Synergistic Osteogenesis of Biocompatible Reduced Graphene Oxide

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Tissue Engineering and Regenerative Medicine

Synergistic osteogenesis of biocompatible reduced graphene oxide with methyl vanillate in BMSCs Delong Jiao, Lingyan Cao, Yang Liu, Jiannan Wu, Ao Zheng, and Xinquan Jiang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01264 • Publication Date (Web): 22 Mar 2019 Downloaded from http://pubs.acs.org on March 23, 2019

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ACS Biomaterials Science & Engineering

Synergistic osteogenesis of biocompatible reduced graphene oxide with methyl vanillate in BMSCs Delong Jiaoa, Lingyan Caoa, Yang Liub, Jiannan Wua, Ao Zhenga, and Xinquan Jianga a

Department of Prosthodontics, Shanghai Ninth People’s Hospital, College of

Stomatology, Shanghai Jiao Tong University School of Medicine; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, 639 Zhizaoju Road, Shanghai 200011, China. b

The State Key Laboratory of Bioreactor Engineering, East China University of Science

and Technology, 130 Meilong Road, Shanghai 200237, China. *Corresponding author: Xinquan Jiang; email: [email protected]

Abstract:

Methyl vanillate (MV), a recently characterized small molecule, can promote the Wnt/β-catenin signaling pathway and induce osteoblast differentiation both in vitro and in vivo. On the other hand, graphene-based materials have been introduced into the field of biomedical sciences in the past decade, and graphene oxide (GO), which serves as an

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efficient nanocarrier for drug delivery, has attracted great attention from its biomedical applications in tissue engineering. This study aimed to develop a biocompatible gelatin reduced graphene oxide (GOG) for MV delivery, so as to realize the effective osteogenesis for bone repair. Firstly, GOG was prepared, and its morphology as well as properties were then characterized using scanning electron microscope (SEM), transmission electron microscopy (TEM), atomic force microscope (AFM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and thermal gravimetric analysis (TGA), respectively. In addition, the endocytosis of GOG in bone marrow stromal cells (BMSCs) was also investigated with the treatment of Rhodamine 6G (R6G) labeled GOG. Our results found that GOG could be easily absorbed by cells, which was distributed in both nucleus and cytoplasm, thus suggesting the favorable biocompatibility of GOG. Moreover, the effect of MV on osteogenesis was also tested, the results of which indicated that MV could promote BMSCs osteogenesis in a concentration-dependent manner, and significant enhancement could be achieved at the concentration of 1 µg/mL. In addition, the complex containing different concentrations of GOG and an optimal concentration of MV was used to investigate the synergistic effect between GOG and MV on pro-osteogenesis. The results revealed that, the weight ratio of MV:GOG of 1:1000 could attain remarkably enhanced osteoinduction in BMSCs, as evaluated by Alkaline phosphatase (ALP) assay, Alizarin red S (ARS) staining, immunofluorescence staining and gene expression of related osteogenic markers.


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Taken together, these data had provided strong evidence that the complex of MV and GOG could induce osteogenesis, which was promising for bone tissue engineering.

Key word: Methyl vanillate, Graphene oxide, Wnt/β-catenin, Endocytosis

Introduction Bone defects or fractures are quite commonly seen in daily life; however, large bone defects resulting from tumor resection, skeletal trauma or infection can hardly heal by themselves and may frequently lead to non-unions, as a result, they remain the major challenge for orthopedic, reconstructive, and maxillofacial surgeons1. Many strategies have been developed to repair large bone defects, including allografts and tissue engineered scaffolds; nonetheless, they are often associated with high clinical failure rates, which can be attributed to the insufficient osteogenic activity and slow bone regeneration2. Clinically, administration of agents that can regulate bone metabolism has been considered as a typical and effective therapeutic method for bone healing. Besides, it is predictable that introducing such agents into the artificial bone healing materials can promote their osteogenic activity to accelerate bone regeneration. Currently, an increasing number of studies have suggested that, the Wnt/β-catenin signaling pathway can serve as a novel therapeutic target to treat bone defects3. As is wellknown, multiple signaling pathways are involved in the healing process of natural bone, among which, the Wnt/β-catenin signaling pathway has been reported to play an important



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role in bone metabolism4. Moreover, growing evidence suggests that the activation of the Wnt/β-catenin signaling pathway will promote osteoblast differentiation and the subsequent bone formation, in the meantime of suppressing osteoclastogenesis5. Furthermore, neutralizing inhibitors of the Wnt signaling can improve the bone quality and reduce the risk of bone fracture6. As a result, it is useful and efficient to develop an agent to target the Wnt/β-catenin pathway, so as to enhance the osteogenic activity of artificial bone healing materials. Nowadays, natural products, such as plant extracts as well as their individual ingredients, have become increasingly popular in drug development since they are the traditional medicines7, 8 that are regarded to be relatively safe. Typically, the medicinal plants have huge potential market and wide acceptance9,

10

. However, no clinically

available drug is available so far to target the Wnt/β-catenin pathway with regard to bone metabolic diseases, such as osteoporosis11. Choi et al. had screened 350 plants and identified that the Hovenia dulcis Thunb (HDT) extract together with its ingredient methyl vanillate (MV) could activate the Wnt/β-catenin pathway and induce osteoblast differentiation in vitro11. Notably, HDT is well known in folk medicine, which has been used as a therapeutic drug to treat liver disease. However, few study is available so far on the osteogenic effect of MV, and study regarding its effect on the differentiation of bone mesenchymal stem cells (BMSCs) is even rare.


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Typically, the small molecular drugs are associated with certain retention time in vivo, which can be easily removed by the organism. Therefore, it is necessary to adopt a suitable carrier for drug delivery and activity protection, so as to better exert the drug action. Noticeably, the drug delivery system would fundamentally affect the pharmacological profiles of a majority of therapeutic molecules. Over the past few decades, various kinds of nanomaterials have been utilized to develop the advanced drug delivery systems with efficient drug action and targeted delivery or controlled release of drugs, such as nanoparticles12, liposomes13, carbon nanohorns14, carbon nanotubes15 and nanosheet graphene oxide (GO)16. Among the nanomaterial-based drug carrier family, the graphene nanosheet, which is composed of a flat monolayer of sp2 hybridized carbon atoms, has displayed great potentials in the application of drug delivery since its first report in 200417. As a two-dimensional plane structural material, graphene can provide a larger specific surface area than those of other commonly used materials and can form the strong π-π conjugation with the drug molecules, which can therefore act as a favorable candidate for drug loading18. Besides, it is also reported that, graphene can be taken up by cells by means of endocytosis19; as a result, it is supposed to be potentially used as a drug carrier that can rapidly deliver drugs to the site surrounding or inside cells or into the nucleus, thus better exerting the effect of drugs. On the other hand, graphene and graphene-based materials have displayed great potentials in inducing the osteogenic differentiation of mesenchymal stem cells20-22. However, there is few report on the direct application of graphene or GO for drug delivery until now. Graphene is likely to form aggregations due to its poor



solubility and stability in physiological environments, which has limited its applications in many fields; therefore, it is necessary to modify graphene or graphene oxide. To compensate the disadvantage of graphene and reduce the strong combination between layers, covalent or non-covalent modifications have been applied to introduce the functional groups into their molecular structures, so as to promote the dispersion in fluids23. It is reported that, the chemically reduced GO is a kind of modified graphene with superior solubility and stability, and this approach is also the most commonly used to prepare graphene nanosheets24-26, which can be ascribed to its low cost and mass production. Typically, the reduced GO (rGO) has different physicochemical features compared with those of graphene; for instance, the 60-90% optical transmittance versus 97.7% optical transmittance of graphene, 0.14–0.87 W/mK thermal conductivity versus ~ 5000 W/mK in graphene, and 200-35000 S/cm electrical conductivity versus 104 S/cm in graphene, depending on the reduction agent and fabrication method27. As a kind of graphene-based nanomaterial, the reduced GO was more suitable for biomedical applications, such as drug and gene delivery, antibacterial agents, biosensor, bioimaging and tissue engineering, which can be ascribed to the modification from reduction. Besides, the high surface-tovolume ratio, as well as great π–π stacking, hydrogen bonding and electrostatic or hydrophobic interactions of the reduced GO contribute to loading different drugs with high efficiency (Table 1).

Table 1. Absorption of drugs on reduced graphene oxide (rGO).


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rGO

Reducing reagent

Drugs

Absorption mechanism

Reference

rGO

Sodium borohydride

Sodium diclofenac drug

Hydrogen bonding, electrostatic and π–π interactions

Jauris et al.28

Ciprofloxacin

Hydrogen bonding, π– π interactions and intra-particle diffusion

Ma et al.29

π-π stacking and hydrophobic interactions

Kim et al.30

Liu et al.31

Boruah et al.32

GH

PNTrGO

Ascorbic acid

Hydrazine monohydrate and Doxorubicin ammonia solution

Glucose

Doxorubicin

π–π stacking and hydrophobic interactions

Fe3O4/rGO

Ascorbic acid

Triazine pesticides

Electrostatic, hydrophobic and π–π interactions

PSSG

Hydrazine hydrate

nrGO

Epirubicin

π–π stacking, hydrogen bonding and physical Wang et al.33 absorption

Recently, biological reduction of GO has become increasingly popular for more medical applications. Specifically, the reducing sugar, L-ascorbic acid (AA), or bovine serum albumin (BSA)34, 35 has been selected as the reducing reagent to modify the graphene nanosheets. However, such approach is associated with the limitations of long reaction time (48 h) or strong alkalinity (pH 12). Hence, a nontoxic and environmental friendly



reducing reagent is urgently needed. Interestingly, gelatin has possessed obvious reducibility, since abundant amino side chains on the backbone of its molecular chain can be oxidized to nitrite36. Meanwhile, gelatin is also known as a biocompatible polypeptide, which is the product of the partially hydrolyzed collagen37. Furthermore, gelatin can also be used as an efficient dispersing agent for many colloids38. Importantly, previous reports have revealed that the gelatin-reduced graphene oxide (GOG) is capable of forming the π– π conjugation and the hydrogen bonding interactions between GOG and drugs39. They found that it could act as an ideal carrier which resided mainly in the cytoplasm of MCF-7 breast cancer cell line for drug delivery and the gelatin-mediated sustained release process may have potential clinical advantages in increasing therapeutic efficacy of the anticancer drug doxorubicin. It was hypothesized in this study that, GOG could serve as a delivery vehicle and a specific enhancer of osteogenesis if it was fabricated as a carrier for MV loading. GOG was prepared in this study, and its endocytosis and distribution in BMSCs were also investigated. To the best of our knowledge, this was the first study to investigate the application of GOG in efficiently delivering the Wnt/β-catenin activator into the nucleus of BMSCs. Firstly, the cytotoxic and osteogenic effects of MV were tested at different concentrations. Later, GOG was used to load MV and the release profiles were examined. Afterwards, the synergistic effects between MV and GOG were investigated by detecting the biocompatibility and the pro-osteogenic ability; in addition, the best weight


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ratio of MV to GOG in their complex was also optimized. Finally, immunofluorescence staining of the related osteogenic markers was carried out, and gene expression was detected, so as to evidence the effect of MV/GOG complex on the osteogenic differentiation of BMSCs. Our results showed that GOG was widely located in BMSCs; besides, the proper GOG/MV ratio could enhance the effect of MV on osteogenesis, rendering GOG a promising material in drug delivery.

Materials and methods Isolation and culture of BMSCs BMSCs were isolated and cultured as previously described40. Briefly, primary BMSCs were harvested from Sprague–Dawley (SD) rats. All the experimental protocols regarding the use of animals in this study followed the procedure for Animal Experimental Ethical Inspection of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, and were approved by the Institutional Animal Care and Use Committee. In addition, bone marrow was rinsed with Dulbecco's modified Eagle's medium (DMEM; HyClone, USA) supplemented with 1% penicillin/streptomycin (HyClone, USA). Afterwards, blood cells were removed by centrifugation, and the collection was mixed with complete DMEM containing 10% fetal bovine serum (FBS; Gibco, USA), followed by plating into a tissue culture flask. Subsequently, trypsin digestion and passage of cells were carried out when large colonies were formed and



became confluent. Specifically, BMSCs acquired from Passages 2 to 3 were used in our experiments.

Preparation of gelatin reduced graphene oxide (GOG) In this study, gelatin was used to reduce graphene oxide (GO, XFNANO, China), so as to obtain GOG. To chemically reduce GO into GOG, according to the protocol described by K. Liu et al39, 1 g gelatin was first added into 50 mL water and stirred at 80 °C for 1 h. Then, 50 mL of 0.2 mg/mL GO aqueous dispersion was prepared and added dropwise into 50 mL homogeneous gelatin solution at 80 °C. Later, the mixture was stirred for 1 h before reaction at 95 °C for 24 h under stirring, and the mixture had become the stable black dispersion after reaction. To remove the excess gelatin, the resulting liquid was centrifuged at 20000 rpm and washed with hot water for three times. Finally, the obtained GOG was re-suspended in water at a final concentration of 1.0 mg/mL and stored at 4 °C until use.

Physical characterization of GO and GOG The stability of GO and GOG was investigated based on digital images after suspended in ddH2O, phosphate buffer saline (PBS), alcohol, DMEM and simulated body fluid (SBF) for 0 h, 2 h and 6 h, respectively. In addition, the particle size and zeta potential of GO and GOG were tested using a Nicomp 380 particle sizer (Z3000, PSS, USA), respectively. Moreover, the morphology of GO and GOG was examined using a field


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emission scanning electron microscope (FE-SEM; S4800; Hitachi, Tokyo, Japan) at an accelerating voltage of 15 kV through gold sputter coating. Additionally, the morphology and microstructure of GO and GOG were further characterized by high-resolution transmission electron microscopy (TEM; JEM-2100; JEOL, Japan) at a voltage of 200 kV. Meanwhile, the elements on GO and GOG were analyzed via energy-dispersive X-ray spectrometry (EDS-XRS; QUANTAX 400-30, BRUKER, Germany). Typically, the atomic force microscope (AFM; XE7, Park systems, Korea) was operated in a tapping mode with GO and GOG samples on the fleshly cleaved mica. All Raman spectroscopy of GO and GOG were performed on Horiba LabRam HR Evolution Raman spectrometer using dry samples on a glass substrate. A 50x objective was used for all measurements and the system was used unpolarised with a 514 nm argon ion laser. All spectra were gathered over 10 s exposures using 514 nm wavelength. For spectral analysis, the Gapp peaks of GO and GOG were fitted to a two-Lorentzian peak fit using PeakFit and the fits all achieved an R2 greater than 0.98. X-ray photoelectron spectrum (XPS) of the substrate was recorded on Kratos Axis Ultra DLD surface analysis instrument using an Al Kα radiation (1484.6 eV) as primary excitation. The binding energies were calibrated by C1s (284.8eV). XPS elemental spectra were acquired in the fixed analyzer transmission (FAT) mode with 0.1 eV energy steps at a pass energy of 40 eV. X-ray diffraction (XRD) data were recorded on a Rigaku D/Max-2550V diffractometer using Cu Kα radiation (40 kV and 40 mA, wavelength of 1.5406 Å), while Fourier transform infrared spectroscopy (FTIR) of GO and GOG was measured on Nicolet 380 (Thermo, USA). Besides, thermal gravimetric analysis



(TGA) was performed on a STA 449C simultaneous thermal analyzer (Netzsch) from room temperature to 900 °C at a heating rate of 4 °C/min under a nitrogen atmosphere.

Endocytosis of GOG in BMSCs To visualize the endocytosis of GOG in BMSCs, GOG was labeled with Rhodamine 6G (R6G) in advance. Briefly, 2 mL R6G solution (2 mg/mL) was added into 1 mg GOG to incubate for 2 h under shaking at room temperature. The mixture was then centrifuged at 20000 rpm for 10 min and the R6G labeled GOG was employed to incubate BMSCs. Specifically, BMSCs were seeded onto the glass bottom cell culture dish at a density of 5×104 cells/mL, and the medium containing R6G labeled GOG was subsequently added to replace the culture medium 24 h later. After incubation with the R6G labeled GOG for 2 h, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) solution for 10 min and washed with PBS; afterwards, cytoskeletal β-actin was stained with FITC-labeled phalloidin, whereas the nuclei were stained with DAPI to examine the morphology of adherent cells. Then, the distribution of R6G labeled GOG and its endocytosis in BMSCs were recorded through LEICA TCS SP8 confocal laser scanning microscopy (CLSM) (LEICA, Germany).

MV loading-release kinetics and characterization of MV loaded GOG The ultraviolet-visible (UV-vis) spectra of GO, GOG, MV and MV loaded GOG (M/G) were recorded using the Spectrophotometers (SPARK 10M, Tecan, Austria). To


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load MV onto GOG, 5 μL MV solution (1mg/mL) was incubated with increasing amounts of 100 mg/mL GOG (0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μL) in the total volume of 300 μL PBS buffer for 2 h under shaking at room temperature. After incubation, the mixture was centrifuged at 14000 rpm for 10 min, and the supernatant was collected to monitor the changes in MV concentration after loaded onto GOG, which were determined by UV-vis spectrophotometry at 280 nm. The resulting MV loaded GOG composite was stored at 4 °C for further release kinetic study. Subsequently, 30 mg MV loaded GOG with saturated adsorption of MV was added into 1 mL PBS (or acetate buffer solution) at 37 °C with different pH values (7, 5 and 2) to release MV from the MV loaded GOG. After shaking for different time periods (1, 3, 5, 7, 9, 22, 30, 45, 60, 70, 100, 124, 148, 172 and 268 h), the mixture was centrifuged at 14000 rpm for 10 min, and the supernatant was collected to determine the amount of released MV through the absorbance (OD) value at 280 nm tested by UV-vis spectrophotometry using Spectrophotometers (SPARK 10M, Tecan, Austria). Furthermore, SEM, EDS-XRS, TEM, AFM, Raman, XPS, XRD, FTIR and TGA were also employed to characterize the M/G according to the same methods described above in characterizing GO and GOG, so as to display the changes in GOG after loading with MV.

Osteogenic effect of MV on BMSCs



Cell viability and proliferation after MV treatment at different concentrations (0, 0.01, 0.1, 1, and 10 µg/mL) supplemented in DMEM medium and osteoinductive culture medium (OIM) were assessed by CCK-8 kit. In brief, BMSCs were plated at a density of 5×103 cells into the 96-well plate, and then treated with different concentrations of MV in DMEM medium and OIM, respectively. The culture medium was replaced once at an interval of two days, and BMSCs were incubated for 1, 3 and 7 days according to the CCK8 kit protocol. Finally, the OD value was tested at 450 nm to examine the cell viability. In addition, the live/dead stains of different concentrations of MV were also tested to observe the cytotoxicity of MV using the Calcein-AM/PI Double Stain kit (Yeasen, China) under CLSM (LEICA, Germany). On the other hand, the BMSCs were seeded into the 24-well tissue culture plates (TCPs) at a density of 10×104 cells/well, and cultured with various concentrations of MV in DMEM medium and OIM according to the groups described in CCK-8 assay. Typically, OIM was used as the positive control to evaluate the osteogenic effect of MV on BMSCs. Besides, the alkaline phosphatase (ALP) activity quantitative assay of BMSCs was also performed on days 4 and 7, respectively. In brief, samples from all groups were incubated with para-nitrophenyl phosphate (Sigma-Aldrich, USA) for 60 min at room temperature, and the OD values at 405 nm were recorded to evaluate the ALP activity. Meanwhile, the total protein content was determined using the BCA protein assay kit (Sigma-Aldrich, USA), and the OD values were then normalized to the BSA standard curve at 590 nm.


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Concretely, the relative ALP activity of each group was assessed as the OD value at 405 nm per milligram of total protein, and then all the OD values were normalized to the fold changes, with the control group without MV treatment as the reference (set to 1). The ALP staining was tested according to the manufacture’s instruction (Beyotime, China), and the stained cells were examined under microscope (Eclipse Ti; Nikon, Japan).

Effect of the complex of MV and GOG (M/G) on cell viability The cell viability and proliferation effects of M/G were assessed using the CCK-8 kit. In brief, BMSCs were plated at a density of 5×103 cells/well into the 96-well plate, and then treated with M/G at various weight ratios of 1:0 (M), 1:10 (M/G1), 1:102 (M/G2), 1:103 (M/G3) and 1:104 (M/G4) (at the same concentration of MV at 1 μg/mL, while different concentrations of GOG at 0, 0.01, 0.1, 1 and 10 mg/mL, respectively). The culture medium was replaced once every two days, and BMSCs were incubated for 1, 3 and 7 days according to the CCK-8 protocol. Finally, the OD value was tested at 450 nm to examine the cytotoxicity of M/G. Afterwards, the live/dead staining of different concentrations of M/G was tested to detect the cytotoxicity of different complexes using the Calcein-AM/PI Double Stain kit (Yeasen, China), which was observed by CLSM (LEICA, Germany).

In vitro osteogenesis of the complex of MV and GOG (M/G) Firstly, BMSCs were seeded into the 24-well TCPs at a density of 1×105 cells/well, and then the medium with different concentrations of GOG (0.01, 0.1, 1 and 10 mg/mL)



was replaced in the MV-free control medium, so as to evaluate the effect of GOG on the osteogenic differentiation of BMSCs by ALP staining on days 4 and 7, while the group treated by medium containing 1 μg/mL MV was used as a positive control (MV). Secondly, BMSCs were also seeded into the 24-well TCPs at a density of 1×105 cells/well, and then the medium with different M/Gs was replaced in DMEM medium on the following day, so as to study the synergistic effect of GOG and MV on the osteogenesis of BMSCs by ALP and ARS assays, respectively. All the M/Gs groups had contained 1 μg/mL MV at different weight ratios of MV/GOG, including 1:10 (M/G1), 1:102 (M/G2), 1:103 (M/G3) and 1:104 (M/G4), while GOG concentrations were set at 0.01, 0.1, 1 and 10 mg/mL, respectively. Notably, the DMEM medium supplemented with 1 μg/mL MV but without GOG (M group) served as the control to investigate the stimulating effect of GOG on MV. On days 4 and 7, each plate was fixed with 4% paraformaldehyde for 10 min; afterwards, cells were rinsed with PBS for three times and subjected to ALP staining (Beyotime, China) according to the manufacture's instruction. The stained cells were then examined under a microscope (Eclipse Ti; Nikon, Japan); meanwhile, the ALP activity quantitative assay was also performed according to the same procedure described above. In addition, on days 14 and 21, the fixed cells were stained with 1% Alizarin red S (Sigma-Aldrich) for 10 min, rinsed with water and viewed under a microscope (Eclipse Ti; Nikon, Japan). After the photos of nodules were taken, 10% cetylpyridinium chloride was used to dissolve the nodules for 1 h, and the absorbance was examined at 562 nm. Notably, the culture medium was replaced every other day, and all experiments were performed in triplicate.


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For immunofluorescence staining of Runx2 on day 7 and Col I on day 14, the cultured cells with different M/G complexes were fixed using 4% phosphate paraformaldehyde (Sigma-Aldrich, USA) solution for 10 min, and permeabilized with the freshly prepared 0.5% TritonX-100 (R&D Systems, USA) solution in PBS for 3 min. Subsequently, cells were blocked with 5% BSA for 1 h at 37 °C, followed by incubation with primary antibody (ab23981 for Runx2 and ab90395 for Col I; Abcam, USA) in BSA overnight at 4 °C. Afterwards, cells were incubated with the fluorophore-conjugated goat anti-rabbit (for Runx2) or goat anti-mouse (for Col I) Alexa Fluor® 594 (Abcam, USA) for 1 h at room temperature, and rinsed with PBST for three times among all steps. Typically, DAPI was used to stain the nuclei (blue) while FITC-labeled phalloidin was adopted to stain the cytoskeletal β-actin, and then all cells were subject to CLSM (LEICA, Germany). On the other hand, rat BMSCs were plated into the 12-well plates at a density of 2×105 cells/well and incubated for 24 h, and then the medium containing different M/G complexes was replaced for the osteogenic differentiation of BMSCs. At the same time, cells cultured in the 12-well TCPs containing MV alone were used as the blank controls. Afterwards, the total RNA was isolated from BMSCs on days 4 and 7, and was isolated with the TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Later, RNA concentrations were determined using a NanoDrop spectrophotometer (ND-1000; Thermo, Wilmington, DE, USA), and cDNA was synthesized by a cDNA synthesis reverse transcription kit (Takara, Tokyo, Japan).



Then, cDNA synthesis was performed using a Prime Script First-Strand cDNA Synthesis kit (Takara), and quantitative real-time PCR analysis was carried out using the ROCHE480 real-time PCR system (LightCycler480, Roche, Rotkreuz, Switzerland) to detect the osteogenic markers of RUNX2, ALP, COL I, and OCN, with β-Actin being used as an internal reference. Data were analyzed according to the comparative Ct (2−∆∆Ct) method and were expressed as the fold changes with respect to the control. The primer sequences used in this study were synthesized commercially (Shengong, China). The specific primer sets are listed in Table 2. All experiments were performed in triplicate. The relative expression levels of bone marker genes were calculated on days 4 and 7 compared with those of the controls, and all values were normalized to β-Actin.

Table 2. List of primers used and the respective forward and reverse sequences Gene

Forward and reverse sequences

Runx2-F

5′-ATCCAGCCACCTTCACTTACACC-3′

Runx2-R

5′-GGGACCATTGGGAACTGATAGG-3′

OCN-F

5′-GCCCTGACTGCATTCTGCCTCT-3′

OCN-R

5′-TCACCACCTTACTGCCCTCCTG-3′

ALP-F

5′-TATGTCTGGAACCGCACTGAAC-3′


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ALP-R

5′-CACTAGCAAGAAGAAGCCTTTGG-3′

Col I-F

5′-CAGGCTGGTGTGATGGGATT-3′

Col I-R

5′-CCAAGGTCTCCAGGAACACC-3′

β-Actin-F

5′-GTAAAGACCTCTATGCCAACA-3′

β-Actin-R

5′-GGACTCATCGTACTCCTGCT-3′

Statistical analysis All quantitative data were expressed as means ± SD, and were analyzed using Origin 8.0 (OriginLab Corporation Northampton, MA, USA). Meanwhile, analysis of variance was performed for statistical comparisons. The confidence levels of >95% (p 25; C/O > 500, the subtraction of (D’inf – Gapp) in both GO and GOG, as well as the C/O ratio in GO had all fitted the criterion, with the values of -6.25 cm-1, 2.50 cm-1 and 0.705, respectively. However, the C/O ratio in GOG was 1.081 within the defined range of (1) GO, and such inconsistent result might be related to the small C/O ratio of gelatin and the high O content in GOG after reduction. Consequently, the higher C/O ratio of GOG than GO was still within the range of (1) GO, but the successful reduction of GO to GOG could be verified by the XRD pattern.

Figure 4. Raman and XPS analysis of GO and GOG. (a) Raman spectra and (b) two-peak fits applied to the Gapp peak of GO. (c) the XPS spectra of GO. (d) Raman spectra and (e) two-peak fits applied to the Gapp peak of GOG. (f) the XPS spectra of GOG.



The XRD pattern was investigated to monitor the reduction of GO, as shown in Figure. 5(a), which revealed the disappearance of a characteristic diffraction peak of GO at 10o, indicating the complete reduction of GO by gelatin. Besides, FTIR spectroscopy (Figure. 5(b)) was also employed to investigate the difference in the molecular structure between GO and GOG. As could be observed, the spectra of GO had revealed the presence of oxygen functionality, such as the peaks of –OH (3438.5 cm-1), C=O (1724.5 cm-1), C=C (1628.5 cm-1) and C–O (1051.5 cm-1)51. After reduction, such adsorption bands of oxygen functionality on GO were dramatically decreased or disappeared, which indicated that GOG had been successfully generated. Moreover, compared with the spectra of gelatin, the characteristic peaks at 1540.5 cm-1, 1378.5 cm-1, 1219.5 cm-1 and 610 cm-1 were clearly indicative of the functionalization of GO. Moreover, the thermal behaviors of gelatin, GO and GOG were also investigated by TGA, respectively, in a nitrogen atmosphere, so as to verify the functionalization. As shown in Figure. 5(c and d), GO had exhibited 35 wt% of mass losses at the temperature of < 400 oC, and another 20% of mass loss after heating up to 900 oC; by contrast, gelatin had exhibited about 20 wt% of mass loss at < 300 oC, which might result from the removal of the labile oxo-groups, such as CO, CO2, and H2O vapors, and about another 50 wt% of mass loss was discovered at

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