability on DGO/BG.Figure 5(e) showed the cell viability of rBMSCs cultured on various samples at 3 h and 1 day.Cell initial adhesion on 1 wt.%DGO/BG yielded the best results followed by 5 wt.%DGO/BG.There was no significant difference between pure BG and 5 wt.%DGO/BG; both of them facilitate the cell initial adhesion, while the cell initial adhesion showed decrease on pure DGO.

Osteogenic Differentiation of rBMSCs on DGO/BG.
BMP2, BSP, RUNX2, and OPN mRNA expression profiles at 7 days are shown in Figure 6.DGO/BG composites promoted higher expression of selected osteogenic genes.When compared to pure DGO and BG, 1 wt.%DGO/BG yielded the best results.BMP2, OPN, and RUNX2 were chosen to have immunofluorescence staining (Figure 7).All of samples allowed for osteogenic differentiation of rBMSCs but led to different differentiation characteristics.The quantitative results demonstrated that 1 wt.%DGO/BG induced significantly higher expression levels of OPN and RUNX2 compared to other samples.DGO and BG substrates show similar osteogenic differentiation propensity.

Discussion
Since the discovery of GO, much attention has been paid on improvement of its potential risks to biological systems so as to extend its application in biomedicine.The GO resulted hemolysis and cytotoxicity could be directly associated with its surfactant-like chemical structure with hydrophilic edges (ionized carboxyl groups) and hydrophobic plane (unoxidized sp2 carbon skeleton), leading to strong electrostatic and hydrophobic interactions between GO and cell membrane.To enhance the biocompatibility of GO, dopamine (DA), a mussel-adhesive-protein inspired molecule, was used to partly reduce the ionized carboxyl groups on GO in this work.
TEM showed GO and DGO sheets have the typical fold morphology.While following the chemical reduction, G sheets displayed a more crumpled and wrinkled structure than GO and DGO, which is due to the lack of mutually exclusive polar groups; graphene atomic layers can stick together more easily by the van der Waals force.
The XRD showed a decrease of interlayer spacing from 0.78 nm to 0.39 nm, indicating that removal of oxygen and water from the interlayer occurred during reduction and modification.This broad peak also suggested a loss of the long range order in graphene.As indicated in Raman, the increase of the intensity ratio of the D band and G band (ID/IG) was corresponding to high level defects with the insertion of functional groups during the modification of graphite oxide.Furthermore, the broad bands of GO and DGO ranging from 3400 to 3500 cm −1 shown in FTIR were corresponding to the O-H stretching vibration of adsorbed water molecules.The oxygen-containing functional groups of GO were revealed by the bands at 1054, 1226, 1617, and 1719 cm −1 .These bands can be assigned to the C-O stretching vibration, C-O-H deformation vibration, C=C stretching vibration, and C=O stretching vibration of COOH group, respectively [28,29].The bands associated with oxygencontaining functional groups decreased or disappeared significantly after reduction.Peak located at 1635 and 1558 cm −1 in the FTIR spectrum of DGO corresponds to the vibration of amide.Furthermore, the absorption peak of hydroxyl and epoxy group decreased after modification because of the reaction of phenolic hydroxyl group on amide and functional groups on GO and the part reduction of GO by amide.The FTIR results further agree with the XRD analyses results.Taken together, the XRD, Raman, and FTIR spectrums indicated that successful removal of oxygen and water from the interlayer occurred during reduction and high level defects with the insertion of functional groups during the modification of graphite oxide.As an amphiphilic molecule, DA has been modified onto various biopolymers, and the as-prepared biopolymers exhibited excellent adhesive ability to many kinds of solid surfaces.More recently, DA has been found to connect with GO by -stacking.In our experiment, DA modification was found to enhance the dispersion stability of GO, and it might also provide catechol groups to facilitate subsequent nucleation of BG on GO.
To improve the bioactivity of DGO in osteogenesis, BG was used in further surface functionalization due to its excellent osteoinductivity and osteoconductivity [21,30,31].Previously, biopolymers such as polysaccharide, protein, and DNA have been used to promote the bioactivity of GO.However, in these studies, the biopolymer loading ratio, the elution of the adsorbed biopolymer, and the 2D surface morphology were difficult to be controlled.In our studies, the influences of different DGO/BG loading ratio and the sol-gel-melting technique on physical and chemical features of final composites were exquisitely controlled.As indicated in Figure 3, BG particles only emerged above reaction temperature of 600 ∘ C with sol-gel method, which proved the previous studies demonstrating that the nucleation of BG was impeded by low temperature [32].Heterogeneous microscale particles presented on 5 wt.%DGO/BG composite scaffold, while homogeneous distribution of nanospheric particles with diameter ranging from 50 to 100 nm was achieved at 1000 ∘ C on 1 wt.%DGO/BG.EDS spectra indicated that these particles are BG.These results indicated that the addition of DGO had generated the porous inner microstructure and coarse surface morphology for BG, which was attributed to the microphase separation between DGO and BG in the blend solution.During the carbonization and pore structure formation, BG precursors transformed into BG nanoparticles by thermodynamic driving force [33].EDS spectra also showed that DGO/BG scaffold possessed the C element of GO and Si and Ca elements of BG.Raman spectrum demonstrated that DGO/BG composite scaffold has the characteristic peaks of both DGO and BG without apparent shift, suggesting that these characteristic bands were not destroyed during the functionalization process.These results indicated that with unique topographical DGO/BG composite scaffolds were achieved with controlled DGO/BG loading ratio and sol-gel-melting technique.
As the first fundamental step of interaction with scaffold, cell adhesion could significantly impact the spreading morphology and capacity of cell proliferation and differentiation [34,35].Therefore, the cell adhesion and subsequent growth are essentially important markers to determine whether materials could be used as scaffolds in bone regeneration.In this study, to investigate whether the functionalization of BG on DGO would influence the initial behavior of rBMSCs, we examined the cell adhesion and spreading patterns by immunofluorescent staining at 3 h and 1 day.The immunofluorescent staining showed that rBMSCs cultivated on DGO/BG scaffolds are more favorable to rBMSCs than DGO and BG substrates with respect to cell adhesion.The stable adhesion of stem cells on substrates is a fundamental and important requirement for regulating stem cell functions.Furthermore, the qualified morphological analysis suggests that 1 wt.% and 5 wt.%DGO/BG scaffolds present much higher cell spreading area.It is widely accepted that enhanced cell spreading is an important factor to be considered for enhancing differentiation of stem cells.Additionally, cells on the DGO/BG substrates have higher cell viability than those on pure DGO and BG substrates regardless of the incorporated DGO/BG molar ratio.These results suggested that the incorporation of DGO and BG has improved the biocompatibility of both of them, yielding a suitable environment for the rBMSCs growth.
A growing number of studies suggested that the physical geometry as well as chemical composition of substrates could direct specific tissue lineages of stem cells [36].Since as achieved DGO/BG has unique properties including topographic features and bioactive chemical components, we expected that DGO/BG scaffold would influence the differentiation of stem cells.Our study shows that all the DGO, BG, and DGO/BG scaffolds can be used to modulate the osteogenesis of rBMSCs.In particular, 1 wt.%DGO/BG scaffold exhibits significantly highest osteogenic differentiation propensity compared to those on the other substrates, indicating that the 1 wt.%DGO/BG scaffold may have great potential for bone defect healing applications.According to the previous research, the effect of GO substrate on promoting stem cell differentiation was intimately related to its high Young's modulus [37] and the surface oxygen functional groups as well as proteins absorption [10,38,39].On one hand, the favorable osteogenic lineage effect of BG was clarified to be ascribed to the release and exchange of critical concentrations of soluble Si and Ca ions.The BG nanoparticles with large surface area/volume ratio have been reported to facilitate quick ions release and high mineralization and have a significant function in genetic control over the cellular response to favor osteogenesis.Meanwhile, extensive works have demonstrated that the BG concentration is critical in cellular response to BG containing composites [40].Moreover, the growth and differentiation of osteoblast-like cells and the kinetics of Ca-P chemistry on PLGA/BG and PDLA/BG films were shown to depend on BG content.As such, although a combination of factors including stiffness, reactive functional groups, and absorption of biomolecules on DGO/BG composite scaffolds could affect the rBMSCs osteogenic behaviors, the unique nanotopography and high bioactive BG content of 1 wt.%DGO/BG scaffold might provide more chances compared to microtopographical 5 wt.%DGO/BG to promote stem cell osteogenic differentiation.These findings suggest that the DGO/BG composite scaffolds could be used as an enabling tool for modulating the osteogenesis of stem cells.

Conclusions
In this study, from evaluation of the biocompatibility of DGO/BG, we found that DGO/BG hybrid scaffolds containing different content of DGO could enhance rBMSCs' adhesion, spreading, and differentiation.The TEM, XRD, Raman, and FTIR spectrums indicated successful synthesis of the typical fold morphology DGO by insertion of functional groups during the modification of graphite oxide.The dopamine modification enhanced the dispersion stability of GO and might provide catechol groups to facilitate subsequent nucleation of BG on DGO with sol-gel method.Scanning electron microscopy (SEM) showed that unique homogeneous nanospheric topography could be achieved on GO by controlling the DGO/BG molar ratio and reaction temperature.The Raman and EDS characterization confirmed the modification of BG on DGO.Higher cellular activities of cell adhesion and spreading were achieved on DGO/BG scaffolds compared to pure DGO and BG substrates.Enhanced cell adhesion and spreading were achieved on 1 wt.% and 5 wt.%DGO/BG scaffolds, indicating the favorable biocompatibility of DGO/BG.The highest expression level of osteogenic markers including BMP2, OPN, and RUNX2 was observed.The significantly high osteogenic markers expression on DGO/BG scaffold suggests that the proposed DGO/BG scaffolds could promote the osteogenic differentiation of rBMSCs.Taken together, we envision that the DGO/BG scaffold will open avenues for next-generation graphene applications in the realm of functional biomaterial.However, further studies in situ are still needed to evaluate their efficacy in promoting bone regeneration and neovascularization.

Figure 1 :
Figure 1: Schematic of process to synthesis DGO/BG by sol-gelmelting method in this study.

Figure 5 :
Figure 5: Adhesion and spreading patterns of rBMSCs on various samples.Immunostaining of vinculin (green), actin (red), and nuclei (blue) for rBMSCs cultured on the samples for 3 hours (a) and 1 day (b): (A) DGO, (B) 1 wt.%DGO/BG, (C) 5 wt.%DGO/BG, and (D) BG.Scale bars, 20 m.The quantitative analysis of cell spreading area (c) and vinculin (d) on different samples.(e) Initial adhesion was evaluated by counting the number of live cells per unit area on each substrate at 3 hours and 1 day, and each group was repeated for three times ( *  < 0.05, * *  < 0.01).

Table 1 :
Primers sequences utilized for real time RT-PCR.
2in air.Cells were seeded at 2.5 × 10 4 cells per cm 2 after two passages.